Compositions and methods for analyzing antigen-specific immune cells of sample

Abstract

Methods of analyzing the presence or absence of immune cells capable of binding to an antigen peptide in a sample from one or more individuals are provided. Also provided are methods of detecting or treating cancer or a tumor, a pathogen infection, or an autoimmune disease. A display moiety is provided having particles associated with a plurality of MHC-peptide complexes, where at least two MHC-peptide complexes are different. Also provided are bait compositions having a plurality of display portions and methods of analyzing immune cells by using the display portions or bait compositions as screening tools.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 463,886, filed May 3, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Reference to the electronic sequence list The contents of the electronic serial number (230062000440SEQLIST.xml; size: 101,021 bytes; and creation date: May 1, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] This application relates to compositions and methods for analyzing antigen-specific immune cells in samples. Background Technology

[0004] The recognition of class I and II molecules of peptide-major histocompatibility complexes (pMHC or MHC-peptide complexes) by immune cells (e.g., T cells) plays a crucial role in controlling intracellular pathogens and cancer cells through effective immune cell stimulation. The overall affinity of TCRs for pMHC complexes is low, and the dissociation rate of this interaction is rapid, making it difficult to detect antigen-specific T cells using soluble monomeric pMHC complexes. To overcome these limitations, MHC multimers have been prepared using antibody dimerization or tetravalent binding of biotin-streptavidin. These MHC dimers or tetramers exhibit increased affinity for their homologous TCRs. To date, MHC multimers have been successfully used for in vitro visualization of antigen-specific CD8 T cells by flow cytometry.

[0005] Traditional MHC tetramers are constructed by tetramerizing C-terminal biotinylated pMHC monomers with streptavidin molecules. MHC tetramers have demonstrated versatility and practicality in detecting αβ T cells via flow cytometry and in situ staining. The well-defined properties of MHC tetramers and their public and commercial availability have led to their widespread use in T cell research. MHC tetramer staining techniques have been extended to new research areas, such as the enrichment of rare T cells, the construction of large libraries and high-throughput epitope screening, cell counting via time-of-flight mass spectrometry (CyTOF), and combined staining for multiplex detection.

[0006] The MHC pentamer, composed of five pMHC complexes arranged in a planar configuration, is commercially available and exhibits higher sensitivity for staining T cells with low-affinity TCRs due to its higher valence state compared to the dimer or tetramer. Recently, another commercially available pMHC complex linked to a dextran backbone, the pMHC dextran multimer, has also been found to be useful for detecting T cells with low-affinity TCRs due to its increased valence state. See, for example, Mol Cells. 2021 May 31; 44(5): 328–334.

[0007] A variety of assays can be used to characterize immune cell responses (such as T cells) to determine the state and capacity of the immune system. These studies can reveal the fundamental mechanisms behind immunity, aiding in the design of clinical diagnostics, helping to develop interventional therapies, and identifying the characteristics of effective immune responses. However, because antigen-specific immune cells typically constitute a very small percentage of the cells available from samples (such as blood samples), the effective detection and analysis of these cells can be challenging.

[0008] All publications, patents, patent applications, and published patent applications mentioned herein are incorporated herein by reference in their entirety. Summary of the Invention

[0009] This application provides a method for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in a sample from one or more individuals. The method includes: a) immobilizing immune cells from the sample onto a solid support, wherein the immune cells: 1) prior to immobilization, have been contacted with a bait composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; or 2) after immobilization, have been contacted with the bait composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; and c) detecting the detection agent to detect immune cells bound to the display portion. In some embodiments, the immune cells have been contacted with the bait composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after immobilization. In some embodiments, the immune cells are contacted with the bait composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after immobilization.

[0010] On the other hand, this application provides a method for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in samples from one or more individuals, the method comprising: a) contacting immune cells with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; b) contacting immune cells with a detection agent that recognizes the display portion under conditions sufficient to cause the agent to bind to the display portion; c) fixing immune cells from the sample onto a solid support; and d) detecting the detection agent to detect immune cells bound to the display portion.

[0011] In some implementations of any of the methods described above, the sample is obtained from a single individual.

[0012] In some embodiments of the methods described above, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of the multiple individual samples is processed individually before being combined to form the mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by the immune cells) by washing the immune cells at least once. In some embodiments, the method further includes identifying the sample from which the immune cells originated by determining a unique barcode associated with the immune cells that have bound the display portion.

[0013] On the other hand, this application provides a method for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in multiple samples from one or more individuals, comprising: a) processing each of a plurality of individual samples separately such that the immune cells contained therein are associated with a unique sample barcode; b) combining the plurality of processed samples to form a sample mixture; c) contacting the sample mixture with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a decoy-sample mixture; d) detecting immune cells bound to the display portion in the decoy-sample mixture; and e) determining a unique barcode associated with the immune cells bound to the display portion, thereby identifying the sample from which the immune cells originated.

[0014] In some embodiments according to any of the above methods, the immune cells include T cells, optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptide in the bait composition. In some embodiments, the immune cells are selected from cytotoxic T cells, memory T cells, and tumor-infiltrating T cells.

[0015] In some embodiments according to any of the above methods, the immune cells include B cells.

[0016] In some embodiments of any of the above methods, the method further includes removing unbound display portions after the immune cells have come into contact with the reagent that recognizes the display portion and before detecting immune cells that have bound the display portion.

[0017] In some embodiments according to any of the methods described above, the antigenic peptide is associated with a cancer or tumor antigen, optionally wherein the antigenic peptide is a neoantigen peptide. In some embodiments, the sample is obtained from one or more individuals who do not exhibit cancer pathology symptoms.

[0018] In some embodiments of any of the methods described above, the antigenic peptide is associated with a pathogen antigen, optionally wherein the antigenic peptide is associated with a viral, bacterial, or fungal antigen.

[0019] In some embodiments according to any of the above methods, antigenicity is an autoantigen peptide associated with an autoantigen.

[0020] In some embodiments according to any of the methods described above, immune cells are immobilized on a solid matrix such as beads. In some embodiments, the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody.

[0021] In some embodiments according to any of the methods described above, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides. In some embodiments, the two or more antigenic peptides in the display portion are identical.

[0022] In some embodiments of the methods described above, a portion of an antigenic peptide library containing mutations associated with cancer or tumors is shown, wherein the library includes at least about 5, 10, 12, 15, 18, or 20 different neoantigen peptides.

[0023] In some embodiments according to any of the above methods, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM; c) being hydrophobic; and d) having a high content of aromatic residues.

[0024] In some embodiments of the methods described above, the antigenic peptide is a neoantigen peptide containing a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus.

[0025] In some embodiments according to any of the methods described above, the antigenic peptide has low immunogenicity.

[0026] In some embodiments according to any of the above methods, a portion is shown comprising an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, more optionally an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally comprises an HLA-A molecule, more optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and more optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the antigenic peptide is about 8 to about 10 amino acids long. In some embodiments, the MHC comprises an MHC class II molecule. In some embodiments, the MHC class II molecule is selected from HLA-DQ and HLA-DR. In some embodiments, the antigenic peptide is about 10 to about 20 amino acids long. In some embodiments, the display portion includes two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01 and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, HLA-A*33:03.

[0027] In some embodiments according to any of the methods described above, the display portion comprises particles. In some embodiments, the particles in the display portion are selected from: surfaces, nanoparticles, beads, and polymers. In some embodiments, the particles in the display portion are dextran particles. In some embodiments, the particles in the display portion are magnetic nanoparticles or polystyrene nanoparticles. In some embodiments, the particles in the display portion are agarose beads or sepharose beads. In some embodiments, the antigenic peptide or MHC is directly attached to the particles in the display portion. In some embodiments, the antigenic peptide or MHC is attached to the particles in the display portion via a binding pair, the binding pair comprising a first binding component attached to the antigenic peptide and a second binding component bound to the particle.

[0028] In some embodiments according to any of the methods described above, the portion comprises a cell. In some embodiments, the cell comprises a polynucleotide encoding an antigenic peptide. In some embodiments, the polynucleotide encodes multiple antigenic peptides.

[0029] In some embodiments according to any of the methods described above, the display portion further includes a detectable marker. In some embodiments, the detectable marker is a fluorophore.

[0030] In some embodiments of any of the methods described above, the method further includes separating and displaying partially bound immune cells, or removing display portions or immune cells that are not bound to each other.

[0031] In some embodiments of any of the above methods, the method further includes phenotypic analysis of the immune cells bound to the display portion.

[0032] In some embodiments of any of the above methods, the method further includes sequencing one or more nucleic acids in the immune cells that bind to the displayed portion, optionally wherein the method further includes analyzing the sequence of one or more nucleic acids, optionally wherein the one or more nucleic acids are TCR, CD8, CD45 sequences or any specific sequence specific to the captured immune cells, optionally wherein analyzing the sequence of one or more nucleic acids includes whole genome sequencing and / or RNAseq sequencing, or by a series of primer sequence-specific q-PCR and / or RT-PCR.

[0033] In some embodiments of any of the methods described above, the method further includes mass spectrometry analysis of the immune cells bound to the display portion.

[0034] In some embodiments of any of the methods described above, the method further includes identifying one or more epigenetic modifications in immune cells, optionally including DNA methylation and / or histone glycosylation.

[0035] In some implementations of any of the methods described above, the individual is a person. In some implementations, the individual is at least about 50 years old.

[0036] In some implementations of the methods described above, the individual a) has not previously been diagnosed with cancer, optionally wherein the individual is at risk of developing cancer, or b) the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment.

[0037] In some implementations of the methods described above, an individual a) is not diagnosed with a pathogen infection, optionally where the individual is at risk of infection, and / or b) does not exhibit pathological symptoms of a pathogen infection, optionally not exhibiting pathological symptoms of a pathogen infection.

[0038] In some implementations of the methods described above, the individual a) has not been diagnosed with an immune response against an autoantigen, optionally the individual is at risk of developing an immune response against an autoantigen, and / or b) does not exhibit pathological symptoms of an immune response (e.g., an autoimmune disease or condition), optionally not exhibiting pathological symptoms of an immune response against an autoantigen.

[0039] In some embodiments of any of the above methods, the sample is selected from blood, plasma, and peripheral blood mononuclear cell (PBMC) samples.

[0040] In some embodiments according to any of the methods described above, the decoy composition comprises a plurality of different display portions. In some embodiments, each of the plurality of different display portions in the decoy composition contains a different antigenic peptide. In some embodiments, the plurality of different display portions in the decoy composition comprises at least two different display portions, each display portion containing a different MHC molecule. In some embodiments, the plurality of different display portions in the decoy composition comprises at least four different display portions, each display portion containing a different MHC molecule. In some embodiments, the plurality of different display portions in the decoy composition comprises at least 100 different display portions, each display portion containing a different MHC molecule. In some embodiments, each of the different display portions containing different MHC molecules contains a different detectable marker. In some embodiments, the different detectable markers include fluorophores.

[0041] In some embodiments according to any of the methods described above, the agent includes an antibody that binds to the displayed portion.

[0042] On the other hand, this application provides a method for detecting antigen-specific immune cells in an individual, comprising: analyzing a sample from the individual according to any of the methods described above, wherein predetermined characteristics of the immune cells indicate a disease or condition in the individual. In some embodiments, the predetermined characteristics of the immune cells include the presence of immune cells. In some embodiments, the presence of immune cells specifically binding to a bait composition containing one or more neoantigens indicates the presence of cancer. In some embodiments, the presence of immune cells specifically binding to a bait composition containing one or more pathogen (e.g., virus, bacteria, fungus) antigens indicates the presence of pathogen infection. In some embodiments, the presence of immune cells specifically binding to a bait composition containing one or more autoantigens associated with an autoimmune disease or condition indicates the presence of an autoimmune disease or condition. In some embodiments, the predetermined characteristics of the immune cells include the number of immune cells above a threshold level. In some embodiments, the predetermined characteristics of the immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics. In some embodiments, the epigenetic modifications include DNA or RNA methylation, hydroxylation characteristics, and histone acetylation, methylation, and / or glycosylation characteristics.

[0043] On the other hand, this application provides a method for detecting residual cancer in an individual who has previously been treated with anticancer therapy and has not exhibited pathological symptoms of cancer after treatment. The method includes analyzing a post-treatment sample from the individual according to any of the methods described above, wherein predetermined characteristics of immune cells from the post-treatment sample indicate residual cancer in the individual. In some embodiments, the method includes: a) analyzing a pre-treatment sample from the individual before anticancer treatment and a post-treatment sample from the individual according to any of the methods described above; and b) comparing the characteristics of immune cells from the pre-treatment sample and the post-treatment sample; wherein predetermined differences in the characteristics of immune cells from the pre-treatment sample and the post-treatment sample indicate residual cancer in the individual.

[0044] This application also provides methods for treating an individual with cancer, including a) diagnosing the individual with cancer according to some of the methods described above; and b) administering anti-cancer treatment to the individual. In some embodiments, the anti-cancer treatment is not immunotherapy. In some embodiments, the cancer is a solid tumor.

[0045] This application also provides methods for treating an individual infection, including a) diagnosing an individual with a pathogen infection according to some of the methods described above; and b) subjecting the individual to therapy against the pathogen.

[0046] On the other hand, this application provides methods for treating an individual's autoimmune disease or condition, including a) diagnosing an individual with an autoimmune disease or condition according to some of the methods described above; and b) subjecting the individual to treatment for the disease or condition.

[0047] In another aspect, this application provides a composition comprising one or more display portions, the display portions comprising particles associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises not less than or more than 2, 3, 4, 5, 6, 8, 9, 10 or 12 MHC-peptide complexes, wherein each of the MHC-peptide complexes comprises at least one MHC molecule complexed with a peptide, wherein at least two or more MHC-peptide complexes are distinct.

[0048] In some embodiments according to any of the foregoing illustrations, one or more of the plurality of MHC-peptide complexes are MHC-peptide monomers, MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers. In some embodiments, each of the plurality of MHC-peptide complexes is an MHC-peptide monomer, an MHC-peptide dimer, or an MHC-peptide trimer. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0049] In some embodiments according to any of the above-described illustrations, the plurality of MHC-peptide complexes on the particles include at least 20 MHC-peptide complexes, such as at least 50 or 100 MHC-peptide complexes.

[0050] In some embodiments according to any of the foregoing illustrations, the MHC-peptide complex is attached to a particle via a binding pair comprising a first binding component attached to the MHC-peptide complex or a unit thereof (i.e., an MHC-peptide monomer) and a second binding component attached to the particle. In some embodiments, the first binding component comprises biotin, and the second binding component comprises streptavidin. In some embodiments, the second binding component comprises biotin, and the first binding component comprises streptavidin.

[0051] In some embodiments according to any of the above-described sections, the particles are solid.

[0052] In some embodiments according to any of the foregoing illustrations, the particle diameter is at least 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 µm. In some embodiments, the particle diameter is from about 0.5 μm to about 20 μm, such as from about 0.5 μm to about 10 μm, or about 1 μm.

[0053] In some embodiments according to any of the above-described embodiments, the surface area of ​​the particles is approximately 1 µm. 2 Approximately 20 µm 2 .

[0054] In some embodiments according to any of the above-described embodiments, the particles have about 10 to about 10,000 associated second binding components.

[0055] In some embodiments according to any of the above-described embodiments, the particles are spherical. In some embodiments, the particles are beads.

[0056] In some embodiments according to any of the above-described sections, the particles are magnetic.

[0057] In some embodiments of any of the foregoing illustrations, the illustrations comprise at least two different MHC molecules. In some embodiments, the at least two different MHC molecules include two different MHC class I molecules. In some embodiments, the at least two different MHC molecules include two different MHC class II molecules. In some embodiments, the at least two different MHC molecules include both MHC class I and MHC class II molecules.

[0058] In some embodiments according to any of the foregoing illustrated portions, the illustrated portion comprises an HLA-A molecule. In some embodiments, the HLA-A molecule comprises a mutation that reduces its binding to CD8. In some embodiments, the HLA-A molecule comprises an HLA A2 heavy chain with an A245V mutation.

[0059] In some embodiments of any of the foregoing display portions, the display portion comprises at least two MHC class I molecules selected from: a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01 and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, HLA-A*33:03.

[0060] In some embodiments of any of the above-described display portions, the display portion comprises MHC class II molecules selected from HLA-DQ and HLA-DR.

[0061] In some embodiments according to any of the foregoing illustrations, the peptide is associated with a cancer or tumor antigen. In some embodiments, the peptide is associated with a neoantigen.

[0062] In some embodiments according to any of the foregoing illustrations, the peptide is associated with an antigen of the pathogen. In some embodiments, the pathogen is a virus, bacteria, or fungus.

[0063] In some embodiments according to any of the above-described sections, the peptide is associated with an autoantigen.

[0064] In some embodiments of any of the foregoing display portions, the display portion contains at least two different peptides. In some embodiments, the display portion contains at least five different peptides. In some embodiments, the display portion contains at least 10, 20, or 50 different peptides. In some embodiments, at least two different peptides in the display portion do not overlap. In some embodiments, at least two different peptides in the display portion overlap. In some embodiments, at least two different peptides are associated with two different antigens (e.g., derived from two different antigens). In some embodiments, the two different antigens contain two different mutations in one or more genes associated with a disease. In some embodiments, the disease is cancer, and the gene is an oncogene. In some embodiments, the disease is not cancer or a tumor (e.g., an infection, such as an autoimmune disease).

[0065] In some embodiments of any of the above-described display portions, the display portion does not contain fluorescent dyes or fluorophores.

[0066] On the other hand, this application provides a decoy composition comprising one or more of the aforementioned display portions. In some embodiments, the decoy composition comprises multiple of the aforementioned display portions. In some embodiments, the decoy composition comprises at least two different display portions. In some embodiments, the decoy composition comprises HLA-A molecules, HLA-B molecules, and HLA-C molecules. In some embodiments, the decoy composition comprises HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the decoy composition comprises at least 25, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition comprises at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the peptides in the decoy composition are associated with mutations in at least two, three, four, five, or six genes. In some embodiments, the gene is an oncogene. In some embodiments, the decoy composition does not contain a fluorescent dye or fluorophore.

[0067] This application, in another aspect, provides a method for analyzing immune cells in a single sample, comprising a) contacting a plurality of immune cells with one or more display portions or any bait composition described herein, and b) detecting or analyzing one or more immune cells bound to one or more display portions. In some embodiments, the immune cells include T cells. In some embodiments, the immune cells are selected from cytotoxic T cells, memory T cells, and tumor-infiltrating T cells. In some embodiments, the T cells are CD4+ T cells and / or CD8+ T cells. In some embodiments, the immune cells include B cells. In some embodiments, the immune cells comprise a mixture of immune cells, said mixture comprising T cells, B cells, macrophages, or dendritic cells, or combinations thereof. In some embodiments, the sample is selected from blood, plasma, and peripheral blood mononuclear cell (PMBC) samples. In some embodiments, the sample is obtained from diseased tissue. In some embodiments, the diseased tissue is cancerous tissue or tumor tissue. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, the individual is a person. In some embodiments, the individual shows no signs of disease. In some embodiments, detecting or analyzing immune cells bound to one or more display portions includes observing the agglutination of immune cells and MHC multimer units. In some embodiments, detecting or analyzing immune cells bound to one or more display portions includes: 1) fixing immune cells onto a solid matrix before or after step a); 2) contacting the fixed immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; and 3) detecting the detection agent to detect immune cells bound to the display portion. Attached Figure Description

[0068] Figure 1 Examples illustrate novel predicted Kras neoantigens (SEQ ID NO: 57-76) derived from the six most common Kras mutations (Kras G12V, Kras G12D, Kras G12R, Kras G12C, Kras G12I, Kras G12A).

[0069] Figure 2 The four most common HLA-A proteins were expressed and purified from *E. coli*, including HLA-A:0201, HLA-A:2402, HLA-A:0301, HLA-A:1101, and β2m. The target proteins were expressed in insoluble inclusion bodies (DPEs).

[0070] Figure 3The assembly of Kras neoantigen libraries with five common mutant types is shown. Four HLA types (HLA-A: 0201, HLA-A: 2402, HLA-A: 0301, HLA-A: 1101) and the four most common Kras mutations (Kras G12V, KrasG12D, Kras G12R, Kras G12C) are represented. Only a portion of the results for the Kras G12V mutation are shown in the figure.

[0071] Figure 4 This study demonstrates the confirmation of luciferase intensity in a constructed pancreatic tumor cell line. D-luciferin was used as a substrate. The signal was captured 15 minutes after intraperitoneal injection of D-luciferin.

[0072] Figure 5 Depicting vaccination of 1×10 5 Or 1×10 6 Tumor size 12 days after Pano2-Luc-GFP tumor cell treatment (Figure A) and tumor size after subcutaneous attack with 1×10⁶ cells. 6 Tumor growth curve of Pano2-Luc-GFP tumor in C57BL / 6J mice, measured by tumor volume after tumor cell count (Figure B).

[0073] Figure 6 The construction of plasmids overexpressing five common Kras mutations was depicted. The band size was approximately 750 bp. Two equal aliquots were loaded, and bands smaller than 50 bp were considered nonspecific.

[0074] Figure 7A and 7B Depicting in 4×10 5 Or 1×10 6 Kras-mutated neoantigen-specific CD8 was detected in mouse peripheral blood as early as day 4 after intravenous tumor cell attack. + cell.

[0075] Figure 8 Depicting in 1×10 4 Or 1×10 5 Kras-mutated neoantigen-specific CD8 was detected in mouse peripheral blood as early as day 4 after intravenous tumor cell attack. + cell.

[0076] Figure 9A and 9B Depicting in 4×10 5 Kras-mutated neoantigen-specific CD8 was detected in the peripheral blood of mice as early as day 4 after subcutaneous tumor attack. + T cells. As shown in the figure, tetramers were detected in mice challenged with PanO2-n peptide. + CD8+ T cell population (range: 0.042% to 0.22%, median: 0.0866%), but tetramers were not detected in mice challenged with Pan02-EV cells. + CD8 + T cells.

[0077] Figure 10A and 10B The inoculation of 4 × 10⁶ cells was described by bioluminescence assessment. 5 Tumor growth curves of mice with Pan02-EV (a neoantigen that does not express the Kras mutation) and Pan02-n (a neoantigen that expresses the Kras mutation) tumor cells ( Figure 9A Images of mice on day 4 after tumor attack show no tumor observed at the inoculation site (red circle). Figure 9B ).

[0078] Figure 11A-11C The study depicted the Kras mutation-specific neoantigen-specific CD8+ in a Kras mutation-specific tetramer library in the peripheral blood of pancreatic cancer patients (patients #1-13). + The presence or absence of T cells.

[0079] Figure 12 The study depicted CD8 in thirteen patients diagnosed with pancreatic disease and with or without Kras mutations and a specific HLA-A phenotype. + Summary of the presence or absence of T cells. In this experiment, the bait composition contained twenty MHC tetramers associated with the G12D neoantigen peptide, but only four tetramers associated with the G12R neoantigen peptide.

[0080] Figure 13 A schematic diagram and process of MHC class I tetramer (MHC-tetramer) synthesis are depicted. (A) Composition and assembly of MHC peptide monomers. (B) Schematic diagram of MHC-tetramer binding to specific CD8+ T cells in the blood of cancer patients and MHC-tetramer targeting strategies. Exemplary targets are B2M, streptavidin, biotin, and any labeled tags such as PE, APC, or His.

[0081] Figure 14The main procedures of a sandwich ELISA are described. (A) The method is performed stepwise in the order shown. Step 1 involves coating an ELISA plate with a capture antibody. Any antigens in the sample (e.g., blood or cell supernatant) then bind to the capture antibody. Any excess sample is washed off the plate. In step 3, a detection antibody labeled with horseradish peroxidase or alkaline phosphatase is added, which binds to any target antigens already bound to the capture antibody. Finally, a substrate (e.g., a chromogenic agent) is added to the plate to convert the substrate (e.g., TMB or ABTS) into a colored product that can be measured using a plate reader. (B) A detailed ELISA protocol is shown in the figure. (C) An exemplary method. MHC-tetramer binds to specific TCRs in CD8+ T cells in a patient's blood or cells, and the CD8+ cells are then captured by CD8 antibodies coated on a plate or conjugated to magnetic beads. HRP detection antibodies can be used to identify the captured CD8 antibodies using a plate reader. Alternatively, cells can be incubated with CD8 antibodies coated on a plate or conjugated to magnetic beads, and then MHC-tetramer can be added to the resulting cells. HRP detection antibodies (primarily targeting the MHC-tetramer component) can then be added to detect MHC-tetramer-bound CD8+ T cells.

[0082] Figure 15 Schematic diagrams and procedures of exemplary methods for capturing CD8+ T cells that specifically bind to MHG-tetramers are depicted. (AB) For example, MHC-tetramers with different tags are first added to immune cells. Then, magnetic beads are added as shown in step 2. In step 3, the presence of MHC-tetramers binding to CD8+ T cells is identified using an HRP antibody. (C) First, CD8 antibody-conjugated magnetic beads are added to immune cells. Then, MHC-tetramers with different tags are added as shown in step 2. In step 3, the presence of MHC-tetramers binding to CD8+ T cells is identified using an HRP antibody.

[0083] Figure 16 The strategy for clinical sample collection and tumor neoantigen-specific CD8+ T cell identification via ELISA is described.

[0084] Figure 17 The main procedure and results of HRP-β2-microglobulin antibody detection of MHC-tetramer-bound CD8+ T cells are described. (A) The main method is shown step-by-step. Cells are mixed with MHC-tetramer and then incubated with magnetic beads conjugated with CD8 antibody. HRP-β2-microglobulin antibody is a detection antibody used to identify MHC-tetramer-bound CD8+ T cells. (B) Results of detecting MHC-tetramer-bound CD8+ T cells using the method described in (A).

[0085] Figure 18The main procedure and results of detecting MHC tetramers bound to CD8+ T cells using HRP-PE antibody are described. (A) The main method is shown step by step. First, cells are mixed with magnetic beads conjugated with CD8 antibody, and then incubated with MHC tetramers. HRP-PE antibody is a detection antibody used to identify MHC tetramers bound to CD8+ T cells. (B) Results obtained according to the method shown in (A).

[0086] Figure 19 The main procedure and results of detecting MHC-tetramer bound to CD8+ T cells using HRP-PE antibody are described. (A) An improved method for capturing MHC-tetramer bound to CD8+ T cells using HRP-PE antibody. In this protocol, the tube is replaced with a new tube after the MHC-tetramer washing step. The tube can also be replaced during the CD8 conjugated bead washing step. (B) Results obtained using the improved method shown in (A). There is a 3-fold difference between group 1 and group 4. Representative photograph of results (B). Two copies.

[0087] Figure 20 The scheme of the example method is described.

[0088] Figure 21 A high-throughput identification protocol for pancreatic neoantigen-specific immune cells (e.g., CD8+ T cells) in a population was described.

[0089] Figure 22 An exemplary list of HLA subtypes and high-frequency tumor mutations used in the construction of tetramer libraries for analyzing early pancreatic cancer is provided. Figure 23 A schematic diagram depicts antibodies with DNA barcode tags targeting CD45 or CD8 specific T cells.

[0090] Figure 24 A schematic diagram depicts an exemplary method for identifying DNA barcoded CD8+ T cells from multiple pancreatic cancer patients using ELISA.

[0091] Figure 25 A schematic diagram depicts another exemplary method for capturing antigen-specific immune cells (e.g., neoantigen-specific CD8+ T cells) using magnetic beads and DNA barcode-based ELISA assays.

[0092] Figure 26 A schematic diagram depicts another exemplary method for capturing antigen-specific immune cells (e.g., neoantigen-specific CD8+ T cells) via ELISA assays based on tetramer-beads and DNA barcoding.

[0093] Figure 27AA schematic diagram depicts another exemplary method for capturing antigen-specific immune cells (e.g., neoantigen-specific CD8+ T cells) via MHC-tetramer-beads bound to multiple antigenic peptides and for DNA barcoding-based ELISA assays.

[0094] Figure 27B A schematic diagram depicts another exemplary method for capturing antigen-specific immune cells (e.g., neoantigen-specific CD8+ T cells) via MHC-tetramers that bind to multiple antigenic peptides and for DNA barcoding-based ELISA assays.

[0095] Figure 27C Examples are given of Kras neoantigens (SEQ ID NO: xx-xx) derived from Kras mutations (Kras G12V, Kras G12D, Kras G12R).

[0096] Figure 28A The characteristics of MHC-tetramers and MHC-tetramer beads on PAGE were described.

[0097] Figure 28B The method described describes the detection of Kras mutant neoantigen-specific CD8 in mouse peripheral blood using MHC-tetramer-bead assays. + T cells.

[0098] Figure 28C The study depicted the Kras mutant neoantigen-specific CD8 in mouse peripheral blood via MHC-tetramer-beads in an agglutination assay. + Results of T cell activity.

[0099] Figure 28D The study describes the detection of Kras mutant neoantigen-specific CD8 in mouse spleen using MHC-tetramer-bead assays. + T cells.

[0100] Figure 28E The study depicted the specific CD8 antigen of the Kras mutant neoantigen in mouse spleen via MHC-tetramer-beads in agglutination assay. + Results of T cell activity.

[0101] Figure 29A The MHC-β-galactosidase beads and Kras G12D MHC monomers were characterized on PAGE.

[0102] Figure 29B The MHC-β-galactosidase beads and Kras G12D MHC monomers and MHC-tetramers were characterized on PAGE.

[0103] Figure 29CThe method described describes the detection of Kras G12D neoantigen-specific CD8 in mouse peripheral blood via MHC-tetramer-bead assay. + T cells.

[0104] Figures 29D-29E The method described describes the detection of KrasG12D neoantigen-specific CD8 in mouse spleen via MHC-tetramer-bead assay. + T cells.

[0105] Figure 30A The characteristics of MHC monomers bound to multiple antigenic peptides on PAGE were described.

[0106] Figure 30B The characteristics of MHC-tetramers bound to multiple antigenic peptides on PAGE were described.

[0107] Figure 30C Peptide standards for LC-MS / MS detection are described.

[0108] Figure 30D The method for detecting antigenic peptides from PAGE is described.

[0109] Figure 30E The characteristics of MHC-tetramers bound to multiple antigenic peptides were described in LC-MS / MS detection.

[0110] Figure 30F The characteristics of MHC-monomers and MHC-tetramers on PAGE were described.

[0111] Figure 31A The study described the detection of Kras G12V neoantigen-specific CD8 in mouse spleen using MHC monomer beads. + T cells.

[0112] Figure 31B This describes a method for detecting Kras G12D neoantigen-specific CD8 in human peripheral blood using MHC monomer beads. + T cells.

[0113] Figure 31C This describes a method for detecting Kras multispecific CD8 in human peripheral blood using MHC monomer beads containing multiple antigenic peptides. + T cells.

[0114] Figures 31D-31E This describes a method for detecting stimulated Kras multispecific CD8 in human peripheral blood using MHC monomer beads containing multiple antigenic peptides. + T cells. Detailed Implementation

[0115] This application provides a method for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in samples from one or more individuals. In some embodiments, the presence of such immune cells indicates the presence of a disease or ailment in the individual. This application is based, at least in part, on the inventors' unique insight that immune cells activated by specific antigens (e.g., tumor neoantigens) can be detected in an individual even before any pathological symptoms of a disease or ailment (e.g., cancer) appear, thus serving as a useful marker for the early detection of such a disease or ailment.

[0116] As shown in the Examples section, tumor cells expressing Kras mutation-associated neoantigens (down to 10) 4 Individual cells, see, for example Figure 8 In mice that were intravenously or subcutaneously injected, and as early as day 4 post-inoculation (see example...) Figures 9A-9B ), prior to any detection via bioluminescence (see, for example) Figures 10A-10B ), or in pancreatic cancer patients carrying Kras mutations (see, for example) Figure 11A-11C (12) Exemplary Kras mutation-associated neoantigen-specific T cells were successfully detected. These data demonstrate that the presented method can be successfully used for cancer detection, including early cancer detection and for monitoring cancer cells in individuals who may have minimal residual cancer cells.

[0117] This application also provides an innovative method for effectively, cost-effectively, and efficiently detecting the presence of antigen-specific immune cells using immunoassays such as ELISA, even when their abundance in a sample is very low (e.g., less than 1%). The method of this application has been demonstrated to successfully detect neoantigen-specific immune cells in cancer patients and pathogen-specific immune cells in patients infected with pathogens. Some embodiments of the method exhibit surprisingly high sensitivity. Furthermore, the method enables high-throughput analysis of mixed samples with immune cells from multiple individuals (e.g., 10-30 individuals) by associating unique barcodes with immune cells from each individual. See, for example, Examples 3 and 4.

[0118] This application provides a method for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in samples from one or more individuals, the method comprising: a) immobilizing immune cells from the sample onto a solid support, wherein the immune cells: 1) prior to immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; or 2) after immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient to cause the agent to bind to the display portion; and c) detecting the detection agent thereby detecting immune cells bound to the display portion.

[0119] On the other hand, this application provides a method for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in samples from one or more individuals, the method comprising: a) contacting immune cells with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; b) contacting immune cells with a detection agent that recognizes the display portion under conditions sufficient to cause the agent to bind to the display portion; c) fixing immune cells from the sample onto a solid support; and d) detecting the detection agent to detect immune cells bound to the display portion.

[0120] On the other hand, this application provides a method for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in multiple samples from one or more individuals, comprising: a) processing each of a plurality of individual samples separately such that the immune cells contained therein are associated with a unique sample barcode; b) combining the plurality of processed samples to form a sample mixture; c) contacting the sample mixture with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a decoy-sample mixture; d) detecting immune cells bound to the display portion in the decoy-sample mixture; and e) determining a unique barcode associated with the immune cells bound to the display portion, thereby identifying the sample from which the immune cells originated.

[0121] It also provides methods for detecting antigen-specific immune cells in an individual, methods for detecting residual cancer, and methods for treating cancer, infections, or autoimmune diseases.

[0122] On the other hand, this application provides compositions comprising a display portion containing particles associated with a plurality of MHC-peptide complexes. In some embodiments, the plurality of MHC-peptide complexes comprises more than 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises at least one MHC molecule complexed with a peptide, and wherein at least two or more MHC-peptide complexes are distinct. In some embodiments, the MHC-peptide complexes are MHC-peptide monomers. In some embodiments, the particles are solids. In some embodiments, the plurality of MHC-peptide complexes comprises at least 20, 50, or 100 MHC-peptide complexes.

[0123] As demonstrated by examples, the display portions described herein are a powerful tool for simultaneously screening antigen-specific immune cells from an individual capable of recognizing multiple peptides (e.g., neoantigen peptides) with high sensitivity and specificity. These display portions aggregate antigen-specific immune cells by binding multiple heterogeneous MHC-peptide complexes to particles, making them easily observable under a microscope or with the naked eye. Therefore, it provides a powerful, sensitive, cost-effective, and convenient platform technology for screening, detecting, and analyzing antigen-specific immune cells in various contexts (e.g., cancer, autoimmune diseases, infections).

[0124] This application also provides a decoy composition comprising a display portion, a method for analyzing or detecting antigen-specific immune cells using the display portion or the decoy composition (such as those described above), and a method relating to treating a patient using the method for detecting antigen-specific immune cells described herein.

[0125] definition Generally, the terms used in the claims and specification are intended to be interpreted in the ordinary sense as understood by one of ordinary skill in the art. Certain terms are defined below to provide additional clarity. In the event of a conflict between the ordinary sense and the provided definitions, the provided definitions will prevail.

[0126] The term "antigen" used in this article refers to substances that induce an immune response.

[0127] As used herein, the term “neoantigen” is an antigen with at least one alteration that distinguishes it from the corresponding wild-type parent antigen, for example, through mutations in tumor cells or tumor cell-specific post-translational modifications. Neoantigens may include polypeptide sequences. Mutations leading to neoantigens may include frameshift or non-frameshift insertions / deletions, missense or nonsense substitutions, splice site alterations, genomic rearrangements or gene fusions, or any genomic or expression alteration that results in a new ORF. Mutations may also include splice variants. Tumor cell-specific post-translational modifications may include aberrant phosphorylation. Tumor cell-specific post-translational modifications may also include proteasome-generated spliced ​​antigens. See Liepe et al., A large fraction of HLA class I ligands are proteasome-generated spliced ​​peptides; Science. 2016 Oct 21;354 (6310):354-358.

[0128] The term “tumor neoantigen” or “cancer neoantigen” as used in this article refers to neoantigens that are present in the subject’s tumor cells or tissues but not in the subject’s corresponding normal cells or tissues.

[0129] The term “missense mutation” as used in this article refers to a mutation that results in one amino acid being replaced by another.

[0130] The term "speechless mutation" as used in this article refers to a mutation that results in an amino acid being replaced by a stop codon.

[0131] The term “frameshift mutation” as used in this article refers to a mutation that alters the protein framework.

[0132] The term “insertion deletion” as used in this article refers to the insertion or deletion of one or more nucleic acids.

[0133] As used herein, the term percentage “identity” in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences having a specific percentage of identical nucleotide or amino acid residues when compared and aligned using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection to obtain maximum correspondence. Depending on the application, percentage “identity” may be present within a region of the sequences being compared, such as within a functional domain, or across the entire length of the two sequences to be compared.

[0134] For sequence comparisons, a sequence typically serves as a reference sequence to be compared with the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates and algorithm parameters are specified if necessary. The algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the specified parameters. Alternatively, sequence similarity or dissimilarity can be determined by the presence or absence of specific combinations of nucleotides, or, for translated sequences, by the presence or absence of combinations of amino acids at selected sequence positions (e.g., sequence motifs).

[0135] As used in this article, the term "epitope" refers to a specific part of an antigen that is typically bound by an antibody or T-cell receptor.

[0136] As used in this article, “immunogenicity” refers to, for example, the ability to elicit an immune response via T cells, B cells, or both.

[0137] The terms “HLA binding affinity” and “MHC binding affinity” used in this article refer to the binding affinity between a specific antigen and a specific MHC allele.

[0138] As used herein, the term "decoy composition" refers to a composition comprising molecules (e.g., antigenic peptides) for enriching cells that specifically bind to the decoy from a sample. It should be clarified that the term "antigenic peptide" as used herein is not limited to peptides capable of inducing an immune response. It can be used interchangeably with "peptide" to include any peptide that can be presented by MHC molecules.

[0139] The term "variant" as used in this article refers to the difference between the nucleic acids of the subjects and the reference human genome used as a control.

[0140] As used in this article, “allelic gene” refers to a version of a gene, a version of a gene sequence, or a version of a protein.

[0141] The term “HLA type” used in this article refers to the complementation of HLA gene alleles.

[0142] As used in this article, the term "exome" refers to a subset of the genome that encodes proteins. An exome can be a collection of exons in the genome.

[0143] As used in this article, the term "proteome" refers to the collection of all proteins expressed and / or translated by a cell, cell population, or individual.

[0144] The term “dextran multimer” as used in this article is a dextran-based peptide-MHC multimer used for antigen-specific immunocellular staining in flow cytometry.

[0145] As used herein, the term "MHC multimer" is a peptide-MHC complex that comprises multiple peptide-MHC monomer units.

[0146] As used herein, the term "MHC tetramer" is a peptide-MHC complex comprising four peptide-MHC monomer units. As used herein, the term "MHC monomer" is a peptide-MHC complex comprising one peptide-MHC monomer unit. As used herein, the term "MHC dimer" is a peptide-MHC complex comprising two peptide-MHC monomer units. As used herein, the term "MHC trimer" is a peptide-MHC complex comprising three peptide-MHC monomer units.

[0147] As used in this article, the term "latex agglutination assay (LAT)" refers to a class of bioanalytical methods used to detect the presence (qualitative) and sometimes the amount (quantitative) of substances in a sample. In the presence of specific substances (such as antigens, antibodies, or cells), latex particles aggregate to form clumps or particle aggregates.

[0148] As used herein, the term "latex beads" refers to spherical particles, such as those made of polystyrene, poly(methyl methacrylate), polyacrylate, or other similar polymers. These latex beads vary in size, typically ranging from nanometers to micrometers in diameter. Their surfaces can be functionalized or modified to allow the attachment of biomolecules, compounds, or other substances. Latex beads can be used in a variety of applications, including but not limited to diagnostic assays, bioseparation, drug delivery, and as standards or controls in various analytical techniques.

[0149] The term "sample" as used in this article refers to aliquots of bodily fluids or tissues containing immune cells obtained from subjects.

[0150] The term "mammal" encompasses both humans and non-humans, including but not limited to humans, non-human primates, canines, felines, rodents, bovines, equines, and pigs.

[0151] As used herein, “treatment” or “treating” is a method for achieving a beneficial or desired outcome (including clinical outcomes). For the purposes of this application, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing remission of the disease (partial or complete), reducing the dosage of one or more other medications required to treat the disease, delaying the progression of the disease, improving or enhancing quality of life, increasing weight gain, and / or prolonging survival. “Treatment” also encompasses reducing the pathological consequences of cancer (such as, for example, tumor volume). The methods of this application consider any one or more aspects of treatment.

[0152] As used herein, “reference” means any sample, standard, or level used for comparative purposes. References may be obtained from healthy and / or disease-free samples. In some instances, references may be obtained from untreated samples. In some instances, references may be obtained from disease-free or untreated samples of individuals. In some instances, references may be obtained from one or more healthy individuals who are not individuals or patients.

[0153] The terms “subject,” “individual,” or “patient” are used interchangeably herein to refer to mammals, including but not limited to humans, bovines, horses, felines, canines, rodents, or primates. In some embodiments, the individual is a human.

[0154] It should be understood that the embodiments of this application described herein include “consisting of embodiments” and / or “substantially consisting of embodiments”.

[0155] In this document, mentioning "about" a value or parameter includes (and describes) variations relating to that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0156] As used in this article, mentioning "not" a value or parameter usually means and describes something that is "different" from a value or parameter. For example, "the method is not used to treat type X cancer" means that the method is used to treat a type of cancer different from X.

[0157] The term “about XY” as used in this article has the same meaning as “about X to about Y”.

[0158] It should be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” used in the specification and appended claims include a plurality of indicators.

[0159] Any term not directly defined herein should be understood to have the meaning generally associated with it as understood within the field of this invention. Certain terms are discussed herein to provide additional guidance to practitioners in describing compositions, apparatuses, methods, etc., and how they are made or used in relation to aspects of the invention. It should be understood that the same thing can be expressed in more than one way. Therefore, alternative language and synonyms may be used for any one or more of the terms discussed herein. Whether a term is elaborated or discussed herein is not important. Several synonyms or alternative methods, materials, etc., are provided. Unless expressly stated, the use of one or more synonyms or equivalents does not preclude the use of other synonyms or equivalents. The use of examples, including instances of terms, is for illustrative purposes only and does not limit the scope and meaning of aspects of the invention.

[0160] Methods for analyzing samples from one or more individuals This application provides methods for analyzing samples from one or more individuals, such as analyzing the presence or absence of immune cells capable of binding to antigenic peptides. Such methods can be further used, for example, to diagnose diseases or disorders (e.g., cancer, infection, autoimmune cells).

[0161] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) immobilizing immune cells from the samples on a solid support, wherein the immune cells: 1) prior to immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; or 2) after immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; and c) detecting the detection agent to detect immune cells bound to the display portion, wherein the antigenic peptide is associated with a cancer or tumor antigen, optionally wherein the antigenic peptide is a neoantigen peptide. In some embodiments, the samples are obtained from one or more individuals who do not exhibit pathological symptoms of cancer. In some embodiments, the individual a) has not previously been diagnosed with cancer, optionally wherein the individual is at risk of developing cancer, or b) the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment. In some embodiments, the neoantigen peptide contains a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of the multiple individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with a molecule containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once. In some embodiments, before fixation, the immune cells have been contacted with a bait composition containing a display portion containing the antigen peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, the immune cells are contacted with a bait composition containing a display portion containing the antigen peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of the antigenic peptides in the bait composition that specifically bind to them. In some embodiments, the immune cells are immobilized on a solid matrix such as beads, optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides.In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the exhibited portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, more optionally wherein the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises a class I molecule. In some embodiments, the class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the class I molecule optionally comprises an HLA-A molecule, more optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and more optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other, for example by washing the immune cells at least 1, 2, or 3 times.

[0162] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) immobilizing immune cells from the samples onto a solid support, wherein the immune cells: 1) prior to immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; or 2) after immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; and c) detecting the detection agent to detect immune cells bound to the display portion, wherein the antigenic peptide is associated with a pathogen antigen, optionally wherein the antigenic peptide is associated with a viral, bacterial, or fungal antigen. In some embodiments, the individual a) has not been diagnosed with a pathogen infection, optionally wherein the individual is at risk of having said infection, and / or b) does not exhibit pathological symptoms of a pathogen infection, optionally not exhibiting pathological symptoms of a pathogen infection. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of the multiple individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once. In some embodiments, before fixation, the immune cells have been contacted with a bait composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, the immune cells are contacted with a bait composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptide in the bait composition. In some embodiments, immune cells are immobilized on a solid matrix such as beads, optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the display portion contains two or more antigenic peptides, optionally wherein the display portion contains four antigenic peptides.In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the exhibited portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, more optionally wherein the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises a class I molecule. In some embodiments, the class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the class I molecule optionally comprises an HLA-A molecule, more optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and more optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0163] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in multiple samples from one or more individuals, comprising: a) immobilizing immune cells from the samples onto a solid support, wherein the immune cells: 1) prior to immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; or 2) after immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; and c) detecting the detection agent to detect immune cells bound to the display portion, wherein the antigenicity is an autoantigen peptide associated with an autoantigen. In some embodiments, the individual a) is not diagnosed with an immune response to an autoantigen, optionally wherein the individual is at risk of developing an immune response to an autoantigen, and / or b) does not exhibit pathological symptoms of an immune response (e.g., autoimmune disease or condition), optionally not exhibiting pathological symptoms of an immune response to an autoantigen. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of the multiple individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once. In some embodiments, before fixation, the immune cells have been contacted with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, the immune cells are contacted with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of the antigenic peptides in the bait composition that specifically bind to them. In some embodiments, the immune cells are immobilized on a solid matrix such as beads, optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides.In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the exhibited portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, more optionally wherein the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises a class I molecule. In some embodiments, the class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the class I molecule optionally comprises an HLA-A molecule, more optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and more optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0164] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) immobilizing immune cells from the samples onto a solid support, wherein the immune cells: 1) prior to immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; or 2) after immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient to cause the agent to bind to the display portion; and c) detecting the detection agent to detect immune cells bound to the display portion, wherein the immune cells are immobilized on a solid matrix such as beads, and optionally wherein the beads are bound to an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the samples are obtained from a single individual. In some embodiments, the samples are a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of a plurality of individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once. In some embodiments, before fixation, the immune cells have been contacted with a bait composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, the immune cells are contacted with a bait composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptide in the bait composition. In some embodiments, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity for MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity for homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the illustrated portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, and more optionally the MHC multimer is an MHC tetramer or an MHC dextran multimer.In some embodiments, the MHC molecule includes MHC class I molecules. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0165] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in multiple samples from one or more individuals, comprising: a) immobilizing immune cells from the samples onto a solid support, wherein the immune cells: 1) prior to immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; or 2) after immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient to cause the agent to bind to the display portion; and c) detecting the detection agent to detect immune cells bound to the display portion, wherein the display portion comprises a library of antigenic peptides of mutations associated with cancer or tumor, pathogen, or self-antigen, wherein the library comprises at least about 5, 10, 12, 15, 18, or 20 different antigenic peptides (e.g., neoantigen peptides). In some embodiments, the samples are obtained from a single individual. In some embodiments, the samples are a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of a plurality of individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once. In some embodiments, before fixation, the immune cells have been contacted with a bait composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, the immune cells are contacted with a bait composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptide in the bait composition. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity for MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity for homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the demonstrator portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, and more optionally the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule.In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0166] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) immobilizing immune cells from the samples onto a solid support, wherein the immune cells: 1) prior to immobilization, have been contacted with a bait composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; or 2) after immobilization, have been contacted with a bait composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; and c) detecting the detection agent to detect immune cells bound to the display portion, wherein the antigenic peptide 1) has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM for MHC molecules; b) having a binding affinity of about 1 nM to about 5000 nM for homologous TCR molecules; c) being hydrophobic; and d) having a high content of aromatic residues; and / or 2) having low immunogenicity. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of the multiple individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once. In some embodiments, before fixation, the immune cells have been contacted with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, the immune cells are contacted with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of the antigenic peptide in the bait composition. In some embodiments, the illustration portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, further optionally an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule.In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0167] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) immobilizing immune cells from the samples onto a solid support, wherein the immune cells: 1) prior to immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; or 2) after immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient to cause the agent to bind to the display portion; and c) detecting the detection agent to detect immune cells bound to the display portion, wherein the display portion further comprises a detectable marker (e.g., a fluorophore, such as PE or APC), wherein the detection agent binds to the detectable marker. In some embodiments, the samples are obtained from a single individual. In some embodiments, the samples are a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of a plurality of individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once. In some embodiments, before fixation, the immune cells have been contacted with a bait composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, the immune cells are contacted with a bait composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptide in the bait composition. In some embodiments, the illustration portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, more preferably an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally comprises an HLA-A molecule, more preferably wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and more preferably wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation.In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0168] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) contacting immune cells with a decoy composition comprising a display portion containing the antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; c) immobilizing the immune cells from the sample on a solid support; and d) detecting the detection agent to detect immune cells bound to the display portion, wherein the antigenic peptide is associated with a cancer or tumor antigen, optionally wherein the antigenic peptide is a neoantigen peptide. In some embodiments, the sample is obtained from one or more individuals who do not exhibit pathological symptoms of cancer. In some embodiments, the individual a) has not previously been diagnosed with cancer, optionally wherein the individual is at risk of developing cancer, or b) the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment. In some embodiments, the neoantigen peptide contains a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of the multiple individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by the immune cells) by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally containing no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptides in the bait composition. In some embodiments, the immune cells are immobilized on a solid matrix such as beads, optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity for MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity for homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the illustrated portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, and more optionally the MHC multimer is an MHC tetramer or an MHC dextran multimer.In some embodiments, the MHC molecule includes MHC class I molecules. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0169] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) contacting immune cells with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; c) immobilizing the immune cells from the sample on a solid support; and d) detecting the detection agent to detect immune cells bound to the display portion, wherein the antigenic peptide binds to a pathogen antigen, optionally wherein the antigenic peptide binds to a viral, bacterial, or fungal antigen. In some embodiments, the individual a) is not diagnosed with a pathogen infection, optionally wherein the individual is at risk of having said infection, and / or b) does not exhibit pathological symptoms of a pathogen infection, optionally not exhibiting pathological symptoms of a pathogen infection. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of a plurality of individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by the immune cells) by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptides in the bait composition. In some embodiments, the immune cells are immobilized on a solid matrix such as beads, optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity for MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity for homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the demonstrator portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, and more optionally the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule.In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0170] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in multiple samples from one or more individuals, comprising: a) contacting immune cells with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; c) immobilizing the immune cells from the sample on a solid support; and d) detecting the detection agent to detect immune cells bound to the display portion, wherein the antigenicity is an autoantigen peptide associated with an autoantigen. In some embodiments, the individual a) is not diagnosed with an immune response to an autoantigen, optionally said individual is at risk of developing an immune response to an autoantigen, and / or b) does not exhibit pathological symptoms of an immune response (e.g., autoimmune disease or condition), optionally not exhibiting pathological symptoms of an immune response to an autoantigen. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of a plurality of individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by the immune cells) by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptides in the bait composition. In some embodiments, the immune cells are immobilized on a solid matrix such as beads, optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity for MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity for homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the demonstrator portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, and more optionally the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule.In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0171] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) contacting immune cells with a decoy composition comprising a display portion containing the antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; b) contacting the immune cells with a detection agent that recognizes the display portion under conditions sufficient to cause the agent to bind to the display portion; c) immobilizing the immune cells from the sample onto a solid support; and d) detecting the detection agent to detect immune cells bound to the display portion, wherein the immune cells are immobilized on a solid matrix such as beads, and optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of the multiple individual samples is treated individually before being combined to form a mixture such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with a molecule containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally containing no more than about 10%, 5%, 4%, 3%, 2%, or 1% of the antigenic peptides in the bait composition that specifically bind to the immune cells. In some embodiments, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides. In some embodiments, the antigenic peptides have one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM; c) being hydrophobic; and d) having a high content of aromatic residues. In some embodiments, the antigenic peptides have low immunogenicity. In some embodiments, the illustration portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, more preferably an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally comprises an HLA-A molecule, more preferably wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and more preferably wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation.In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0172] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in multiple samples from one or more individuals, comprising: a) contacting immune cells with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; c) immobilizing the immune cells from the samples on a solid support; and d) detecting the detection agent to detect immune cells bound to the display portion, wherein the display portion comprises a library of antigenic peptides of mutations associated with cancer or tumor, pathogen, or self-antigen, wherein the library comprises at least about 5, 10, 12, 15, 18, or 20 different antigenic peptides (e.g., neoantigen peptides). In some embodiments, the samples are obtained from a single individual. In some embodiments, the samples are a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of a plurality of individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by the immune cells) by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of the antigenic peptide in the bait composition that specifically binds to the immune cell. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the illustration portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, more preferably an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally comprises an HLA-A molecule, more preferably wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and more preferably wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation.In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0173] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) contacting immune cells with a decoy composition comprising a display portion containing the antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; c) immobilizing the immune cells from the sample on a solid support; and d) detecting the detection agent to detect immune cells bound to the display portion, wherein the antigenic peptide 1) has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM for MHC molecules; b) having a binding affinity of about 1 nM to about 5000 nM for homologous TCR molecules; c) being hydrophobic; and d) having a high content of aromatic residues; and / or 2) having low immunogenicity. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of a plurality of individual samples is treated individually before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptide in the bait composition. In some embodiments, a portion is shown comprising an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, further optionally an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises MHC class I molecules. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation.In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0174] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) contacting immune cells with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to cause the immune cells to bind to the display portion; b) contacting the immune cells with a detection agent that recognizes the display portion under conditions sufficient to cause the agent to bind to the display portion; c) immobilizing the immune cells from the sample on a solid support; and d) detecting the detection agent to detect immune cells bound to the display portion, wherein the display portion further comprises a detectable marker (e.g., a fluorophore, such as PE or APC), wherein the detection agent binds to the detectable marker. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of the multiple individual samples is treated individually before being combined to form a mixture such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with a molecule containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of the antigenic peptide in the bait composition. In some embodiments, the exhibited portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, further optionally an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises MHC class I molecules. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLAA2 heavy chain with an A245V mutation. In some embodiments, the display portion includes two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01 and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, HLA-A*33:03.In some implementations, the method also includes separating and displaying partially bound immune cells, or removing display portions or immune cells that are not bound to each other.

[0175] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) treating each of a plurality of individual samples separately such that the immune cells contained therein are associated with a unique sample barcode; b) combining the plurality of treated samples to form a sample mixture; c) contacting the sample mixture with a decoy composition comprising a display portion containing the antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a decoy-sample mixture; d) detecting immune cells in the decoy-sample mixture that bind to the display portion; and e) determining a unique barcode associated with the immune cells binding to the display portion, thereby identifying the sample from which the immune cells originated, wherein the antigenic peptide is associated with a cancer or tumor antigen, optionally wherein the antigenic peptide is a neoantigen peptide. In some embodiments, the samples are obtained from one or more individuals who do not exhibit pathological symptoms of cancer. In some embodiments, the individual a) has not previously been diagnosed with cancer, optionally wherein the individual is at risk of developing cancer, or b) the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment. In some embodiments, the neoantigen peptide contains a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus. In some embodiments, prior to fixation, immune cells have been contacted with a bait composition containing a display portion of the antigen peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, immune cells are contacted with a bait composition containing a display portion of the antigen peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally containing no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigen peptide in the bait composition. In some embodiments, the immune cells are fixed on a solid matrix such as beads, optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the display portion contains two or more antigen peptides, optionally wherein the display portion contains four antigen peptides. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity for MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity for homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the demonstrator portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, and more optionally the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule.In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0176] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) treating each of a plurality of individual samples separately such that the immune cells contained therein are associated with a unique sample barcode; b) combining the plurality of treated samples to form a sample mixture; c) contacting the sample mixture with a decoy composition containing a display portion of the antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a decoy-sample mixture; d) detecting immune cells in the decoy-sample mixture that bind to the display portion; and e) determining a unique barcode associated with the immune cells binding to the display portion, thereby identifying the sample from which the immune cells originated, wherein the antigenic peptide is associated with a pathogen antigen, optionally wherein the antigenic peptide is associated with a viral, bacterial, or fungal antigen. In some embodiments, the individual a) is not diagnosed with a pathogen infection, optionally wherein the individual is at risk of having said infection, and / or b) does not exhibit pathological symptoms of a pathogen infection, optionally not exhibiting pathological symptoms of a pathogen infection. In some embodiments, prior to fixation, immune cells are contacted with a bait composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, immune cells are contacted with a bait composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptide in the bait composition. In some embodiments, the immune cells are fixed on a solid matrix such as beads, optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity for MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity for homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the demonstrator portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, and more optionally the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule.In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0177] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in multiple samples from one or more individuals, comprising: a) treating each of a plurality of individual samples separately such that the immune cells contained therein are associated with a unique sample barcode; b) combining the plurality of treated samples to form a sample mixture; c) contacting the sample mixture with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a decoy-sample mixture; d) detecting immune cells in the decoy-sample mixture that bind to the display portion; and e) determining a unique barcode associated with the immune cells binding to the display portion, thereby identifying the sample from which the immune cells originated, wherein the antigenicity is an autoantigen peptide associated with an autoantigen. In some embodiments, the individual a) is not diagnosed with an immune response to an autoantigen, optionally said individual is at risk of developing an immune response to an autoantigen, and / or b) does not exhibit pathological symptoms of an immune response (e.g., autoimmune disease or condition), optionally not exhibiting pathological symptoms of an immune response to an autoantigen. In some embodiments, prior to fixation, immune cells are contacted with a bait composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, immune cells are contacted with a bait composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptide in the bait composition. In some embodiments, the immune cells are fixed on a solid matrix such as beads, optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity for MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity for homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the demonstrator portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, and more optionally the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule.In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0178] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) treating each of a plurality of individual samples separately such that the immune cells contained therein are associated with a unique sample barcode; b) combining the plurality of treated samples to form a sample mixture; c) contacting the sample mixture with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a decoy-sample mixture; d) detecting immune cells bound to the display portion in the decoy-sample mixture; and e) identifying a sample of derived immune cells by determining a unique barcode associated with the immune cells bound to the display portion, wherein the immune cells are immobilized on a solid matrix (e.g., by beads), and optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody. In some embodiments, prior to immobilization, the immune cells have been contacted with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion. In some embodiments, immune cells are contacted with a bait composition containing a display portion comprising an antigenic peptide, under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally containing no more than about 10%, 5%, 4%, 3%, 2%, or 1% of the antigenic peptide in the bait composition that specifically binds to the immune cells. In some embodiments, the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the illustration portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, more preferably an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally comprises an HLA-A molecule, more preferably wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and more preferably wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation.In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0179] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in multiple samples from one or more individuals, comprising: a) treating each of a plurality of individual samples separately such that the immune cells contained therein are associated with a unique sample barcode; b) combining the plurality of treated samples to form a sample mixture; c) contacting the sample mixture with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a decoy-sample mixture; d) detecting immune cells bound to the display portion in the decoy-sample mixture; and e) determining a unique barcode associated with the immune cells bound to the display portion, thereby identifying the sample from which the immune cells originated, wherein the display portion contains a library of antigenic peptides of mutations associated with cancer or tumor, pathogen, or self-antigen, wherein the library contains at least about 5, 10, 12, 15, 18, or 20 different antigenic peptides (e.g., neoantigen peptides). In some embodiments, the immune cells have been contacted with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion prior to fixation. In some embodiments, immune cells are contacted with a bait composition containing a display portion comprising an antigenic peptide, under conditions sufficient to allow immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally containing no more than about 10%, 5%, 4%, 3%, 2%, or 1% of the antigenic peptide in the bait composition that specifically binds to the immune peptide. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM; b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM; c) is hydrophobic; and d) has a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the display portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, further optionally the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule includes MHC class I molecules. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation.In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0180] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) treating each of a plurality of individual samples separately such that the immune cells contained therein are associated with a unique sample barcode; b) combining the plurality of treated samples to form a sample mixture; c) contacting the sample mixture with a bait composition containing a display portion of the antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a bait-sample mixture; d) detecting immune cells in the bait-sample mixture that bind to the display portion; and e) determining a unique barcode associated with the immune cells that bind to the display portion, thereby identifying the sample from which the immune cells originated, wherein the antigenic peptide 1) has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM for MHC molecules; b) having a binding affinity of about 1 nM to about 5000 nM for homologous TCR molecules; c) being hydrophobic; and d) having a high content of aromatic residues; and / or 2) having low immunogenicity. In some embodiments, prior to fixation, immune cells are contacted with a bait composition containing a display portion of the antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, immune cells are contacted with a bait composition containing a display portion of the antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of T cells that specifically bind to the antigenic peptide in the bait composition. In some embodiments, the display portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, further optionally an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally includes an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation.In some embodiments, the display portion comprises two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion also comprises a detectable marker (e.g., a fluorophore). In some embodiments, the method further includes isolating immune cells bound to the display portion, or removing display portions or immune cells that are not bound to each other.

[0181] In some embodiments, a method is provided for analyzing the presence or absence of immune cells capable of binding to an antigenic peptide in multiple samples from one or more individuals, comprising: a) treating each of a plurality of individual samples separately such that the immune cells contained therein are associated with a unique sample barcode; b) combining the plurality of treated samples to form a sample mixture; c) contacting the sample mixture with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a decoy-sample mixture; d) detecting immune cells bound to the display portion in the decoy-sample mixture; and e) determining a unique barcode associated with the immune cells bound to the display portion, thereby identifying the sample from which the immune cells originated, wherein the display portion further comprises a detectable marker (e.g., a fluorophore, such as PE or APC), wherein a detection agent binds to the detectable marker. In some embodiments, the immune cells have been contacted with the decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion before fixation. In some embodiments, the immune cells are contacted with the decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion after fixation. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells comprise T cells (e.g., CD8 T cells), optionally comprising no more than about 10%, 5%, 4%, 3%, 2%, or 1% of the antigenic peptide in the bait composition. In some embodiments, the display portion comprises an MHC molecule complexed with the antigenic peptide, optionally wherein the MHC molecule comprises an MHC multimer, further optionally the MHC multimer is an MHC tetramer or an MHC dextran multimer. In some embodiments, the MHC molecule comprises a MHC class I molecule. In some embodiments, the MHC class I molecule is selected from HLA-A, HLA-B, and HLA-C, wherein the MHC class I molecule optionally comprises an HLA-A molecule, further optionally wherein the HLA-A molecule contains a mutation that reduces its binding to CD8, and further optionally wherein the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion includes two or more different types of MHC class I molecules selected from a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01 and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, HLA-A*33:03.In some implementations, the method also includes separating and displaying partially bound immune cells, or removing display portions or immune cells that are not bound to each other.

[0182] In some embodiments, the term "conditions" in "conditions sufficient to allow immune cells to bind to the display portion" refers to conditions under which specific binding occurs between the immune cells and the display portion. In some embodiments, it refers to conditions involving incubating the immune cells and the display portion for about or at least about 5, 10, 15, 20, 25, or 30 minutes (e.g., at room temperature in a culture medium). In some embodiments, it refers to conditions under which maximum binding occurs between the immune cells and the display portion at at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Maximum binding can be assessed, for example, by exposing the immune cells to the display portion for a sufficient time (e.g., 30 minutes) under appropriate conditions (e.g., room temperature).

[0183] In some embodiments, immune cells are immobilized onto a solid matrix, for example, by coating or conjugating an agent that binds to the immune cells onto the solid matrix. In some embodiments, the agent is an antibody that specifically recognizes a marker on the surface of the immune cells. In some embodiments, the agent is an anti-CD8 antibody and the immune cells are CD8 T cells. In some embodiments, the agent is an anti-CD4 antibody and the immune cells are CD4 T cells. In some embodiments, the agent is an anti-CD3 antibody and the immune cells are T cells. Immobilization can be performed by mixing the immune cells with an agent that specifically binds to the immune cells (or by incubating the immune cells with the agent). In some embodiments, the agent has been coated or conjugated onto a solid matrix such as a plate or magnetic beads. The immune cells are exposed to the agent for a period of time, and then a washing step can be performed to remove cells that have not bound to the agent (e.g., using a magnet). See, for example, Examples 2-4.

[0184] In some embodiments, the solid support (or “solid matrix,” which may be used interchangeably) comprises a plate or particle surface (e.g., a particle surface, such as the inner surface of a porous particle). In some embodiments, the solid support comprises any of the following: beads (e.g., magnetic beads), porous beads, porous matrices, arrays, glass surfaces, silicon surfaces, plastic surfaces, filters, membranes, nylon, silicon wafers, and flow-through chips. In some embodiments, the solid support comprises any of the following: biochips containing electronic devices, microtiter wells, ELISA plates, spin interferometers, nitrocellulose membranes, nitrocellulose-based polymer surfaces, nanoparticles, or microspheres.

[0185] In some embodiments, the sample is a mixture of multiple individual samples. In some embodiments, the multiple individual samples are derived from a single individual. In some embodiments, the multiple individual samples are derived from multiple individuals.

[0186] In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, the method includes treating each of the multiple individual samples individually before combining them to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with a molecule containing a unique sample barcode). In some embodiments, the method further includes removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once, for example. Association can be achieved, for example, by mixing immune cells with molecules (e.g., antibodies that bind to immune cells, such as CD45 antibodies) that include a unique barcode and binding the immune cells to them to bind to each other. For example, a unique barcode can be associated with cells via an antibody, for example, conjugated or fused to a barcode specifically binding to a cell. In some embodiments, the barcode is a nucleic acid barcode (e.g., a DNA barcode). In some embodiments, a unique DNA barcode is a randomized barcode of about 10-20 nucleic acids (e.g., 12-18 nucleic acids) in length. In some embodiments, the barcode is a peptide barcode. In some implementations, the method also includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing immune cells at least 1, 2, or 3 times.

[0187] In some implementations, the method further includes identifying a unique barcode associated with the immune cells binding to the display portion, thereby identifying the sample from which the immune cells originated. For example, nucleic acids can be purified and then sequenced to identify the immune cells associated with the unique DNA barcode. See, for example, Example 4.

[0188] In some embodiments, the particles described herein are dextran particles. In some embodiments, the particles are magnetic nanoparticles or polystyrene nanoparticles. In some embodiments, the particles are agarose beads or sepharose beads. In some embodiments, the antigenic peptide or MHC is directly attached to the particle. In some embodiments, the antigenic peptide or MHC is attached to the particle via a binding pair comprising a first binding component attached to the antigenic peptide and a second binding component bound to the particle. In some embodiments, the displayed portion comprises a cell. In some embodiments, the cell comprises a polynucleotide encoding the antigenic peptide. In some embodiments, the polynucleotide encodes multiple antigenic peptides.

[0189] In some implementations, the individual is a person. In some implementations, the individual is at least about 50 years old (e.g., at least 50, 60, 70, or 80 years old).

[0190] In some embodiments, the sample described herein (including this section and any other part of this application) can be any sample from an individual. In some embodiments, the sample described herein is a blood sample. In some embodiments, the sample described herein is a plasma sample. In some embodiments, the sample described herein includes peripheral blood mononuclear cells (PMBCs). In some embodiments, the sample described herein is a sample obtained from an individual's tissue or organ (e.g., a biopsy sample). In some embodiments, the sample is obtained from an individual's lymph nodes.

[0191] The methods described herein (including methods for analyzing immune cells and methods for detecting antigen-specific immune cells described in the following sections) have high specificity and sensitivity, enabling the detection of antigen-specific immune cells even when present in small quantities (e.g., less than 1%) in a sample. Sensitivity is calculated as the percentage of true positives / (true positives + false negatives). Specificity is calculated as the percentage of true negatives / (true negatives + false positives). In some embodiments, the methods described herein exhibit specificity exceeding about 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some embodiments, the methods described herein exhibit sensitivity exceeding about 50%, 55%, 60%, 65%, 70%, 75%, or 80%. See, for example... Figure 12 In some embodiments, the sample comprises PBMCs and the immune cells comprise, or are T cells or B cells contained within the PBMCs. In some embodiments, the immune cells do not undergo an enrichment step of general immune cells or immune cells specifically binding to the antigenic peptide prior to contact with the bait composition. In some embodiments, the immune cells do not undergo an enrichment step of general immune cells or immune cells specifically binding to the antigenic peptide throughout the method. In some embodiments, the sample contains no more than 1 x 102 8 5x10 7 2x10 7 1x10 7 5x10 6 2x10 6 Or 1x10 6 One PBMC. In some implementations, the sample contains no more than 1x10 6 5x10 5 2x10 5 1x10 5 5x10 4 2x10 4 1x10 4 7x10 3 5x10 3 2x10 3 1x10 3 5x10 22x10 2 Or 1x10 2 Individual immune cells (e.g., CD8 T cells, such as immune cells that bind to the antigenic peptide in the bait composition, such as CD4 or CD8 T cells that bind to the antigenic peptide in the bait composition).

[0192] Methods for analyzing immune cells This application also includes methods for analyzing immune cells in samples from individuals, including a) contacting a plurality of immune cells with one or more display portions or decoy compositions described herein, and b) detecting or analyzing one or more immune cells bound to one or more display portions.

[0193] In some embodiments, methods are provided for analyzing immune cells in samples from an individual, including a) contacting a plurality of immune cells with one or more display portions or the bait composition described herein, and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises particles (e.g., spherical particles, solid particles, beads) associated with multiple MHC-peptide complexes, wherein the multiple MHC-peptide complexes comprise at least more than 12 MHC-peptide complexes, wherein each MHC-peptide complex is an MHC-peptide monomer comprising an MHC molecule associated with the peptide complex, wherein at least two MHC-peptide complexes are distinct. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) distinct MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 distinct peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).

[0194] In some embodiments, methods are provided for analyzing immune cells in samples from an individual, including a) contacting a plurality of immune cells with one or more display portions or the bait composition described herein, and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises particles (e.g., spherical particles, solid particles, beads) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule complexed with a peptide, wherein at least two MHC-peptide complexes are distinct, and wherein the plurality of MHC-peptide complexes comprises at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 50,000, 100,000, 15,000, or 200,000 MHC-peptide complexes. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the decoy composition comprises an HLA-DP molecule, an HLA-DQ molecule, and an HLA-DR molecule. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition comprises at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or the decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0195] In some embodiments, methods are provided for analyzing immune cells in samples from an individual, including a) contacting a plurality of immune cells with one or more display portions or the bait composition described herein, and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4 Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, solid particle, bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule complexed with a peptide, wherein at least two MHC-peptide complexes are distinct, and wherein the surface area of ​​the particle is at least about 1 µm. 2The diameter is at least 0.5 µm. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the decoy composition comprises an HLA-DP molecule, an HLA-DQ molecule, and an HLA-DR molecule. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition comprises at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or the decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0196] In some embodiments, methods are provided for analyzing immune cells in samples from an individual, including a) contacting a plurality of immune cells with one or more display portions or the bait composition described herein, and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., a spherical particle, a solid particle, or a bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule bound to a peptide, wherein at least two MHC-peptide complexes are different, and wherein the plurality of MHC-peptide complexes comprises a) at least two different MHC molecules and b) at least two different peptides. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers.In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0197] In some embodiments, methods are provided for analyzing immune cells in samples from an individual, including a) contacting a plurality of immune cells with one or more display portions or the bait composition described herein, and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, solid particle, bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule bound to a peptide, wherein at least two MHC-peptide complexes are different, and wherein the display portion comprises at least two different MHC class I molecules. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC class I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHCII molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers.In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0198] In some embodiments, methods are provided for analyzing immune cells in samples from an individual, including a) contacting a plurality of immune cells with one or more display portions or the bait composition described herein, and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, solid particle, bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprise at least or more than 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule bound to a peptide, wherein at least two MHC-peptide complexes are different, and wherein the display portion comprises at least two different MHC class II molecules. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHCII molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers.In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0199] In some embodiments, methods are provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions or the bait composition described herein, and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who is at risk of developing a disease or condition (e.g., cancer, infection, or autoimmune disease or condition), such as having a family history of such a disease or being an individual who has been exposed to a pathogen but has not yet received treatment for said disease or condition. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the decoy composition comprises an HLA-DP molecule, an HLA-DQ molecule, and an HLA-DR molecule. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition comprises at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or the decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).

[0200] In some embodiments, methods are provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who is at risk of developing a disease or condition (e.g., cancer, infection, or autoimmune disease or condition), such as having a family history of such a disease or being an individual who has been exposed to a pathogen but has not yet received treatment for said disease or condition. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x105 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4 Individual cells. In some embodiments, each of one or more display portions comprises particles (e.g., spherical particles, solid particles, beads) associated with multiple MHC-peptide complexes, wherein the multiple MHC-peptide complexes comprise at least or more than 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex is an MHC-peptide monomer comprising an MHC molecule associated with the peptide complex, wherein at least two MHC-peptide complexes are distinct. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) distinct MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 distinct peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).

[0201] In some embodiments, methods are provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who is at risk of developing a disease or condition (e.g., cancer, infection, or autoimmune disease or condition), such as having a family history of such a disease or being an individual who has been exposed to a pathogen but has not yet received treatment for said disease or condition. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises particles (e.g., spherical particles, solid particles, beads) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule complexed with a peptide, wherein at least two MHC-peptide complexes are distinct, and wherein the plurality of MHC-peptide complexes comprises at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 50,000, 100,000, 15,000, or 200,000 MHC-peptide complexes. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the decoy composition comprises an HLA-DP molecule, an HLA-DQ molecule, and an HLA-DR molecule. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition comprises at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or the decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0202] In some embodiments, methods are provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who is at risk of developing a disease or condition (e.g., cancer, infection, or autoimmune disease or condition), such as having a family history of such a disease or being an individual who has been exposed to a pathogen but has not yet received treatment for said disease or condition. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4 Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, solid particle, bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule complexed with a peptide, wherein at least two MHC-peptide complexes are distinct, and wherein the surface area of ​​the particle is at least about 1 µm. 2The diameter is at least 0.5 µm. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the decoy composition comprises an HLA-DP molecule, an HLA-DQ molecule, and an HLA-DR molecule. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition comprises at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or the decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0203] In some embodiments, methods are provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who is at risk of developing a disease or condition (e.g., cancer, infection, or autoimmune disease or condition), such as having a family history of such a disease or being an individual who has been exposed to a pathogen but has not yet received treatment for said disease or condition. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., a spherical particle, a solid particle, or a bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule bound to a peptide, wherein at least two MHC-peptide complexes are different, and wherein the plurality of MHC-peptide complexes comprises a) at least two different MHC molecules and b) at least two different peptides. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers.In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0204] In some embodiments, methods are provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who is at risk of developing a disease or condition (e.g., cancer, infection, or autoimmune disease or condition), such as having a family history of such a disease or being an individual who has been exposed to a pathogen but has not yet received treatment for said disease or condition. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, solid particle, bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule bound to a peptide, wherein at least two MHC-peptide complexes are different, and wherein the display portion comprises at least two different MHC class I molecules. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC class I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHCII molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers.In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0205] In some embodiments, methods are provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who is at risk of developing a disease or condition (e.g., cancer, infection, or autoimmune disease or condition), such as having a family history of such a disease or being an individual who has been exposed to a pathogen but has not yet received treatment for said disease or condition. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, solid particle, bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprise at least or more than 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule bound to a peptide, wherein at least two MHC-peptide complexes are different, and wherein the display portion comprises at least two different MHC class II molecules. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHCII molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers.In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0206] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, comprising a) contacting a plurality of immune cells with one or more display portions or the decoy composition described herein, and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual, wherein the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual cancer. In some embodiments, minimal residual cancer is visible after the cancer has been surgically removed or cured. In some embodiments, the minimal residual disease is too small to be detected by imaging instruments (e.g., conventionally used or standard imaging instruments for detecting cancer). In some embodiments, the location of the minimal residual disease is diverse. In some embodiments, the minimal residual cancer is a result of immune escape or treatment resistance. In some embodiments, the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 102 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the decoy composition comprises an HLA-DP molecule, an HLA-DQ molecule, and an HLA-DR molecule. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition comprises at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or the decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0207] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who has minimal residual disease (MRD). In some embodiments, the individual has minimal residual cancer. In some embodiments, minimal residual cancer is visible after the cancer has been surgically removed or cured. In some embodiments, the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises particles (e.g., spherical particles, solid particles, beads) associated with multiple MHC-peptide complexes, wherein the multiple MHC-peptide complexes comprise at least or more than 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex is an MHC-peptide monomer comprising an MHC molecule associated with the peptide complex, wherein at least two MHC-peptide complexes are distinct. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) distinct MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 distinct peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).

[0208] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who has minimal residual disease (MRD). In some embodiments, the individual has minimal residual cancer. In some embodiments, minimal residual cancer is visible after the cancer has been surgically removed or cured. In some embodiments, the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises particles (e.g., spherical particles, solid particles, beads) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule complexed with a peptide, wherein at least two MHC-peptide complexes are distinct, and wherein the plurality of MHC-peptide complexes comprises at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 50,000, 100,000, 15,000, or 200,000 MHC-peptide complexes. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the decoy composition comprises an HLA-DP molecule, an HLA-DQ molecule, and an HLA-DR molecule. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition comprises at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or the decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0209] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who has minimal residual disease (MRD). In some embodiments, the individual has minimal residual cancer. In some embodiments, minimal residual cancer is visible after the cancer has been surgically removed or cured. In some embodiments, the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4 Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, solid particle, bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule complexed with a peptide, wherein at least two MHC-peptide complexes are distinct, and wherein the surface area of ​​the particle is at least about 1 µm. 2The diameter is at least 0.5 µm. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the decoy composition comprises an HLA-DP molecule, an HLA-DQ molecule, and an HLA-DR molecule. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition comprises at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or the decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0210] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who has minimal residual disease (MRD). In some embodiments, the individual has minimal residual cancer. In some embodiments, minimal residual cancer is visible after the cancer has been surgically removed or cured. In some embodiments, the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., a spherical particle, a solid particle, or a bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule bound to a peptide, wherein at least two MHC-peptide complexes are different, and wherein the plurality of MHC-peptide complexes comprises a) at least two different MHC molecules and b) at least two different peptides. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers.In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0211] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who has minimal residual disease (MRD). In some embodiments, the individual has minimal residual cancer. In some embodiments, minimal residual cancer is visible after the cancer has been surgically removed or cured. In some embodiments, the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, solid particle, bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least or more 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule bound to a peptide, wherein at least two MHC-peptide complexes are different, and wherein the display portion comprises at least two different MHC class I molecules. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC class I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers.In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0212] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, including a) contacting a plurality of immune cells with one or more display portions, and b) detecting or analyzing one or more immune cells bound to the one or more display portions, wherein the immune cells are obtained from a sample (e.g., PBMCs) from an individual who has minimal residual disease (MRD). In some embodiments, the individual has minimal residual cancer. In some embodiments, minimal residual cancer is visible after the cancer has been surgically removed or cured. In some embodiments, the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment. In some embodiments, the number of cells in the sample (e.g., PBMCs) does not exceed about 5 x 10-1 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4Individual cells. In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, solid particle, bead) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprise at least or more than 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule bound to a peptide, wherein at least two MHC-peptide complexes are different, and wherein the display portion comprises at least two different MHC class II molecules. In some embodiments, one or more display portions are contained in a bait composition. In some embodiments, the bait composition comprises a) at least two (or at least three) different MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises a) at least two, three, or four HLA-A molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, wherein the two, three, or four HLA-A molecules are selected from HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the decoy composition comprises a) at least two (or at least three) different MHC II molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the decoy composition comprises HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR molecules. In some embodiments, the decoy composition comprises HLA-DP, HLA-DQ, and HLA-DR molecules. In some embodiments, the peptides in the decoy composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the decoy composition contains at least 2, 3, 4, 5, 6, or 7 different overlapping peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or decoy composition does not contain a fluorescent dye or fluorophore. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample was obtained. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers.In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0213] In some embodiments, immune cells are immobilized onto a solid matrix, for example, by coating or conjugating an agent that binds to the immune cells onto the solid matrix. In some embodiments, the agent is an antibody that specifically recognizes a marker on the surface of the immune cells. In some embodiments, the agent is an anti-CD8 antibody and the immune cells are CD8 T cells. In some embodiments, the agent is an anti-CD4 antibody and the immune cells are CD4 T cells. In some embodiments, the agent is an anti-CD3 antibody and the immune cells are T cells. Immobilization can be performed by mixing the immune cells with an agent that specifically binds to the immune cells (or by incubating the immune cells with the agent). In some embodiments, the agent has been coated or conjugated onto a solid matrix such as a plate or magnetic beads. The immune cells are exposed to the agent for a period of time, and then a washing step can be performed to remove cells that have not bound to the agent (e.g., using a magnet). See examples.

[0214] In some implementations, the number of immune cells does not exceed approximately 10. 8 5x10 7 10 7 5x10 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4 Each cell.

[0215] In some embodiments, the solid matrix comprises a plate or surface of particles (e.g., a particle surface different from the particles used as supports in the display section, such as the inner surface of porous particles). In some embodiments, the solid matrix comprises any of the following: beads (e.g., magnetic beads), porous beads, porous matrices, arrays, glass surfaces, silicon surfaces, plastic surfaces, filters, membranes, nylon, silicon wafer chips, and flow-through chips. In some embodiments, the solid matrix comprises any of the following: biochips containing electronic devices, microtiter wells, ELISA plates, spin interference disks, nitrocellulose membranes, nitrocellulose-based polymer surfaces, nanoparticles, or microspheres.

[0216] In some embodiments, multiple immune cells are contacted with the display portion or bait composition under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, the term "conditions" in "conditions sufficient to allow the immune cells to bind to the display portion" refers to conditions under which specific binding occurs between the immune cells and the display portion. In some embodiments, it refers to conditions involving incubating the immune cells and the display portion for about or at least about 5, 10, 15, 20, 25, or 30 minutes (e.g., at room temperature in a culture medium). In some embodiments, it refers to conditions under which maximum binding occurs between the immune cells and the display portion at at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Maximum binding can be assessed, for example, by contacting the immune cells with the display portion for a sufficient time (e.g., 30 minutes) under appropriate conditions (e.g., room temperature).

[0217] In some embodiments, the sample is a mixture of multiple individual samples. In some embodiments, the multiple individual samples are derived from a single individual. In some embodiments, the multiple individual samples are derived from multiple individuals.

[0218] In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, the method includes treating each of the multiple individual samples individually before combining them to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with a molecule containing a unique sample barcode). In some embodiments, the method further includes removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once, for example. Association can be achieved, for example, by mixing immune cells with molecules (e.g., antibodies that bind to immune cells, such as CD45 antibodies) that include a unique barcode and binding the immune cells to them to bind to each other. For example, a unique barcode can be associated with cells via an antibody, for example, conjugated or fused to a barcode specifically binding to a cell. In some embodiments, the barcode is a nucleic acid barcode (e.g., a DNA barcode). In some embodiments, a unique DNA barcode is a randomized barcode of about 10-20 nucleic acids (e.g., 12-18 nucleic acids) in length. In some embodiments, the barcode is a peptide barcode. In some implementations, the method also includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing immune cells at least 1, 2, or 3 times.

[0219] In some implementations, the method further includes identifying a unique barcode associated with the immune cells binding to the display portion, thereby identifying the sample from which the immune cells originated. For example, nucleic acids can be purified and then sequenced to identify the immune cells associated with the unique DNA barcode.

[0220] In some implementations, the individual is a person. In some implementations, the individual is at least about 50 years old (e.g., at least 50, 60, 70, or 80 years old).

[0221] In some embodiments, the sample described herein (including this section and any other part of this application) may be any sample from an individual. In some embodiments, the sample described herein is a blood sample. In some embodiments, the sample described herein is a plasma sample. In some embodiments, the sample described herein includes peripheral blood mononuclear cells (PMBCs). In some embodiments, the number of PBMCs does not exceed about 10. 8 5x10 7 10 7 5x10 6 2x10 6 10 6 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4 Individual cells. In some embodiments, the sample described herein is a sample obtained from an individual's tissue or organ (e.g., a biopsy sample). In some embodiments, the sample is obtained from an individual's lymph nodes.

[0222] The method described herein has high specificity and sensitivity, enabling the detection of antigen-specific immune cells even when the sample contains very few (e.g., less than 1%). In some embodiments, the sample comprises PBMCs and the immune cells comprise, or are T cells or B cells contained within the PBMCs. In some embodiments, the immune cells do not undergo an enrichment step against general immune cells or immune cells against specific binding peptides prior to contact with the bait composition. In some embodiments, the immune cells do not undergo an enrichment step against general immune cells or immune cells against specific binding peptides throughout the method. In some embodiments, the sample contains no more than 1 x 102 8 5x10 7 2x10 7 1x10 7 5x10 6 2x10 6 Or 1x10 6 One PBMC. In some implementations, the sample contains no more than 1x10 6 5x10 5 2x10 5 1x10 5 5x104 2x10 4 1x10 4 7x10 3 5x10 3 2x10 3 1x10 3 5x10 2 2x10 2 Or 1x10 2 Individual immune cells (e.g., CD8 T cells, such as immune cells that bind to peptides in the bait composition, such as CD4 or CD8 T cells that bind to peptides in the bait composition).

[0223] Methods for detecting antigen-specific immune cells in an individual In some embodiments, a method is provided for detecting antigen-specific immune cells in an individual (e.g., an individual who does not exhibit pathological symptoms of cancer or tumor, infection with a specific pathogen, or an autoimmune disease or condition associated with an autoantigen, such as an individual at risk of developing a disease or condition like cancer, infection, or autoimmune disease or condition, such as an individual not receiving treatment for a disease or condition, or an individual who has received treatment for a disease or condition (e.g., cancer) and is suspected of having minimal residual cancer cells), comprising: analyzing a sample from said individual according to any of the methods described herein, wherein predetermined characteristics of the immune cells indicate a disease or condition in said individual. In some embodiments, the predetermined characteristics of the immune cells include the presence of immune cells. In some embodiments, the presence of immune cells that specifically bind to a decoy composition containing one or more neoantigens indicates the presence of cancer. In some embodiments, the presence of immune cells that specifically bind to a decoy composition containing one or more pathogen (e.g., viral, bacterial, fungal) antigens indicates the presence of a pathogen infection. In some embodiments, the presence of immune cells that specifically bind to a decoy composition containing one or more autoantigens associated with an autoimmune disease or condition indicates the presence of an autoimmune disease or condition. In some embodiments, the predetermined characteristics of the immune cells include the number of immune cells above a threshold level. In some embodiments, predetermined characteristics of immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics. In some embodiments, characteristic epigenetic modifications include DNA methylation characteristics and histone glycosylation characteristics.

[0224] In some embodiments, a method is provided for detecting antigen-specific immune cells in an individual (e.g., an individual who does not exhibit pathological symptoms of cancer or tumor, infection with a specific pathogen, or an autoimmune disease or disorder associated with an autoantigen, such as an individual at risk of developing a disease or disorder such as cancer, infection, or autoimmune disease or disorder, such as an individual who has not received treatment for a disease or condition, or an individual who has received treatment for a disease or condition (such as cancer) and is suspected of having minimal residual cancer cells), comprising: a) immobilizing immune cells from a sample onto a solid support, wherein the immune cells: 1) prior to immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to bind the immune cells to the display portion; or 2) after immobilization, have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to bind the immune cells to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient to bind the agent to the display portion; and c) detecting the detection agent, thereby detecting the immune cells bound to the display portion. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, each of the multiple individual samples is treated individually before being combined to form the mixture, such that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with molecules containing a unique sample barcode). In some embodiments, the method further includes, for example, removing unbound barcode-containing molecules (molecules not bound by immune cells) by washing the immune cells at least once. In some embodiments, before fixation, the immune cells have been contacted with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion. In some embodiments, the immune cells are contacted with a decoy composition containing a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation.

[0225] In some embodiments, a method is provided for detecting antigen-specific immune cells in an individual (e.g., an individual who does not exhibit pathological symptoms of cancer or tumor, infection with a specific pathogen, or an autoimmune disease or disorder associated with an autoantigen, such as an individual at risk of developing a disease or disorder such as cancer, infection, or autoimmune disease or disorder, such as an individual not receiving treatment for a disease or condition, or an individual who has received treatment for a disease or condition (such as cancer) and is suspected of having minimal residual cancer cells), comprising: a) contacting the immune cells with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) contacting the immune cells with a detection agent that recognizes the display portion under conditions sufficient to allow the agent to bind to the display portion; c) immobilizing the immune cells from the sample on a solid support; and d) detecting the detection agent, thereby detecting the immune cells bound to the display portion. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some implementations, each of the multiple individual samples is processed separately before being combined to form a mixture, such that the immune cells contained therein are associated with a unique sample barcode.

[0226] In some embodiments, a method is provided for detecting antigen-specific immune cells in a plurality of individuals (e.g., a plurality of individuals) who a) do not exhibit pathological symptoms of cancer or tumor, infection with a specific pathogen, or autoimmune disease or condition associated with an autoantigen, b) are at risk of developing said disease or condition (e.g., cancer, infection, or autoimmune disease or condition), c) are not receiving treatment for said disease or condition, or d) are patients who have received treatment for a disease or condition such as cancer and are suspected of having minimal residual cancer cells, comprising: a) processing each of a plurality of individual samples separately such that the immune cells contained therein are associated with a unique sample barcode, b) combining the plurality of processed samples to form a sample mixture, c) contacting the sample mixture with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a decoy-sample mixture, d) detecting immune cells bound to the display portion in the decoy-sample mixture; and e) determining a unique barcode associated with the immune cells bound to the display portion, thereby identifying the sample from which the immune cells originated.

[0227] In some embodiments, a method is provided for detecting antigen-specific immune cells in an individual (e.g., an individual who does not exhibit pathological symptoms of cancer or tumor, infection with a specific pathogen, or an autoimmune disease or condition associated with an autoantigen, such as an individual at risk of developing a disease or condition such as cancer, infection, or autoimmune disease or condition, such as an individual who has not received treatment for a disease or condition, or an individual who has received treatment for a disease or condition (e.g., cancer) and is suspected of having minimal residual cancer cells), comprising: a) contacting a sample with a bait composition containing a display portion containing an antigenic peptide under conditions sufficient to cause immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; b) isolating immune cells associated with the display portion; and c) analyzing the immune cells, wherein predetermined characteristics of the immune cells indicate a disease or condition in the individual, and wherein the predetermined characteristics of the immune cells include the presence of immune cells, optionally wherein the predetermined characteristics of the immune cells include an amount of immune cells above a threshold level. In some embodiments, the predetermined characteristics of the immune cells also include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and histone glycosylation characteristics). In some embodiments, the display portion comprises two or more (e.g., four) antigenic peptides. In some embodiments, the two or more antigenic peptides in the display portion are identical. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus; d) being hydrophobic; and e) having a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the display portion comprises an MHC molecule complexed with the antigenic peptide. In some embodiments, the MHC molecules are MHC class I molecules and / or MHC class II molecules. In some embodiments, the immune cells are isolated single immune cells. In some embodiments, the immune cells are a mixture of immune cells. In some embodiments, the immune cell mixture is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or combinations thereof. In some embodiments, further analysis of the immune cells includes quantifying the immune cells. In some embodiments, further analysis of the immune cells includes sequencing one or more nucleic acids in the immune cells, optionally including analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences).In some embodiments, further analysis of immune cells includes identifying one or more epigenetic modifications in the immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications, such as acetylation, methylation, and glycosylation). In some embodiments, the method also includes generating a report containing information on an individual's cancer status. In some embodiments, the cancer status information includes: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; likelihood of cancer progression; likelihood of the presence of one or more cancer symptoms; molecular diagnostics; NGS pathology; and / or the individual's treatment regimen. In some embodiments, the decoy composition comprises multiple different display moieties. In some embodiments, each of the multiple different display moieties in the decoy composition contains a different antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display moieties, each containing a different MHC molecule). In some embodiments, each different display moieties containing a different MHC molecule contains a different detectable marker (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells associated with each different display moiety containing different MHC molecules into different populations, optionally further including contacting each of the multiple different display mocies separately with a sample from an individual and separating the immune cells associated with each different display moiety. In some embodiments, the method further includes culturing the immune cells prior to the analysis step. In some embodiments, the display moiety comprises particles (e.g., particles selected from: surfaces, nanoparticles, beads, and polymers). In some embodiments, the display moiety also comprises a detectable marker. In some embodiments, the detectable marker is a fluorophore. In some embodiments, the analysis of the sequence of one or more nucleic acids includes whole-genome sequencing, RNA-seq sequencing, and / or mass spectrometry analysis of the immune cells.

[0228] In some embodiments, a method is provided for detecting antigen-specific immune cells in an individual (e.g., an individual who does not exhibit pathological symptoms of cancer or tumor, infection with a specific pathogen, or an autoimmune disease or condition associated with an autoantigen, such as an individual at risk of developing a disease or condition such as cancer, infection, or autoimmune disease or condition, such as an individual not receiving treatment for a disease or condition, or an individual who has received treatment for a disease or condition (e.g., cancer) and is suspected of having minimal residual cancer cells), comprising: a) contacting a sample with a bait composition containing a display portion containing antigenic peptides under conditions sufficient to cause immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; b) isolating immune cells associated with the display portion; and c) analyzing the immune cells, wherein predetermined characteristics of the immune cells indicate cancer in the individual, and wherein the predetermined characteristics of the immune cells include gene expression profile characteristics. In some embodiments, the predetermined characteristics of the immune cells also include gene mutation profile characteristics and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and histone glycosylation characteristics). In some embodiments, the display portion contains two or more (e.g., four) antigenic peptides. In some embodiments, the two or more antigenic peptides in the display portion are identical. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus; d) being hydrophobic; and e) having a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the display portion comprises an MHC molecule complexed with the antigenic peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the immune cells are isolated single immune cells. In some embodiments, the immune cells are a mixture of immune cells. In some embodiments, the immune cell mixture is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or combinations thereof. In some embodiments, further analysis of the immune cells includes detecting and / or quantifying the immune cells. In some embodiments, further analysis of the immune cells includes sequencing one or more nucleic acids in the immune cells, optionally including analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences).In some embodiments, further analysis of immune cells includes identifying one or more epigenetic modifications in the immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications, such as acetylation, methylation, and glycosylation). In some embodiments, the method also includes generating a report containing information on an individual's cancer status. In some embodiments, the cancer status information includes: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; likelihood of cancer progression; likelihood of the presence of one or more cancer symptoms; molecular diagnostics; NGS pathology; and / or the individual's treatment regimen. In some embodiments, the decoy composition comprises multiple different display moieties. In some embodiments, each of the multiple different display moieties in the decoy composition contains a different antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display moieties, each containing a different MHC molecule). In some embodiments, each different display moieties containing a different MHC molecule contains a different detectable marker (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells associated with each different display moiety containing different MHC molecules into different populations, optionally further including contacting each of the multiple different display mocies separately with a sample from an individual and separating the immune cells associated with each different display moiety. In some embodiments, the method further includes culturing the immune cells prior to the analysis step. In some embodiments, the display moiety comprises particles (e.g., particles selected from: surfaces, nanoparticles, beads, and polymers). In some embodiments, the display moiety also comprises a detectable marker. In some embodiments, the detectable marker is a fluorophore. In some embodiments, the analysis of the sequence of one or more nucleic acids includes whole-genome sequencing, RNA-seq sequencing, and / or mass spectrometry analysis of the immune cells.

[0229] In some embodiments, a method is provided for detecting cancer in an individual (e.g., an individual not yet diagnosed with cancer, an individual at risk of developing cancer, or an individual who has received cancer treatment), comprising: a) contacting a sample with a bait composition containing a display portion comprising a cancer antigenic peptide under conditions sufficient to cause immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; b) isolating immune cells associated with the display portion; and c) analyzing the immune cells, wherein predetermined characteristics of the immune cells indicate cancer in the individual, and wherein the predetermined characteristics of the immune cells include genetic mutation profile characteristics and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and histone glycosylation characteristics). In some embodiments, the display portion comprises two or more (e.g., four) antigenic peptides. In some embodiments, the two or more antigenic peptides in the display portion are identical. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus; d) being hydrophobic; and e) having a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the exhibited portion comprises an MHC molecule complexed with the antigenic peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the immune cell is a single, isolated immune cell. In some embodiments, the immune cells are a mixture of immune cells. In some embodiments, the immune cell mixture is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or combinations thereof. In some embodiments, further analysis of the immune cells includes detecting and / or quantifying the immune cells. In some embodiments, further analysis of the immune cells includes sequencing one or more nucleic acids in the immune cells, optionally including analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences). In some embodiments, further analysis of the immune cells also includes identifying one or more epigenetic modifications in the immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications, such as acetylation, methylation, glycosylation). In some embodiments, the method also includes generating a report containing information on an individual's cancer status.In some embodiments, cancer status information includes: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; likelihood of cancer progression; likelihood of the presence of one or more cancer symptoms; molecular diagnostics; NGS pathology; and / or the individual's treatment regimen. In some embodiments, the decoy composition comprises multiple distinct display moieties. In some embodiments, each of the multiple distinct display moieties in the decoy composition comprises a distinct antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 distinct display moieties, each containing a distinct MHC molecule). In some embodiments, each distinct display moiety containing a distinct MHC molecule comprises a distinct detectable marker (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells associated with each distinct display moiety containing a distinct MHC molecule into distinct populations, optionally further including contacting each of the multiple distinct display moieties separately with a sample from the individual and separating the immune cells associated with each distinct display moiety. In some embodiments, the method further includes culturing the immune cells prior to the analytical step. In some embodiments, the display portion includes particles (e.g., particles selected from surfaces, nanoparticles, beads, and polymers). In some embodiments, the display portion also includes a detectable marker. In some embodiments, the detectable marker is a fluorophore. In some embodiments, the analysis of the sequence of one or more nucleic acids includes whole-genome sequencing, RNA-seq sequencing, and / or mass spectrometry analysis of immune cells.

[0230] In some embodiments, a method is provided for detecting residual cancer in an individual, wherein the individual has previously been treated with an anticancer therapy and has not exhibited pathological symptoms of cancer after treatment, the method comprising analyzing post-treatment samples from the individual. In some embodiments, the method further comprises a) analyzing pre-treatment samples from the individual prior to anticancer treatment and post-treatment samples from the individual according to any of the methods described herein, and b) comparing the characteristics of immune cells from the pre-treatment samples and isolated immune cells from the post-treatment samples. In some embodiments, a predetermined difference in the characteristics of immune cells from the pre-treatment samples and immune cells from the post-treatment samples indicates residual cancer in the individual. In some embodiments, the predetermined characteristics of immune cells include the presence of immune cells, optionally including a certain amount of immune cells above a threshold level. In some embodiments, the predetermined characteristics of immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and / or histone glycosylation characteristics). In some embodiments, the method includes: a) contacting a pre-treatment and / or post-treatment sample with a decoy composition containing a display portion of a cancer antigen peptide under conditions sufficient to cause immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; b) isolating immune cells associated with the display portion; and c) analyzing the immune cells. In some embodiments, the display portion contains two or more (e.g., four) antigen peptides. In some embodiments, the two or more antigen peptides in the display portion are identical. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus; d) being hydrophobic; and e) having a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the exhibited portion comprises an MHC molecule complexed with the antigenic peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the immune cell is a single, isolated immune cell. In some embodiments, the immune cells are a mixture of immune cells. In some embodiments, the immune cell mixture is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or combinations thereof.In some embodiments, further analysis of immune cells includes detecting and / or quantifying immune cells. In some embodiments, further analysis of immune cells includes sequencing one or more nucleic acids in the immune cells, optionally including analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences). In some embodiments, further analysis of immune cells also includes identifying one or more epigenetic modifications in the immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications, such as acetylation, methylation, and glycosylation). In some embodiments, the method also includes generating a report containing information on an individual's cancer status. In some embodiments, the cancer status information includes: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; likelihood of cancer progression; likelihood of the presence of one or more cancer symptoms; molecular diagnostics; NGS pathology; and / or the individual's treatment regimen. In some embodiments, the decoy composition comprises multiple different display moieties. In some embodiments, each of the multiple different display moieties in the decoy composition comprises a different antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display moieties, each comprising a different MHC molecule). In some embodiments, each different display moiety containing a different MHC molecule contains a different detectable marker (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells associated with each different display moiety containing a different MHC molecule into different populations, optionally further including contacting each of the multiple different display mocies separately with a sample from an individual and separating the immune cells associated with each different display moiety. In some embodiments, the method further includes culturing immune cells prior to the analysis step. In some embodiments, the display moiety contains particles (e.g., particles selected from: surfaces, nanoparticles, beads, and polymers). In some embodiments, the display moiety also contains a detectable marker. In some embodiments, the detectable marker is a fluorophore. In some embodiments, the analysis of the sequence of one or more nucleic acids includes whole-genome sequencing, RNA-seq sequencing, and / or mass spectrometry analysis of immune cells.

[0231] Aggregation test In some embodiments, the methods described herein (e.g., methods for analyzing immune cells or detecting antigen-specific immune cells) involve (e.g., including) performing an agglutination assay. In some embodiments, the agglutination assay includes observing cell agglutination under a microscope or with the naked eye. In some embodiments, the number of immune cells does not exceed about 10 8 5x10 7 10 7 5x10 6 2x10 6 106 5x10 5 2x10 5 10 5 5x10 4 4x10 4 2x10 4 Or 10 4 Each cell.

[0232] Agglutination assays are a class of bioanalytical methods used to detect the presence (qualitative) of a substance in a sample and sometimes to detect the amount of that substance (quantitative). The process typically involves particles agglomerating or clustering in the presence of a specific substance (such as an antigen, antibody, or cell). In some implementations, the agglutination reaction occurs using specially designed particles and is commonly used in a variety of diagnostic applications, including blood typing, pathogen detection, and measurement of antibody levels.

[0233] In some embodiments, in agglutination assays, antibodies or other binding agents are mixed with a sample containing a potential target antigen. If the antigen is present, it binds to the antibody, causing particles to aggregate or agglutinate. This agglutination can be observed directly or quantified using various detection methods. In some embodiments, the reaction is controlled to avoid nonspecific agglutination, which can lead to false positive results. In some embodiments, various buffers and additives can be used to achieve the desired reaction conditions, including pH, ionic strength, and temperature. In some embodiments, the test can be performed in various forms, such as slide agglutination, tube agglutination, or microplate agglutination, depending on the sample type, target concentration, and required throughput.

[0234] In some embodiments, latex beads are used as carrier particles for the binder in a latex bead-based agglutination assay (LBAT). In some embodiments, these latex beads offer several advantages, including uniform size, shape, and surface properties that can be precisely controlled to optimize test performance. In some embodiments, the latex beads can be functionalized with various chemical groups and conjugated with appropriate reagents (e.g., antibodies or antigens), or they can passively bind to these reagents.

[0235] In some embodiments, LBAT includes bead functionalization. The latex beads are functionalized using a desired binder. This may involve chemical activation, coupling chemistry, and purification steps. In some embodiments, LBAT includes sample preparation. The sample is prepared according to the specific requirements of the test and may include dilution, filtration, or other pretreatment steps. In some embodiments, LBAT includes reaction mixing: the functionalized beads are mixed with the sample in a controlled environment. Buffer solutions, stabilizers, or other additives may be used to ensure optimal reaction conditions. In some embodiments, LBAT includes incubation. The reaction mixture is incubated to allow time for the agglomeration reaction to occur. Temperature, time, and stirring may be adjusted to achieve the desired reaction kinetics. In some embodiments, LBAT includes detection. Agglomeration is observed or quantified using appropriate detection methods. This may include visual inspection, microscopic examination, or instrumental analysis, such as spectrophotometry.

[0236] In some implementations, the use of latex beads in agglutination tests allows for customized beads to meet the unique requirements of different applications, including infectious disease diagnosis, immunological research, food testing, etc.

[0237] Latex beads, commonly known as granules and developed in the mid-20th century, have played a significant role in advancing diagnostics and molecular biology. In some embodiments, these beads are synthesized via emulsion polymerization, where monomers polymerize in an aqueous phase to form polymer beads. In some embodiments, the process typically involves the use of stabilizers and surfactants to control the size and shape of the particles. In some embodiments, subsequent improvements in synthesis techniques have resulted in greater uniformity and control over the physical properties of the particles, such as size, shape, and surface charge.

[0238] In some embodiments, the latex beads have a core made of a synthetic polymer, with polystyrene being the most common core material. In some embodiments, it is known for its structural strength and chemical and physical inertness when interacting with biological systems. In some embodiments, other variants of the latex beads have been synthesized using polymers such as poly(methyl methacrylate) (PMMA) and poly(lactic-co-glycolic acid) (PLGA), PLGA being a biodegradable polymer known for its use in drug delivery applications. In some embodiments, the size of the latex beads can be finely controlled, and the diameter is typically in the range of about 20 nanometers to about 100 micrometers. In some embodiments, the core of the beads typically contains a crosslinking agent, such as divinylbenzene (DVB). In some embodiments, crosslinking imparts a three-dimensional network within the polymer, greatly enhancing the structural integrity and solvent resistance of the beads.

[0239] In some embodiments, the diameter of the latex beads is at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 μm. In some embodiments, the diameter of the latex beads is about 0.01 to about 150 μm, or 0.1 to about 100 μm (e.g., about 0.1 to about 50 μm, about 0.5 μm to about 20 μm, about 0.5 μm to about 10 μm, about 0.5 μm to about 5 μm, or about 0.5 μm to about 2.5 μm). In some embodiments, the diameter of the latex beads is about 0.1 to about 10 µm. In some embodiments, the diameter of the latex beads is about 0.5 to about 2 µm. In some embodiments, the diameter of the latex beads is about 1 µm.

[0240] In some embodiments, surface modification of latex beads is crucial to their utility in a variety of applications, as it allows them to be customized to specific testing requirements. In some embodiments, beads can be designed to conjugate a variety of biological targets, such as antibodies, proteins, carbohydrates, DNA, or RNA, by introducing different surface functional groups. In some embodiments, carboxylated beads, for example, provide a reactive surface for the covalent conjugation of biomolecules, making them ideal for immobilizing proteins or antibodies via carbodiimide chemistry. In some embodiments, amine-functionalized beads contain primary amine groups that can react with a variety of target molecules and are commonly used for oligonucleotide and peptide conjugation. In some embodiments, aldehyde-functionalized beads are capable of reacting with amine groups to form Schiff bases, making them suitable for protein coupling, while hydroxyl beads can be used for additional modification or as a blank control. In some embodiments, specialized functionalization options include magnetic beads, fluorescently labeled beads, and biotinylated beads that can bind to streptavidin or avidin, thereby enhancing the flexibility and adaptability of latex beads in a wide range of applications.

[0241] Treatment In some embodiments, a method for treating cancer in an individual (e.g., an individual not diagnosed with cancer, an individual at risk of developing cancer, or an individual who has received cancer treatment) is provided, comprising a) diagnosing the individual with cancer according to the method described herein; and b) administering anticancer treatment to the individual. In some embodiments, diagnosing the individual comprises a) obtaining a sample from the individual; b) contacting the sample with a bait composition containing a display portion comprising a cancer antigen peptide, under conditions sufficient to cause immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; c) isolating immune cells associated with the display portion; and d) analyzing the immune cells, wherein predetermined characteristics of the immune cells indicate cancer in the individual. In some embodiments, the predetermined characteristics of the immune cells include the presence of immune cells. In some embodiments, the predetermined characteristics of the immune cells include the number of immune cells above a threshold level. In some embodiments, the predetermined characteristics of the immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and / or histone glycosylation characteristics). In some embodiments, the display portion contains two or more (e.g., four) antigen peptides. In some embodiments, the two or more antigenic peptides in the display portion are identical. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus; d) being hydrophobic; and e) having a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the display portion comprises an MHC molecule complexed with the antigenic peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the immune cells are isolated single immune cells. In some embodiments, the immune cells are a mixture of immune cells. In some embodiments, the immune cell mixture is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or combinations thereof. In some embodiments, further analysis of the immune cells includes detecting and / or quantifying the immune cells. In some embodiments, further analysis of the immune cells includes sequencing one or more nucleic acids in the immune cells, optionally including analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences).In some embodiments, further analysis of immune cells includes identifying one or more epigenetic modifications in the immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications, such as acetylation, methylation, and glycosylation). In some embodiments, the method also includes generating a report containing information on an individual's cancer status. In some embodiments, the cancer status information includes: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; likelihood of cancer progression; likelihood of the presence of one or more cancer symptoms; molecular diagnostics; NGS pathology; and / or the individual's treatment regimen. In some embodiments, the decoy composition comprises multiple different display moieties. In some embodiments, each of the multiple different display moieties in the decoy composition contains a different antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display moieties, each containing a different MHC molecule). In some embodiments, each different display moieties containing a different MHC molecule contains a different detectable marker (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells associated with each different display moiety containing different MHC molecules into different populations, optionally further including contacting each of the multiple different display mocies separately with a sample from an individual and separating the immune cells associated with each different display moiety. In some embodiments, the method further includes culturing the immune cells prior to the analysis step. In some embodiments, the display moiety comprises particles (e.g., particles selected from: surfaces, nanoparticles, beads, and polymers). In some embodiments, the display moiety also comprises a detectable marker. In some embodiments, the detectable marker is a fluorophore. In some embodiments, the analysis of the sequence of one or more nucleic acids includes whole-genome sequencing, RNA-seq sequencing, and / or mass spectrometry analysis of the immune cells.

[0242] In some embodiments, the anticancer therapy is a standard or commonly used agent or therapy for treating cancer (e.g., a specific cancer). In some embodiments, the anticancer therapy includes chemotherapy agents. In some embodiments, the anticancer therapy includes surgery. In some embodiments, the anticancer therapy includes radiation therapy. In some embodiments, the anticancer therapy includes immunotherapy. In some embodiments, the anticancer therapy includes cell therapy (e.g., cell therapy comprising immune cells (e.g., CAR T cells)). In some embodiments, the anticancer therapy includes angiogenesis inhibitors.

[0243] In some implementation schemes, cancer therapy is not immunotherapy.

[0244] In some embodiments, the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual cancer. In some embodiments, minimal residual cancer is visible after the cancer has been surgically removed or cured. In some embodiments, the minimal residual disease is too small to be detected by imaging instruments (e.g., conventional or standard imaging instruments used for cancer detection). In some embodiments, the location of the minimal residual disease is diverse. In some embodiments, minimal residual cancer is a result of immune evasion or treatment resistance. In some embodiments, the individual has previously received cancer treatment and has not exhibited pathological symptoms of cancer after treatment.

[0245] In some embodiments, a method for treating an infection in an individual (e.g., an individual not yet diagnosed with an infection by a pathogen such as a virus, bacteria, or fungus, such as an individual at risk of developing an infection, such as an individual already receiving treatment for an infection) is provided, comprising a) diagnosing the individual with an infection according to the methods described herein; and b) administering anti-infective treatment to the individual. In some embodiments, diagnosing an individual comprises a) obtaining a sample from the individual; b) contacting the sample with a bait composition containing a display portion, the display portion containing an infectious antigenic peptide, under conditions sufficient to cause immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; c) isolating immune cells associated with the display portion; and d) analyzing the immune cells, wherein predetermined characteristics of the immune cells indicate an infection in the individual. In some embodiments, the predetermined characteristics of the immune cells include the presence of immune cells. In some embodiments, the predetermined characteristics of the immune cells include the number of immune cells above a threshold level. In some embodiments, the predetermined characteristics of the immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and / or histone glycosylation characteristics). In some embodiments, the display portion comprises two or more (e.g., four) antigenic peptides. In some embodiments, the two or more antigenic peptides in the display portion are identical. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus; d) being hydrophobic; and e) having a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the display portion comprises an MHC molecule complexed with the antigenic peptide. In some embodiments, the MHC molecules are MHC class I molecules and / or MHC class II molecules. In some embodiments, the immune cells are isolated single immune cells. In some embodiments, the immune cells are a mixture of immune cells. In some embodiments, the immune cell mixture is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or combinations thereof. In some embodiments, further analysis of the immune cells includes detecting and / or quantifying the immune cells. In some embodiments, further analysis of the immune cells includes sequencing one or more nucleic acids in the immune cells, optionally including analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences).In some embodiments, further analysis of immune cells includes identifying one or more epigenetic modifications in the immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications, such as acetylation, methylation, and glycosylation). In some embodiments, the method further includes generating a report that includes information about the infection status in an individual. In some embodiments, the information about the infection status includes: infection classification; infection type; nature of infection; source of infection; stage of infection; likelihood of infection development; likelihood of the presence of one or more infection symptoms; molecular diagnosis; NGS pathology; and / or treatment options for the individual. In some embodiments, the decoy composition includes a plurality of different display moieties. In some embodiments, each of the plurality of different display moieties in the decoy composition contains a different antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display moieties, each containing a different MHC molecule). In some embodiments, each different display moieties containing a different MHC molecule contains a different detectable marker (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells associated with each different display moiety containing different MHC molecules into different populations, optionally further including contacting each of the multiple different display mocies separately with a sample from an individual and separating the immune cells associated with each different display moiety. In some embodiments, the method further includes culturing the immune cells prior to the analysis step. In some embodiments, the display moiety comprises particles (e.g., particles selected from: surfaces, nanoparticles, beads, and polymers). In some embodiments, the display moiety also comprises a detectable marker. In some embodiments, the detectable marker is a fluorophore. In some embodiments, the analysis of the sequence of one or more nucleic acids includes whole-genome sequencing, RNA-seq sequencing, and / or mass spectrometry analysis of the immune cells.

[0246] In some embodiments, a method is provided for treating an individual (e.g., an individual not diagnosed with an autoimmune disease or disorder, such as an individual at risk of developing an autoimmune disease or disorder, such as an individual already receiving treatment for an autoimmune disease or disorder) with an autoimmune disease or disorder, comprising a) diagnosing the individual with an autoimmune disease or disorder according to the methods described herein; and b) administering anti-autoimmune disease or disorder treatment to the individual. In some embodiments, diagnosing an individual comprises a) obtaining a sample from the individual; b) contacting the sample with a bait composition containing a display portion, the display portion containing an autoimmune disease or disorder antigenic peptide, under conditions sufficient to cause immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; c) isolating immune cells associated with the display portion; and d) analyzing the immune cells, wherein predetermined characteristics of the immune cells indicate an autoimmune disease or disorder in the individual. In some embodiments, the predetermined characteristics of the immune cells include the presence of immune cells. In some embodiments, the predetermined characteristics of the immune cells include the number of immune cells above a threshold level. In some embodiments, predetermined characteristics of the immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and / or histone glycosylation characteristics). In some embodiments, the display portion contains two or more (e.g., four) antigenic peptides. In some embodiments, the two or more antigenic peptides in the display portion are identical. In some embodiments, the antigenic peptide has one or more of the following characteristics: a) a binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) a binding affinity to homologous TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) a mutation relative to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus; d) being hydrophobic; and e) having a high content of aromatic residues. In some embodiments, the antigenic peptide has low immunogenicity. In some embodiments, the exhibited portion comprises an MHC molecule complexed with the antigenic peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the immune cell is a single, isolated immune cell. In some embodiments, the immune cells are a mixture of immune cells. In some embodiments, the immune cell mixture is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or combinations thereof. In some embodiments, further analysis of the immune cells includes detecting and / or quantifying the immune cells.In some embodiments, further analysis of immune cells includes sequencing one or more nucleic acids in the immune cells, optionally including analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences). In some embodiments, further analysis of immune cells also includes identifying one or more epigenetic modifications in the immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications, such as acetylation, methylation, and glycosylation). In some embodiments, the method also includes generating a report that includes information about an individual's autoimmune disease or condition status. In some embodiments, the information about the autoimmune disease or condition status includes: classification of the autoimmune disease or condition; type of the autoimmune disease or condition; nature of the autoimmune disease or condition; origin of the autoimmune disease or condition; stage of the autoimmune disease or condition; likelihood of progression of the autoimmune disease or condition; likelihood of the presence of one or more symptoms of the autoimmune disease or condition; molecular diagnostics; NGS pathology; and / or treatment options. In some embodiments, the decoy composition includes multiple different display portions. In some embodiments, each of the plurality of different display moieties in the decoy composition contains a different antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display moieties, each containing a different MHC molecule). In some embodiments, each different display moiety containing a different MHC molecule contains a different detectable marker (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each different display moiety containing a different MHC molecule into different populations, optionally further including contacting each of the plurality of different display moieties with a sample from an individual and separating the immune cells associated with each different display moiety. In some embodiments, the method further includes culturing immune cells prior to the analysis step. In some embodiments, the display moieties contain particles (e.g., particles selected from: surfaces, nanoparticles, beads, and polymers). In some embodiments, the display moieties also contain a detectable marker. In some embodiments, the detectable marker is a fluorophore. In some embodiments, the analysis of the sequence of one or more nucleic acids includes whole-genome sequencing, RNA-seq sequencing, and / or mass spectrometry analysis of immune cells.

[0247] bait composition, display portion, MHC-peptide complex and antigenic peptide In some embodiments, this application provides compositions comprising one or more display portions or decoy compositions. The decoy compositions described herein comprise one or more display portions containing one or more antigenic peptides (e.g., containing one or more MHC-peptide complexes), wherein immune cells (e.g., T cells) bind to the display portion and / or the antigenic peptide (e.g., homologous binding). Any of these decoy compositions and display portions can be used in the methods described herein for analyzing samples, detecting antigen-specific immune cells, and therapeutic methods.

[0248] Demonstration section (i.e., MHC multimer unit) In some embodiments, the illustrated portion described herein comprises an MHC molecule complexed with an antigenic peptide (e.g., a neoantigen peptide, such as a backbone neoantigen peptide).

[0249] This application provides an illustration comprising a support (e.g., particles) associated with multiple MHC-peptide complexes (e.g., MHC-peptide monomers), wherein at least two MHC-peptide complexes are different.

[0250] In some embodiments, the display portion includes a support (e.g., particles) that bind to a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes include not less than or more than 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex includes at least one MHC molecule complexed with a peptide, wherein at least two or more MHC-peptide complexes are distinct.

[0251] In some implementations, the display portion does not contain fluorescent dyes or fluorophores.

[0252] In some embodiments, this application provides an illustration portion comprising particles (e.g., spherical particles, solid particles, or beads) bound to a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex is an MHC-peptide monomer comprising an MHC molecule complexed with the peptide, and wherein at least two MHC-peptide complexes are distinct. In some embodiments, the plurality of MHC-peptide complexes comprises at least 20 MHC-peptide complexes. In some embodiments, the plurality of MHC-peptide complexes comprises 20, 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 50,000, 100,000, 15,000, or 200,000 MHC-peptide complexes. In some embodiments, the MHC-peptide complex is attached to the particle via a binding pair comprising a first binding component binding to the MHC-peptide complex or its units (i.e., MHC-peptide monomers) and a second binding component binding to the particle. In some embodiments, the first binding component comprises biotin, and the second binding component comprises streptavidin. In some embodiments, the particle diameter is at least 0.5 µm (e.g., at least 1 µm). In some embodiments, the particle diameter is from about 0.01 µm to about 100 µm (e.g., from 0.5 µm to about 100 µm, or from 0.5 µm to about 20 µm). In some embodiments, the particle diameter is from about 0.5 µm to about 10 µm. In some embodiments, the particle diameter is about 1 µm. In some embodiments, the particle surface area is about 0.01 μm. 2 Approximately 1000 μm 2 (e.g., approximately 0.1 μm) 2 Approximately 100 μm 2 Approximately 1 μm 2 Approximately 20 μm 2In some embodiments, the particle has about 10 to about 200,000 (e.g., about 100 to about 20,000, or about 10 to about 10,000) of a second binding component associated with it. In some embodiments, the display portion contains at least two different MHC class I molecules. In some embodiments, the display portion contains at least two different MHC class II molecules. In some embodiments, the display portion contains at least one MHC class I molecule and at least one MHC class II molecule. In some embodiments, the display portion contains an HLA-A molecule. In some embodiments, the HLA-A molecule contains a mutation that reduces its binding to CD8. In some embodiments, the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion includes at least two MHC class I molecules selected from the following: a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion includes MHC class II molecules selected from HLA-DP, HLA-DQ, and HLA-DR. In some embodiments, the peptide is associated with a cancer or tumor antigen. In some embodiments, the peptide is associated with a neoantigen. In some embodiments, the peptide is associated with a pathogen antigen. In some embodiments, the pathogen is a virus, bacteria, or fungus. In some embodiments, the peptide is associated with an autoantigen. In some embodiments, the display portion contains at least two different peptides (e.g., at least 2, 5, 10, 20, 30, 40, 50, 100, 200, 500, or 1000 different peptides). In some embodiments, at least two different peptides are associated with two different antigens. In some embodiments, the two different antigens contain two different mutations in one or more genes (e.g., oncogenes) associated with a disease (e.g., cancer). In some embodiments, the oncogene has a driver mutation. In some embodiments, the oncogene includes one or more of KRAS, EGFR, POLE, TP53, SMAD, BRCA1, CDKN2A, TNN, MUC16, BRAF, and ALK. In some embodiments, the oncogene includes TP53, KRAS, MUC16, SMAD, CDKN2A, and TNN. In some embodiments, at least two different peptides overlap (e.g., at least 2, 3, 4, 5, or 6 amino acids overlap).In some embodiments, at least two different peptides do not overlap. In some embodiments, the display portion does not contain fluorescent dyes or fluorophores.

[0253] In some embodiments, this application provides an illustration portion comprising particles (e.g., spherical particles, solid particles, or beads) bound to a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule complexed with a peptide, wherein at least two MHC-peptide complexes are distinct, and wherein the plurality of MHC-peptide complexes comprises at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 50,000, 10,000, 15,000, 20,000, 50,000, 100,000, 200,000, 500,000, or 1,000,000 MHC-peptide complexes. In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers. In some embodiments, at least one of the plurality of MHC-peptide complexes has two distinct MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, MHC-peptide trimer, or MHC-peptide tetramer. In some embodiments, the MHC-peptide complex is attached to a particle via a binding pair comprising a first binding component binding to the MHC-peptide complex or its units (i.e., MHC-peptide monomers) and a second binding component binding to the particle. In some embodiments, the first binding component comprises biotin, and the second binding component comprises streptavidin, or vice versa. In some embodiments, the particle diameter is at least 0.5 µm (e.g., at least 1 µm). In some embodiments, the particle diameter is from about 0.01 µm to about 100 µm (e.g., from 0.5 µm to about 100 µm, or from 0.5 µm to about 20 µm). In some embodiments, the particle diameter is from about 0.5 µm to about 10 µm. In some embodiments, the particle diameter is about 1 µm. In some embodiments, the particle surface area is about 0.01 μm. 2 Approximately 1000μm 2 (e.g., approximately 0.1 μm) 2 Approximately 100 μm 2 Approximately 1 μm 2 Approximately 20 μm 2In some embodiments, the particle has about 10 to about 200,000 (e.g., about 100 to about 20,000, or about 10 to about 10,000) of a second binding component associated with it. In some embodiments, the display portion contains at least two different MHC class I molecules. In some embodiments, the display portion contains at least two different MHC class II molecules. In some embodiments, the display portion contains at least one MHC class I molecule and at least one MHC class II molecule. In some embodiments, the display portion contains an HLA-A molecule. In some embodiments, the HLA-A molecule contains a mutation that reduces its binding to CD8. In some embodiments, the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion includes at least two MHC class I molecules selected from the following: a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion includes MHC class II molecules selected from HLA-DP, HLA-DQ, and HLA-DR. In some embodiments, the peptide is associated with a cancer or tumor antigen. In some embodiments, the peptide is associated with a neoantigen. In some embodiments, the peptide is associated with a pathogen antigen. In some embodiments, the pathogen is a virus, bacteria, or fungus. In some embodiments, the peptide is associated with a self-antigen. In some embodiments, the display portion contains at least two different peptides (e.g., at least 2, 5, 10, 20, 30, 40, 50, 100, 200, 500, or 1000 different peptides). In some embodiments, at least two different peptides are associated with two different antigens. In some embodiments, the two different antigens contain two different mutations in one or more genes (e.g., oncogenes) associated with a disease (e.g., cancer). In some embodiments, the oncogene has a driver mutation. In some embodiments, the oncogene includes one or more of KRAS, EGFR, POLE, TP53, SMAD, BRCA1, CDKN2A, TNN, MUC16, BRAF, and ALK. In some embodiments, the oncogene includes TP53, KRAS, MUC16, SMAD, CDKN2A, and TNN. In some embodiments, at least two different peptides overlap. In some embodiments, at least two different peptides do not overlap. In some implementations, the display portion does not contain fluorescent dyes or fluorophores.

[0254] In some embodiments, this application provides an illustration portion comprising particles (e.g., spherical particles, solid particles, beads) associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises at least 2, 3, 4, 5, 6, 8, 9, 10, or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises an MHC molecule complexed with a peptide, wherein at least two MHC-peptide complexes are distinct, and wherein the surface area of ​​the particles is at least about 1 µm. 2 The diameter is at least 0.5 µm. In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer. In some embodiments, the MHC-peptide complex is attached to the particle via a binding pair comprising a first binding component binding to the MHC-peptide complex or its units (i.e., MHC-peptide monomers) and a second binding component binding to the particle. In some embodiments, the first binding component comprises biotin, and the second binding component comprises streptavidin. In some embodiments, the particle diameter is from about 0.5 µm to about 100 µm (e.g., 0.5 µm to about 100 µm, e.g., 1 µm to about 20 µm). In some embodiments, the particle diameter is from about 1 µm to about 10 µm. In some embodiments, the particle diameter is about 1 µm. In some embodiments, the particle surface area is about 1 μm. 2 Approximately 1000 μm 2 (e.g., about 1 μm) 2 Approximately 100 μm 2 Approximately 1 μm 2 Approximately 20 μm 2In some embodiments, the particle has about 10 to about 200,000 (e.g., about 100 to about 20,000, or about 10 to about 10,000) of a second binding component associated with it. In some embodiments, the display portion contains at least two different MHC class I molecules. In some embodiments, the display portion contains at least two different MHC class II molecules. In some embodiments, the display portion contains at least one MHC class I molecule and at least one MHC class II molecule. In some embodiments, the display portion contains an HLA-A molecule. In some embodiments, the HLA-A molecule contains a mutation that reduces its binding to CD8. In some embodiments, the HLA-A molecule contains an HLA A2 heavy chain with an A245V mutation. In some embodiments, the display portion includes at least two MHC class I molecules selected from the followi...

Claims

1. A method for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in samples from one or more individuals, the method comprising: a) Immobilizing immune cells from the sample onto a solid support, wherein the immune cells: 1) Prior to fixation, the bait composition containing a display portion of the antigenic peptide has been contacted under conditions sufficient to allow immune cells to bind to the display portion; or 2) After fixation, contact the bait composition containing the display portion of the antigenic peptide with the bait composition under conditions sufficient to allow immune cells to bind to the display portion; b) Contact the fixed immune cells with a detection agent that recognizes the displayed portion under conditions sufficient to allow the agent to bind to the displayed portion; as well as c) Detect the detection agent to detect immune cells bound to the displayed portion. Optionally, the method further includes, prior to step c), separating immune cells bound to the display portion, or removing the display portion or immune cells that are not bound to each other.

2. A method for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in samples from one or more individuals, the method comprising: a) Contact the immune cells with a bait composition containing a display portion of antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion; b) Contact the immune cells with a detection agent that recognizes the displayed portion under conditions sufficient to allow the agent to bind to the displayed portion; c) Immobilize the immune cells from the sample onto a solid support, and d) Detect the detection agent to detect immune cells bound to the displayed portion. Optionally, the method further includes, prior to step d), separating immune cells bound to the display portion, or removing the display portion or immune cells that are not bound to each other.

3. The method of claim 1 or claim 2, wherein the sample is obtained from a single individual.

4. The method of claim 1 or claim 2, wherein the sample is a mixture of a plurality of individual samples, each obtained from a different individual, optionally wherein each of the plurality of individual samples is individually processed before being combined to form the mixture such that the immune cells contained therein are associated with a unique sample barcode, optionally wherein the method further includes removing unbound molecules containing the barcode, optionally wherein the method further includes determining the unique barcode associated with the immune cells bound to the displayed portion, thereby identifying the sample from which the immune cells originated.

5. The method of any one of claims 1 and 3-4, wherein a) prior to the fixation, the immune cells have been contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion, and b) the immune cells are contacted with a decoy composition comprising a display portion containing an antigenic peptide under conditions sufficient to allow the immune cells to bind to the display portion after fixation.

6. A method for analyzing the presence or absence of immune cells capable of binding to antigenic peptides in multiple samples from one or more individuals, comprising: a) Process each of multiple individual samples separately, so that the immune cells contained within are associated with a unique sample barcode. b) Combining the plurality of processed samples to form a sample mixture. c) The sample mixture is contacted with a bait composition containing a display portion of the antigenic peptide under conditions sufficient to allow immune cells to bind to the display portion, thereby forming a bait-sample mixture. d) Detect immune cells in the bait-sample mixture that bind to the displayed portion; as well as e) Identify a unique barcode associated with the immune cells in connection with the displayed portion, thereby identifying the sample from which the immune cells originated. Optionally, the method further includes, prior to step d), separating immune cells bound to the display portion, or removing the display portion or immune cells that are not bound to each other.

7. The method of any one of claims 1-6, wherein the immune cells comprise T cells, optionally the immune cells comprise no more than about 10%, 5%, 4%, 3%, 2% or 1% of T cells that specifically bind to the antigenic peptide in the bait composition, optionally wherein the immune cells are selected from: cytotoxic T cells, memory T cells and tumor-infiltrating T cells.

8. The method of any one of claims 1-7, wherein the method further comprises removing unbound display portions after contacting the immune cells with the agent recognizing the display portion and before detecting immune cells bound to the display portion.

9. The method of any one of claims 1-8, wherein the antigenic peptide is associated with a cancer or tumor antigen, optionally wherein the antigenic peptide is a neoantigen peptide, and optionally wherein the sample is obtained from one or more individuals who do not exhibit cancer pathological symptoms.

10. The method of any one of claims 1-5 and 7-9, wherein the immune cells are immobilized on the solid matrix such as beads, optionally wherein the beads are associated with an antibody that recognizes the immune cells, optionally wherein the antibody is a CD8 antibody.

11. The method of any one of claims 1-10, wherein the display portion comprises two or more antigenic peptides, optionally wherein the display portion comprises four antigenic peptides, and optionally wherein two or more antigenic peptides in the display portion are identical.

12. The method of claims 9-11, wherein the display portion comprises a library of antigenic peptides associated with cancer or tumor mutations, wherein the library comprises at least about 5, 10, 12, 15, 18, or 20 different neoantigen peptides.

13. The method of any one of claims 1-12, wherein the displayed portion comprises an MHC molecule (i.e., an MHC-peptide complex) complexed with the antigenic peptide, wherein the MHC molecule comprises an MHC class I molecule, optionally wherein the MHC molecule comprises an MHC polymer, and further optionally the MHC polymer is an MHC dimer, an MHC trimer, an MHC tetramer, or an MHC dextran polymer.

14. The method of claim 13, wherein the display portion comprises two or more different types of MHC class I molecules, the MHC class I molecules being selected from... a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01 and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, HLA-A*33:

03. The method of any one of claims 1-13, wherein the display portion comprises particles, optionally wherein the particles in the display portion are selected from: surfaces, nanoparticles, beads, and polymers.

15. The method of claim 14, wherein the particles comprise beads, wherein the diameter of the beads is at least about 0.1 μm, 0.25 μm, 0.5 μm or 1 μm.

16. The method of any one of claims 14-15, wherein the antigenic peptide or MHC a) is directly attached to the particles in the display portion, or b) is attached to the particles in the display portion via a binding pair, the binding pair comprising a first binding component attached to the antigenic peptide or MHC and a second binding component bound to the particles.

17. The method of any one of claims 1-16, wherein the method further comprises a) separating immune cells bound to the display portion or removing the display portion or immune cells that are not bound to each other, and / or b) performing phenotypic analysis on the immune cells bound to the display portion.

18. The method of any one of claims 1-17, wherein the individual is a person.

19. The method of any one of claims 9-18, wherein the individual a) Individuals who have not been previously diagnosed with cancer, optionally wherein said individual is at risk of developing cancer, or b) The individual had previously received cancer treatment and did not exhibit any pathological symptoms of cancer after the treatment.

20. The method of any one of claims 1-19, wherein the sample is selected from blood, plasma, and peripheral blood mononuclear cell (PBMC) samples.

21. The method of any one of claims 1-20, wherein the decoy composition comprises a plurality of different display portions, optionally wherein the plurality of different display portions in the decoy composition comprises at least two different display portions, each display portion comprising a different MHC molecule.

22. A method for detecting antigen-specific immune cells in an individual, comprising: Analyze samples from individuals according to any one of claims 1-21. The predetermined characteristics of the immune cells indicate an individual's disease or condition.

23. The method of claim 22, wherein the predetermined characteristics of the immune cells include the presence of the immune cells, optionally wherein the presence of immune cells specifically binding to a bait composition containing one or more neoantigens indicates the presence of cancer, b) the presence of immune cells specifically binding to a bait composition containing one or more pathogen (e.g., virus, bacteria, fungus) antigens indicates the presence of pathogen infection, or c) the presence of immune cells specifically binding to a bait composition containing one or more autoantigens associated with an autoimmune disease or condition indicates the presence of an immune disease or condition.

24. A method for detecting residual cancer in an individual, wherein the individual has previously been treated with anticancer therapy and has not exhibited pathological symptoms of cancer after treatment, the method comprising analyzing a post-treatment sample from the individual according to any one of claims 9-21. The predetermined characteristics of immune cells from the post-treatment sample indicate residual cancer in the individual.

25. A method for treating cancer in an individual, the method comprising: a) The method according to claim 23 diagnoses the individual as having cancer; and b) subject the individual to anticancer therapy.

26. A method for treating an infection in an individual, the method comprising: a) The method according to claim 23 diagnoses the individual as having a pathogen infection; and b) subject the individual to treatment targeting the pathogen.

27. A method for treating an individual's autoimmune disease or condition, the method comprising: a) The method according to claim 23 for diagnosing an individual with an autoimmune disease or condition; and b) subject the individual to treatment for the disease or condition.

28. A display portion comprising particles associated with a plurality of MHC-peptide complexes, wherein the plurality of MHC-peptide complexes comprises not less than or more than 2, 3, 4, 5, 6, 8, 9, 10 or 12 MHC-peptide complexes, wherein each MHC-peptide complex comprises at least one MHC molecule complexed with a peptide, wherein at least two or more MHC-peptide complexes are distinct.

29. The illustration portion of claim 28, wherein one or more of the plurality of MHC-peptide complexes are MHC-peptide monomers, MHC-peptide dimers, MHC-peptide trimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextran polymers, or MHC-peptide dodecamers.

30. The illustration portion of claim 28 or claim 29, wherein at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers, optionally the at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer or an MHC-peptide tetramer.

31. The illustration portion of any one of claims 28-30, wherein the plurality of MHC-peptide complexes comprises at least 20 MHC-peptide complexes, and optionally wherein the plurality of MHC-peptide complexes comprises at least 50 or 100 MHC-peptide complexes.

32. The illustration portion of any one of claims 28-31, wherein the MHC-peptide complex is attached to the particle via a binding pair, the binding pair comprising a first binding component attached to the MHC-peptide complex or a unit thereof and a second binding component bound to the particle, optionally wherein the first binding component comprises biotin and the second binding component comprises streptavidin.

33. The display portion as claimed in any one of claims 28-32, wherein the particles a) It is a solid; b) The diameter is from about 0.1 µm to about 50 µm (e.g., at least 0.1 µm, 0.2 µm, 0.25 µm, 0.5 µm, 0.75 µm or 1 µm, optionally the diameter of the particle is from about 0.5 µm to about 20 µm, optionally the diameter of the particle is from about 0.5 µm to about 10 µm, and further optionally the diameter of the particle is about 1 µm; c) Surface area is approximately 1 μm 2 Approximately 20 μm 2 ; d) having a number of about 10 to about 10,000 associated second binding components; e) the particles are spherical, optionally wherein the particles are beads; and / or f) is magnetic.

34. The display portion as claimed in any one of claims 28-33, wherein the display portion comprises at least two different MHC molecules, optionally wherein a) The at least two different MHC molecules include two different MHC class I molecules; b) The at least two different MHC molecules include two different MHC class II molecules; and / or c) The at least two different MHC molecules include MHC class I molecules and MHC class II molecules.

35. The display portion as claimed in any one of claims 28-34, wherein: a) The display portion comprises an HLA-A molecule, further optionally wherein the HLA-A molecule comprises a mutation that reduces its binding to CD8, further optionally wherein the HLA-A molecule comprises an HLA A2 heavy chain having an A245V mutation; b) The display portion contains at least two MHC class I molecules selected from the following: a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01; or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03; and / or c) The display portion contains MHC class II molecules selected from HLA-DQ and HLA-DR.

36. The display portion as claimed in any one of claims 28-35, wherein: a) The peptide is associated with a cancer or tumor antigen, optionally wherein the peptide is associated with a neoantigen; b) The peptide is associated with an antigen of a pathogen, optionally wherein the pathogen is a virus, bacteria, or fungus; c) The peptide is associated with an autoantigen; d) The display portion contains at least two different peptides; optionally, the display portion contains at least five different peptides, and optionally, the display portion contains at least 2, 5, or 10 different peptides; and / or e) The display portion does not contain fluorescent dyes or fluorophores.

37. A decoy composition comprising the display portion as described in any one of claims 28-36.

38. The bait composition of claim 37, wherein: a) The bait composition comprises at least two distinct display portions; b) The bait composition contains HLA-A molecules, HLA-B molecules, and HLA-C molecules; c) The bait composition comprises HLA-A*24:02, HLA-A*11:01, HLA-A*02:01 and HLA-a*03:01; d) The decoy composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules; e) The bait composition contains at least 25, 50, 100, 150, 200 or 250 different peptides; f) The peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225 or 250 different antigens; g) The bait composition comprises at least 2, 3, 4, 5, 6 or 7 different overlapping peptides associated with gene mutations, optionally wherein the gene is an oncogene; h) The peptide in the bait composition is associated with mutations in at least two, three, four, five, or six genes, optionally said genes being oncogenes; and / or i) The decoy composition does not contain fluorescent dyes or fluorophores.

39. A method for analyzing immune cells in a sample from an individual, comprising: a) Contacting multiple immune cells with the display portion of any one of claims 28-36 or the bait composition of claim 37 or 38. b) Detect or analyze one or more immune cells that are bound to one or more of the displayed portions.

40. The method of claim 39, wherein: a) The immune cells include T cells; b) The immune cells are selected from: cytotoxic T cells, memory T cells and tumor-infiltrating T cells, optionally wherein the T cells are CD4+ T cells and / or CD8+ T cells; c) The immune cells include B cells; and / or d) The immune cells comprise a mixture of immune cells, including T cells, B cells, macrophages, or dendritic cells or combinations thereof.

41. The method of claim 39 or claim 40, wherein: a) The samples are selected from: blood, plasma, and peripheral blood mononuclear cell (PMBC) samples; and / or b) The sample is obtained from diseased tissue, optionally wherein the diseased tissue is cancerous tissue or tumor tissue.

42. The method of any one of claims 39-41, wherein a) the sample is obtained from a single individual, or b) the sample is a mixture of multiple individual samples, each obtained from a different individual.

43. The method of any one of claims 39-42, wherein the individual a) is a person, and / or b) has no signs of disease, optionally wherein the disease is cancer.

44. The method of any one of claims 39-43, wherein detecting or analyzing the immune cells bound to one or more display portions comprises observing the agglutination of the immune cells and one or more display portions.

45. The method of any one of claims 39-44, wherein detecting or analyzing the immune cells bound to one or more display portions comprises: 1) Before or after step a), fix the immune cells onto a solid matrix; 2) The immobilized immune cells are brought into contact with a detection agent that recognizes the displayed portion under conditions sufficient to allow the agent to bind to the displayed portion; 3) Detect the detection agent to detect immune cells that are bound to the displayed portion.

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