Determining antigen recognition through barcoding of MHC multimers

Abstract

To provide a high-throughput method for detection, isolation and / or identification of specific antigen responsive cells such as antigen specific T-cells, the method being capable of detection, isolation and / or identification of multiple species of specific antigen responsive cells, for example T-cells, in a single sample.SOLUTION: The present invention describes the use of nucleic acid barcodes as specific labels for MHC multimers to determine the antigen responsiveness in biological samples. After cellular selection the barcode sequence will be revealed by sequencing. This technology allows for detection of multiple (potentially over 1000) different antigen-specific cells in a single sample. The technology can be used for T-cell epitope mapping, immune recognition discovery, diagnostic tests and measuring immune reactivity after vaccination or immune-related therapies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antigen recognition by nucleic acid-tagged MHC multimers. [Background technology]

[0002] The adaptive immune system is driven by specific interactions between immune cells and antigen-presenting cells (e.g., dendritic cells, B cells, monocytes, and macrophages) or target cells (e.g., virus-infected cells, bacteria-infected cells, or cancer cells). An important field of immunology concerns understanding the molecular interactions between immune cells and target cells.

[0003] Specifically, in T lymphocytes (T cells), this interaction is mediated by binding between the T cell receptor (TCR) and major histocompatibility complex (MHC) class I or class II. MHC molecules carry peptide cargo, and this peptide plays a crucial role in T cell recognition. In 1996, Atman et al. (1) achieved a dramatic technological breakthrough in our understanding of T cell recognition when they discovered that multimerizing a single peptide-MHC molecule into a tetramer provided sufficient binding strength (avidity) between the peptide-MHC molecule and the TCR, allowing this interaction to be revealed by fluorescent labels attached to the MHC multimer. These fluorescently labeled MHC multimers (both class I and class II molecules) are now widely used to determine T cell specificity. The fluorescence of the MHC multimers can be determined, for example, by flow cytometry or microscopy, or T cells can be sorted based on this fluorescent label, for example, by flow cytometry or bead sorting. However, a limitation imposed on this method is the number of different fluorescent labels that can be used, since each fluorescent label serves as a specific signature for the peptide-MHC in question.

[0004] Therefore, this strategy does not adequately compensate for the enormous diversity of T cell recognition. For the most prevalent subset of T cells (αβTCR T cells), the number of different TCRs in an individual is approximately 10 7 (3), but the number of possible different αβ TCRs is approximately 10 15 (2). Consequently, considerable effort has been devoted to expanding the complexity of T cell characterization to allow for the detection of multiple distinct T cell specificities in a single sample. A relatively recent invention for the multiplexed detection of antigen-specific T cells is the use of encoded MHC multimer combinations. This technique employs combinatorial fluorescent labeling and, when first published, allowed for the detection of 28 distinct T cell populations in a single sample (4, 5). It was later expanded by combining novel instrumentation with heavy metal labels, allowing for the detection of approximately 100 distinct T cell populations in a single sample (6).

[0005] The need for new technologies that can more comprehensively analyze antigen-specific T cell responses is evident from the efforts of several groups to develop so-called MHC microarrays, which would spatially encode T cell specificity rather than fluorescently (7, 8). Although promising, MHC microarrays have not yet been widely adopted, and no studies have demonstrated their value for multiplexed measurements of T cell responses, such as epitope identification.

[0006] In view of the above, there is a need for high throughput methods in the field of detection, isolation and / or identification of specific antigen-responsive cells, such as antigen-specific T cells.

[0007] Furthermore, given that the amount of sample available is often limited, there is a need in the art for methods that can detect, isolate, and / or identify multiple types of specific antigen-responsive cells, such as T cells, in a single sample. Summary of the Invention

[0008] The present invention is the use of nucleic acid barcodes to determine and track the antigen specificity of immune cells.

[0009] In one embodiment of the present invention, nucleic acid barcodes serve as specific labels for a given peptide-MHC molecule that multimerizes to form an MHC multimer. The multimers can be composed of MHC class I, class II, CD1, or other MHC-like molecules. Therefore, hereafter, the term "MHC multimer" includes any MHC-like molecule. MHC multimers are formed by the multimerization of peptide-MHC molecules via different backbones. A barcode is simultaneously attached to the multimer, serving as a specific label for a particular peptide-MHC complex. In this manner, up to 1,000-10,000 (or potentially even more) different peptide-MHC multimers can be mixed and allowed to specifically interact with T cells from a biological sample, such as blood, followed by washing away unbound MHC-multimers and sequencing the DNA barcodes. When sorting a cell population of interest, the sequence of the barcodes present above background levels provides a fingerprint for identifying antigen-responsive cells present in a given cell population. The number of sequence reads for each specific barcode correlates with the frequency of specific T cells, which can be estimated by comparing the frequency of reads with the input frequency of T cells. This strategy may broaden our understanding of T cell recognition.

[0010] The DNA barcode serves as a unique tag for antigen-specific T cells and can be used to determine T cell specificity, for example, after single-cell sorting, functional analysis, or phenotypic evaluation. In this way, antigen specificity can be linked to both the T cell receptor sequence (which can be revealed by single-cell sequencing methods) and the functional and phenotypic characteristics of the antigen-specific cells.

[0011] Furthermore, this strategy may allow multiple different (sequence-related) peptide-MHC multimers to be bound to a given T cell, with the binding affinity of the given peptide-MHC multimer determining the relative contribution of each peptide-MHC multimer to binding with the cell surface TCR. Applying this feature, it is possible to determine the precise specificity / consensus recognition sequence of a given TCR by using overlapping peptide libraries or alanine-substituted peptide libraries. Such determination is not possible with currently used MHC multimer-based technologies.

[0012] Thus, one aspect of the present invention is two or more MHCs linked by a main chain molecule; and at least one nucleic acid molecule linked to the backbone, The nucleic acid molecule relates to a multimeric major histocompatibility complex (MHC) that includes a central stretch of nucleic acid (barcode region) designed to be amplified, for example, by PCR.

[0013] Another aspect of the present invention relates to a composition comprising subsets of multimeric major histocompatibility complexes (MHC) according to the present invention, each set of MHC having a different peptide that plays a crucial role in T cell recognition and a unique "barcode" region in the DNA molecule.

[0014] Another aspect of the present invention is a method for producing a semiconductor device comprising: a composition according to the present invention; one or more sets of primers that amplify the nucleic acid molecule; The purpose of the present invention is to provide a kit of parts including:

[0015] Another aspect of the invention is a method for detecting antigen-responsive cells in a sample, comprising the steps of: Providing one or more multimeric major histocompatibility complex (MHC) molecules according to the invention or a composition according to the invention; contacting the multimeric MHC with the sample; detecting binding of the multimeric MHC to the antigen-responsive cells to detect cells responsive to antigens present in the MHC set; wherein said binding is detected by amplifying a barcode region of said nucleic acid molecule linked to one or more MHC.

[0016] Further aspects relate to various uses. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the production of barcode-labeled MHC multimers. [Figure 2] FIG. 1 shows the generation of a library of barcode-labeled MHC multimers. [Figure 3] FIG. 1 shows detection of antigen-responsive cells in a single sample. [Figure 4] FIG. 1 shows the possibility of linking antigen specificity (tracked by barcode) with other properties. [Figure 5] FIG. 1 shows a set of experimental data demonstrating the experimental feasibility of the present invention. [Figure 6] FIG. 1 shows experimental data from Example 2 onward. [Figure 7] FIG. 1 shows experimental data from Example 2 onward. [Figure 8] FIG. 1 shows experimental data from Example 2 onward. [Figure 9] FIG. 1 shows experimental data from Example 2 onward. [Figure 10] FIG. 1 shows experimental data from Example 2 onward. [Figure 11] FIG. 1 shows experimental data from Example 2 onward. [Figure 12] FIG. 1 shows experimental data from Example 2 onward. DETAILED DESCRIPTION OF THE INVENTION

[0018] (Detailed Description of the Invention) The present invention will now be described in more detail below.

[0019] definition Before describing the present invention in further detail, we first define the following terms and conventions.

[0020] TCR: T-cell receptor MHC: major histocompatibility complex multimeric MHC: multimeric major histocompatibility complex

[0021] Nucleic acid barcode In this context, a nucleic acid barcode is a unique oligonucleotide sequence ranging from 10 to over 50 nucleotides. Barcodes share amplified sequences at the 3' and 5' ends and a unique central sequence that can be revealed by sequencing and serves as a specific barcode for a given molecule.

[0022] Sequencing In this embodiment, it is understood that sequencing also relates to, for example, deep sequencing or next-generation sequencing, which repeats the sequencing of amplified barcodes (PCR products) multiple times (total number of reads is, for example, 100,000).Since all DNA barcodes have almost the same amplification characteristics, the number of reads of each barcode sequence is related to its abundance in the amplification product, which further represents the abundance before amplification.Therefore, the number of reads of a specific barcode sequence relative to the total number of reads correlates with the presence of antigen-responsive cells in the test sample.

[0023] Now, more specifically, the present invention is described in FIG. 1, which illustrates a method for assembling peptide-MHC molecules, nucleic acid (DNA) barcodes, and (optional) fluorescent labels to form a library of MHC multimers, each with a DNA barcode specific to a given peptide-MHC molecule contained within it. The barcodes in FIG. 1A are designed to have unique sequences that can be determined by DNA sequencing. Additionally, the barcodes share a common amplification end that allows all DNA barcodes to be simultaneously amplified in a PCR reaction. The DNA barcodes are attached to an MHC-multimerization backbone (e.g., via a biotin linker that binds to streptavidin on the multimer backbone). FIG. 1B depicts a multimer backbone, which can be any backbone that allows for the multimerization of macromolecules. The backbone can (optionally) carry a fluorescent label (indicated by an asterisk) that tracks the entire pool of MHC multimers that bind to cells, regardless of the specificity of the peptide-MHC multimers. Figure 1C depicts a peptide-MHC molecule of interest with a specific peptide cargo (horizontal line), and Figure 1D depicts an assembled peptide-MHC multimer with a DNA barcode.

[0024] Multimeric major histocompatibility complex (MHC) One aspect of the present invention is two or more MHCs linked by a main chain molecule; at least one nucleic acid molecule linked to the backbone; wherein the nucleic acid molecule comprises a central stretch of nucleic acid (barcode region) designed to be amplified, for example by PCR.

[0025] Various types of backbones may be used. Thus, in one embodiment, the backbone molecule is selected from the group consisting of polysaccharides, e.g., glucans such as dextran, streptavidin or streptavidin multimers. Other alternative backbones may be identified by those skilled in the art.

[0026] The MHC may be attached to the backbone by a variety of means. Thus, in one embodiment, the MHC may be attached to the backbone via a streptavidin-biotin or streptavidin-avidin bond. Other moieties may also be used. Specific attachment may be via a specific attachment point. In another embodiment, the MHC is linked to the backbone via the MHC heavy chain. The MHC consists of various components, some of which can be expressed and purified in cell lines (e.g., the MHC heavy chain and β-2-microglobulin components). Alternatively, the components can be chemically synthesized. Preferably, specific peptides are chemically synthesized.

[0027] All three elements are required to create a stable MHC (complex), therefore in one embodiment the MHC is artificially assembled.

[0028] The multimeric MHC may comprise a varying number of MHCs. Thus, in a further embodiment, the multimeric major histocompatibility complex (MHC) consists of at least four MHCs, such as at least eight, such as at least 10, such as 2-30, 2-20, such as 2-10 or such as 4-10 MHCs.

[0029] The nucleic acid component (preferably DNA) has a specific structure. Thus, in one embodiment, at least one nucleic acid molecule comprises at least a 5' first primer region, a central region (barcode region), and a 3' second primer region. In this manner, the central region (barcode region) can be amplified with a primer set. The length of the nucleic acid molecule can also vary. Thus, in another embodiment, the at least one nucleic acid molecule has a length ranging from 20 to 100 nucleotides, for example, 30 to 100 nucleotides, for example, 30 to 80 nucleotides, for example, 30 to 50 nucleotides. The bond between the nucleic acid molecule and the backbone can also vary. Thus, in a further embodiment, the at least one nucleic acid molecule is linked to the backbone via a streptavidin-biotin bond and / or a streptavidin-avidin bond. Other binding moieties may also be used.

[0030] In a further embodiment, at least one nucleic acid molecule comprises or consists of DNA, RNA and / or artificial nucleotides, such as PLA or LNA, etc. DNA is preferred, but nucleotides other than DNA may also be included, for example to increase stability.

[0031] Various types of MHC can form part of a multimer. Thus, in one embodiment, the MHC is selected from the group consisting of class I MHC, class II MHC, CD1, or MHC-like molecules. In MHC class I, the presented peptide is a 9- to 11-mer peptide; in MHC class II, the presented peptide is a 12- to 18-mer peptide. In alternative MHC molecules, it can be a fragment of a presented lipid or sugar molecule.

[0032] In addition, it would be advantageous to be able to determine the entire pool of bound multimers when incubated with a (cell) sample. Therefore, in a preferred embodiment, the backbone further comprises one or more linked fluorescent labels. Having such a linkage allows for better quantification. Similarly, labels may be used for cell sorting.

[0033] composition Figure 2 illustrates the creation of a complete barcode library. This library, Figure 2A, consists of a large number of potentially more than 1000 different peptide-MHC multimers, each with a specific DNA barcode. Thus, barcode number 1 encodes peptide-MHC complex number 1, barcode number 2 encodes peptide-MHC complex number 2, barcode number 3 encodes peptide-MHC complex number 3, and so on, until a mixture of thousands of different specificities, each with a specific barcode, is possible. Figure 2B depicts the final reagent, which is a mixture of many different MHC-multimers, each with a specific DNA barcode as a label for each peptide-MHC specificity.

[0034] As mentioned above, a pool (library) of different sets of multimeric major histocompatibility complexes (MHC) can be used to analyze the peptide specificity of an entire cell population. Therefore, another aspect of the present invention relates to a composition comprising a subset of multimeric major histocompatibility complexes (MHC) according to the present invention, each set of MHC having a different peptide that plays a crucial role in T cell recognition and a unique "barcode" region in the DNA molecule. In this context, it should be understood that each specific multimeric major histocompatibility complex is present in a certain number in the composition, and that different subsets of multimeric major histocompatibility complexes are present in the composition.

[0035] Preferably, each region specific to each multimeric MHC can be determined with only a small number of primer sets, preferably only one primer set. Thus, in one embodiment, the primer regions in the DNA molecule are identical for each set of MHCs. In this way, only one primer set is required. In another embodiment, multimeric MHCs are grouped with different primer sets, allowing for the proliferation of different sets of multimeric MHCs. In this way, background noise is limited while information on specific binding can be recovered. Therefore, different primer sets can be used for different sets of MHCs.

[0036] The number of individual multimeric MHC sets can vary. Thus, in one embodiment, the composition comprises at least 10 different multimeric MHC sets, such as at least 100, such as at least 500, at least 1000, at least 5000, such as in the range of 10-50000, such as 10-1000 or such as 50-500 MHC sets.

[0037] kit of parts The compositions of the invention may form part of a kit. Thus, a further aspect of the invention is a composition according to the present invention; one or more primer sets for amplifying the DNA molecule; Kit of parts, including:

[0038] Method for detecting antigen-responsive cells in a sample Figure 3 illustrates how this library can be used to stain antigen-responsive cells in a single sample. In Figure 3A, cells in a single-cell suspension (which can be derived from bodily fluids, including, but not limited to, peripheral blood and tissue biopsies) are mixed with the peptide library shown in Figure 2B. In Figure 3B, after staining, the cells are sequentially washed and centrifuged to remove any remaining MHC multimers not bound to the cell surface. Specific cell populations, such as T cells (CD8- or CD4-restricted), other immune cells, or MHC multimer-specific T cells, can be isolated using cell sorting / selection techniques, including flow cytometry. In Figure 3C, DNA barcode oligonucleotide sequences isolated from the cell population are amplified by PCR. In Figure 2D, the amplified products are sequenced by deep sequencing (which can generate 10 to 100,000 reads). After sorting, DNA barcodes bound to cells in a sample appear more frequently than sequences due to nonspecific binding of MHC multimers, revealing their specific barcode sequences through sequencing. Random binding of any nonspecific MHC multimer to 1 / 1000 different barcodes (depending on library size) cancels out the "signal-to-noise" ratio, making the method even more sensitive than standard multimer staining.

[0039] Analysis of barcode sequence data allows for the determination of antigen specificity of cells in a specimen. If DNA barcode #1 is detected above background read levels, it indicates that peptide-MHC multimer #1 preferentially bound to the sorted cell type. The same is true for barcodes #2, 3, 4, 5, and so on, up to over 1000 possible combinations (not limited to this specific number). If the number of input cells is known, for example, if the cell population of interest is captured via fluorescent signals associated with the same multimers using a capture / sorting method such as flow cytometry-based sorting, the frequency of barcode reads can be compared to the number of sorted T cells to calculate the frequency of specific T cells.

[0040] Thus, the multimeric MHC and / or compositions according to the invention may be used for a variety of purposes. Accordingly, a further aspect of the invention is a method for detecting antigen-responsive cells in a sample, comprising the steps of: Providing one or more multimeric major histocompatibility complex (MHC) molecules or compositions according to the present invention; contacting the multimeric MHC with the sample; detecting binding of the multimeric MHC to the antigen-responsive cells to detect cells responsive to antigens present in the MHC set; wherein said binding is detected by amplifying a barcode region of said nucleic acid molecule linked (via its backbone) to one or more MHCs.

[0041] In one embodiment, the method comprises providing a (biological) sample.

[0042] As known to those skilled in the art, it is preferable to remove unbound molecules. Thus, in one embodiment, unbound (multimeric) MHC is removed prior to amplification, e.g., after washing and / or centrifugation, e.g., by removing the supernatant.

[0043] The type of sample may also vary. In one embodiment, the sample is a biological sample. In one embodiment, the sample is a blood sample, such as a peripheral blood sample, a blood-derived sample, a tissue biopsy, or another body fluid, such as cerebrospinal fluid or saliva. The source of the sample may also vary. Thus, in a further embodiment, the sample is obtained from a mammal, such as a human, a mouse, a pig, and / or a horse.

[0044] Additionally, it may be advantageous to be able to sort the cells. Thus, in one embodiment, the method further comprises cell sorting by flow cytometry, e.g., FACS. This may be performed, for example, when the backbone is equipped with a fluorescent marker. Thus, unbound cells may also be removed / sorted.

[0045] As known to those skilled in the art, it is preferable to compare measured value with reference level.Therefore, in one embodiment, detecting said binding comprises comparing measured value with reference level, for example, negative control and / or total response level of sample.In a further embodiment, said amplification is PCR, such as QPCR.

[0046] As mentioned above, detecting the barcode comprises sequencing the amplified barcode region. Thus, in one embodiment, detecting the barcode region comprises sequencing said barcode region, such as by deep sequencing or next generation sequencing.

[0047] Use of multimeric major histocompatibility complex Figure 4 illustrates how this technology can be used to correlate various properties with the antigen specificity of a cell population. Figure 4A shows how cells can be bound to a barcoded MHC multimer library and then exposed to a particular stimulus. Cell populations can be sorted based on their functional response to this stimulus (e.g., a number of measures, including but not limited to, cytokine secretion, phosphorylation, and calcium release). After sorting into responsive or non-responsive populations (following the steps in Figure 2), the DNA barcodes can be sequenced to decipher antigen responsiveness and determine the antigen specificity involved in a given response.

[0048] Figure 4B illustrates how cells can be sorted based on phenotype and correlated with antigen responsiveness for a particular set of phenotypic characteristics.

[0049] Figure 4C shows that single-cell sorting of MHC multimer-binding cells is possible based on the fluorescent labels simultaneously attached to the MHC multimers. Single-cell sorting allows the antigen specificity of a given cell to be determined at the single-cell level by sequencing the associated barcode label. This can be linked to the TCR, which can also be sequenced at the single-cell level, as recently described (10). Thus, the present invention provides a link between single-cell properties, including TCR sequence, and antigen specificity, potentially enabling the use of barcode-labeled MHC multimer libraries to reveal antigen-specific TCRs in a mixture of thousands of different specificities.

[0050] Figure 4D shows the use of a barcode-labeled MHC multimer library to quantitatively assess binding of MHC multimers to a given T cell clone or TCR-transduced / transfected cells. Because sequencing of barcode labels allows for simultaneous determination of multiple different labels on the same cell population, this strategy can be used to determine the affinity of a given TCR for a library of related peptide-MHC multimers. For each different peptide-MHC multimer in the library, the relative contribution of different DNA barcode sequences in the final readout is determined based on the quantitative contribution of TCR binding. Incremental analysis can reveal the quantitative binding characteristics of TCRs for large libraries of peptide-MHC multimers, all combined into a single sample. For this specific purpose, the MHC multimer library can specifically contain related peptide sequences or alanine-substituted peptide libraries.

[0051] Figure 5 shows experimental data on the feasibility of combining DNA barcodes with MHC multimers and amplifying specific sequences after T cell staining. Figure 5A shows staining of cytomegalovirus (CMV)-specific T cells in peripheral blood samples. A specific CMV-derived peptide-MHC multimer was labeled with a barcode (barcode number 1) and mixed with an irrelevant / nonspecific peptide-MHC multimer labeled with a barcode (barcode number 2), and then mixed with 998 other nonspecific MHC multimers without barcode labeling. This data demonstrates the feasibility of staining CMV-specific T cells in a mixture of 1,000 other MHC multimers. Data from three different staining protocols are shown. Figure 5B shows the readout of specific barcode sequences by quantitative PCR. Barcode number 1 (B number 1), which identifies CMV-specific T cells, was detected in all three staining protocols, while barcode number 2 (B number 2), an irrelevant / nonspecific barcode signal, was undetectable.

[0052] Overall, there may be various uses for multimeric MHC or compositions comprising such MHC sets. Thus, one aspect relates to the use of multimeric major histocompatibility complex (MHC) or compositions according to the present invention for detecting antigen-responsive cells in a sample.

[0053] Another aspect relates to the use of a multimeric major histocompatibility complex (MHC) or composition according to the invention for the diagnosis of a disease or condition, preferably cancer and / or infectious disease.

[0054] A further aspect relates to the use of a multimeric major histocompatibility complex (MHC) or composition according to the invention in the development of immunotherapeutic agents.

[0055] A further aspect relates to the use of a multimeric major histocompatibility complex (MHC) or composition according to the invention in vaccine development.

[0056] Another aspect relates to the use of a multimeric major histocompatibility complex (MHC) or composition according to the invention for epitope identification.

[0057] In summary, advantages of the present invention include the ability to detect a large number (potentially, but not limited to, more than 1000) of different antigen-responsive cells in a single sample. This technology can be used for, but not limited to, T cell epitope mapping, immune recognition discovery, diagnostic testing, and measuring immune reactivity after vaccination or immune-related therapy.

[0058] This level of complexity allows us to move from model antigens to determining epitope-specific immune reactivity across whole organisms, viral genomes, cancer genomes, entire vaccine components, etc. They can be personalized and modified according to individual MHC expression and can be used for immune-related diseases such as diabetes and rheumatoid arthritis.

[0059] For example, biological materials are analyzed to monitor natural immune responses, such as potential immune responses to infectious diseases or cancer. Additionally, biological materials are analyzed for the effects of immunotherapeutic agents, including vaccines, on the immune response. As used herein, immunotherapeutic agents are defined as active ingredients used in medical interventions aimed at enhancing, suppressing, or modifying the immune response, including vaccines, nonspecific immunostimulatory agents, immunosuppressants, cell-based immunotherapeutic agents, and combinations thereof.

[0060] The present invention can be used, but is not limited to, in the development of diagnostic kits that can reveal the fingerprint of immune responses associated with a given disease in any biological specimen. Such diagnostic kits can be used to determine exposure to bacterial or viral infections or autoimmune diseases, for example, but not limited to, those associated with tuberculosis, influenza, and diabetes. Similar methods can be used for immunotherapeutics, where immune responsiveness can serve as a biomarker for treatment response. Analysis of barcode-labeled MHC multimer libraries allows for high-throughput evaluation of a large number of antigen-responsive cells in a single sample.

[0061] Furthermore, barcoded MHC-multimers can be used in combination with single-cell sorting and TCR sequencing, where TCR specificity can be determined by simultaneously attached barcodes. This allows for the simultaneous identification of TCR specificities for potentially over 1000 different antigen-responsive T cells from the same sample and allows matching of TCR sequences with antigen specificity. A potential future for this technology is its ability to predict antigen responsiveness based on TCR sequence. This is of particular interest, as changes in TCR usage have previously been linked to immunotherapy (11, 12).

[0062] Furthermore, there is a growing need to identify TCRs involved in target cell recognition (e.g., but not limited to, in the context of cancer recognition). The successful use of TCRs in cancer therapy (13) is likely to lead to further expansion of clinical initiatives along these lines. The complexity of barcoded MHC multimer libraries will likely allow for the individual selection of relevant TCRs for a given individual.

[0063] By providing a barcode sequence readout, barcode-labeled MHC multimer technology allows for the interaction of multiple different peptide-MHC complexes on the surface of a single cell while still maintaining a valid readout. When a single T cell binds to many different peptide-MHC complexes in a library, the number of reads for a given sequence can determine its relative contribution to T cell binding. This feature allows for the precise specificity / consensus sequence of a TCR to be determined. Each TCR may be capable of recognizing many different peptide-MHC complexes, each with different affinities (14). The importance of such quantitative assessment increases with the clinical use of TCRs, and a lack of knowledge can have fatal consequences, as seen in recent clinical trials in which two patients suffered fatal heart failure due to cross-recognition of sequence-related peptides (15, 16). Therefore, this specific feature of quantitative assessment of TCR binding to peptide-MHC molecules associated with the present invention may provide an effective solution for preclinical testing of TCRs aimed at clinical use.

[0064] Related to the above, it also allows for the determination of antigen responsiveness to overlapping libraries or highly similar peptides, something that is not possible with current multiplexing technologies such as combinatorial coding principles, allowing for the mapping of immune reactivity to variants of viruses such as, but not limited to, HIV. In a general embodiment, the invention is the use of barcoded MHC multimers for the high-throughput assessment of large numbers of antigen-responsive cells in a single sample, linking antigen responsiveness to functional and phenotypic characteristics, TCR specificity and determining quantitative binding of large peptide-MHC libraries to a given TCR.

[0065] The above description of the invention will enable one skilled in the art to make and use what is presently believed to be the best mode thereof, but will understand and appreciate that there are variations, combinations, and equivalents of the specific embodiments, methods, and examples herein. Accordingly, the present invention should not be limited by the above-described embodiments, methods, and examples, but by all embodiments and methods that fall within the scope and spirit of the invention.

[0066] Other items of this invention: Item 1: Use of barcode-labeled MHC multimers for the multiplexed detection of different T cell specificities in a single sample, potentially allowing for the simultaneous detection of over 1000 different T cell specificities, with the specificities revealed by sequencing of the barcode labels.

[0067] Item 2: The use of barcoded MHC multimers in combination with single cell sorting and TCR sequencing, whereby TCR specificity can be determined by simultaneously attached barcodes, allowing for the identification of TCRs specific to a mixture of many (potentially, but not limited to, more than 1000) different peptide-MHC multimers, and the matching of TCR sequences with antigen specificity.

[0068] Item 3: Use of barcoded MHC multimers to determine the affinity and binding motif of a given TCR. A barcode labeling strategy allows multiple different (sequence-related) peptide-MHC multimers to bind to a given T cell with binding affinities that dictate the relative contribution of each peptide-MHC multimer. This allows mapping the precise specificity / consensus recognition sequence of a given TCR through the use of overlapping peptide libraries or, for example, alanine substitution libraries.

[0069] Item 4: Use of barcoded MHC multimers to map antigen responsiveness to sequence-related / similar peptides within the same library, e.g., mutational changes in HIV infection, which has not been possible with previous MHC multimer-based technologies.

[0070] Item 5: Use of barcoded MHC multimers to link any functional characteristic of specific T cells or pools of specific T cells to antigen (peptide-MHC) recognition, for example, to determine which T cell specificities within a larger pool secrete cytokines, release calcium, or other functional measures after a specific stimulus.

[0071] It should be noted that embodiments and features described in connection with one aspect of the invention also apply to other aspects of the invention. All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

[0072] The invention will now be described in further detail in the following non-limiting examples and sections.

[0073] item 1. A multimeric major histocompatibility complex (MHC), two or more MHCs linked by a main chain molecule; At least one nucleic acid molecule linked to the backbone; Including, the nucleic acid molecule comprises a central stretch of nucleic acid (barcode region) designed to be amplified, for example by PCR, Multimeric major histocompatibility complex (MHC).

[0074] 2. The multimeric major histocompatibility complex according to item 1, wherein the backbone molecule is selected from the group consisting of a polysaccharide, e.g. a glucan such as dextran, streptavidin or a streptavidin multimer.

[0075] 3. A multimeric major histocompatibility complex according to item 1 or 2, wherein the MHC is linked to the backbone via a streptavidin-biotin bond, streptavidin-avidin.

[0076] 4. The multimeric major histocompatibility complex according to any one of items 1 to 3, wherein the MHC is linked to the main chain via an MHC heavy chain.

[0077] 5. The multimeric major histocompatibility complex (MHC) according to any one of items 1 to 4, wherein the MHC is artificially assembled.

[0078] 6. The multimeric major histocompatibility complex (MHC) according to any of items 1 to 5, which consists of at least four MHCs, such as at least 8, for example at least 10, 2 to 30, 2 to 20, for example 2 to 10 or such as 4 to 10 MHCs.

[0079] 7. The multimeric major histocompatibility complex (MHC) according to any one of items 1 to 6, wherein at least one nucleic acid molecule consists of at least a 5' first primer region, a central region (barcode region), and a 3' second primer region.

[0080] 8. The multimeric major histocompatibility complex (MHC) according to any of items 1 to 7, wherein at least one nucleic acid molecule has a length in the range of 20 to 100 nucleotides, such as 30 to 100 nucleotides, such as 30 to 80 nucleotides, such as 30 to 50 nucleotides.

[0081] 9. The multimeric major histocompatibility complex (MHC) according to any of items 1 to 8, wherein at least one nucleic acid molecule is linked to the backbone via a streptavidin-biotin bond and / or a streptavidin-avidin bond.

[0082] 10. The multimeric major histocompatibility complex (MHC) according to any of items 1 to 9, wherein at least one nucleic acid molecule comprises or consists of DNA, RNA and / or artificial nucleotides, such as PLA or LNA.

[0083] 11. The multimeric major histocompatibility complex (MHC) according to any of items 1 to 10, wherein the MHC is selected from the group consisting of class I MHC, class II MHC, CD1 or an MHC-like molecule.

[0084] 12. The multimeric major histocompatibility complex (MHC) according to any of items 1 to 11, wherein the backbone further comprises one or more linked fluorescent labels.

[0085] 13. A composition comprising a subset of multimeric major histocompatibility complexes (MHCs) according to any one of items 1 to 12, wherein each set of MHCs has a different peptide that plays a crucial role in T cell recognition and a unique "barcode" region in the DNA molecule.

[0086] 14. The composition according to item 13, wherein the primer regions in the DNA molecule are identical for each set of MHC.

[0087] 15. The composition according to item 13 or 14, comprising at least 10 different MHC sets, such as at least 100, for example at least 500, at least 1000, at least 5000, such as in the range of 10-50000, for example 10-1000 or such as 50-500 MHC sets.

[0088] 16. The composition according to any one of items 13 to 15, one or more primer sets for amplifying a nucleic acid molecule; Kit of parts, including:

[0089] 17. A method for detecting antigen-responsive cells in a sample, comprising: Providing one or more multimeric major histocompatibility complex (MHC) molecules according to any one of items 1 to 12 or a composition according to any one of items 13 to 15; contacting the multimeric MHC with the sample; detecting binding of the multimeric MHC to the antigen-responsive cells to detect cells responsive to antigens present in the MHC set; Including, wherein the binding is detected by amplifying a barcode region of the nucleic acid molecule linked to one or more MHC. method.

[0090] 18. The method according to item 17, wherein unbound MHC is removed prior to amplification, for example by washing and / or centrifugal separation.

[0091] 19. The method according to item 17 or 18, wherein the sample is a blood sample, such as a peripheral blood sample, a blood-derived sample, a tissue biopsy or another body fluid, such as cerebrospinal fluid or saliva.

[0092] 20. The method according to any one of items 17 to 19, wherein the sample is collected from a mammal, such as a human, mouse, pig and / or horse.

[0093] 21. The method according to any of items 17 to 20, further comprising cell sorting by flow cytometry, e.g., FACS.

[0094] 22. The method according to any of items 17 to 21, wherein detecting binding comprises comparing the measured value with a reference level, such as a negative control and / or a total response level.

[0095] 23. The method according to any one of items 17 to 22, wherein the amplification is PCR, such as QPCR.

[0096] 24. The method according to any one of items 17 to 13, wherein detecting the barcode region comprises sequencing the region, such as deep sequencing or next-generation sequencing.

[0097] 25. Use of a multimeric major histocompatibility complex (MHC) according to any of items 1 to 12 or a composition according to any of items 13 to 16 for detecting antigen-responsive cells in a sample.

[0098] 26. Use of a multimeric major histocompatibility complex (MHC) according to any of items 1 to 12 or a composition according to any of items 13 to 16 for the diagnosis of a disease or pathology, preferably cancer and / or an infectious disease.

[0099] 27. Use of a multimeric major histocompatibility complex (MHC) according to any one of items 1 to 12 or a composition according to any one of items 13 to 16 in the development of an immunotherapeutic agent.

[0100] 28. Use of a multimeric major histocompatibility complex (MHC) according to any of items 1 to 12 or a composition according to any of items 13 to 16 in vaccine development.

[0101] 29. Use of a multimeric major histocompatibility complex (MHC) according to any of items 1 to 12 or a composition according to any of items 13 to 16 for epitope identification.

[0102] Example Example 1 Results that serve as proof of principle for the claimed invention are shown in Figure 5. Figure 5A shows flow cytometry data from peripheral blood mononuclear cells (PBMCs) from a healthy donor.

[0103] material and method PBMCs were stained with CMV-specific peptide-MHC multimers conjugated with specific nucleotide-barcodes. In addition to the CMV peptide-MHC reagent, negative control reagents were also used: an HIV-peptide-MHC multimer conjugated with another specific barcode label and an additional negative control peptide-MHC reagent without a barcode (p * ) (all multimers were additionally labeled with a PE fluorescent label). To obtain an indication of whether background staining interferes with true-positive signals, the amount of MHC multimers used to stain PBMCs was varied to represent 1000 different peptide-MHC specificities: 1× oligo-labeled CMV-specific MHC multimer, 1× oligo-labeled HIV-specific MHC multimer, and 998× unlabeled peptide. *The volume was the same as that required for staining MHC multimers. After removing the MHC multimers, an additional wash step was performed (either 0 min (A), 30 min (B), or 60 min (C)), and data from all experiments are shown. PE-MHC-multimer-positive cells were sorted by fluorescence-activated cell sorting (FACS).

[0104] Figure 5B shows the crossover threshold (Ct) obtained from multiplex qPCR of sorted PE-MHC-multimer-positive cells. We used qPCR to evaluate whether it was feasible to detect specific cell specificities using barcode-labeled peptide-MHC-multimers. During staining, the reagents bound to the positive control (CMV) barcode and the negative control (HIV) barcode were present, but the negative control (HIV) barcode-peptide-MHC multimers should be washed away.

[0105] Examples of nucleic acid sequences are as follows: DNA barcode oligos for CMV MHC multimer binding: 5GAGATACGTTGACCTCGTTGAANNNNNNTCTATCCATTCCATCCAGCTCACTTAAGCTCTTGGTTGCAT DNA barcode oligos for HIV MHC multimer binding: 5GAGATACGTTGACCTCGTTGAANNNNNNTCTATAGGTGTCTACTACCTCACTTAAGCTCTTGGTTGCAT 5=biotin-TEG.

[0106] result The results showed detectable Ct values ​​only for barcodes bound to CMV peptide-MHC multimers, but not for barcodes bound to HIV-peptide-MHC multimers.

[0107] conclusion This experiment is representative of several similar experiments performed with other antigen specificities. Collectively, these data show that it is possible to: 1) staining with 1000 different MHC-multimers in a single sample while still maintaining a specific signal; 2) binding of DNA barcodes to MHC multimers; 3) amplifying the DNA barcodes after the cell sorting step; 4) Reading the barcode using barcode-specific probes in QPCR; 5) Obtaining a specific signal corresponding to the antigen-specific T cell population present in the sample while not detecting the barcodes of non-specific MHC multimers.

[0108] Taken together, these data (and similar data available) demonstrate the feasibility of the steps described in Figures 1, 2 and 3.

[0109] Example 2 This embodiment relates to: i) The stability of the DNA oligonucleotides used in one embodiment of the present invention in blood samples, and ii) An embodiment of the invention that increases the number of specific tagged dextramers (detection molecules in which the binding molecule is a peptide-MHC complex and the label is a DNA oligonucleotide), allowing the identification of dextramers with binding specificity for specific cells (subpopulations of cells) in a test cell sample.

[0110] In i), it is shown that the DNA oligos are stable to manipulation in PBMCs and blood for the time it takes to amplify the DNA tag after staining, washing and isolating the T cells.

[0111] ii) We demonstrate that a model system consisting of DNA-tagged dextramers with MHC specificity for CMV, influenza and negative control peptides localizes to relevant T cell specificities, which can then be captured / sorted and identified by PCR amplification and / or sequencing.

[0112] A. Stability of Single- and Double-Stranded Oligonucleotides in Blood Specimens DNA tag oligo design A 69-nucleotide biotinylated test oligo was prepared, consisting of a 5' primer region (22 nt, yellow) - a random barcode region (6 × N-nt) - a codon region (21 nt, green / underlined) - a 3' primer region (20 nt, blue): [ka] [ka] TIFF2025128229000003.tif72161

[0113] The stability of the oligo tag was analyzed by Q-PCR under conditions suitable for T cell isolation:

[0114] Test oligos 1-6 were incubated in anticoagulated EDTA blood. After incubation, the amount of each test oligo was determined by Q-PCR using the primers and probes listed above. Oligo tags were quantified by Q-PCR using SYBR® Green JumpStart™ Taq ReadyMix™ in combination with any capillary QPCR instrument (e.g., Roche LightCycler or Agilent Mx3005P) according to the manufacturer's protocol.

[0115] Because test oligos 1-6 have different ends, this also served to test the stability of unmodified DNA oligo tags compared to HEG-modified 5' and HEG-modified 5' and 3' (test oligos -01, -02, and -03, respectively).

[0116] The results are shown in Figure 6. It can be concluded that the stability of the tested oligos, in all variants, is sufficient to carry out the present invention.

[0117] B. Generation and screening of a three-member DNA-tagged MHC dextramers library for screening antigen-specific T cells in lymphoid cell samples. In this experiment, three DNA-tagged dextramers, each with its own unique specificity, were used: Dextramer 1: Influenza (HLA-A * 0201 / GILGFVFTL / MP / influenza) Dextramer 2: CMV (HLA-A * 0201 / NLVPMVATV / pp65 / CMV) Dextramer 3: Negative (HLA-A * 0201 / ALIAPVHAV / Neg.Control) Prepare the following.

[0118] Thus, each of the above dextramers has a unique pMHC specificity (i.e., the three dextramers have different binding molecules), and each dextramer has a unique label (a DNA oligonucleotide) specific for that one pMHC specificity.

[0119] A library of DNA-tagged dextramers is screened on a lymphoid cell sample, such as anticoagulated EDTA blood, or a peripheral blood mononuclear cell (PBMC) sample. The amount of dextramers that bind to the cells in the cell sample is increased relative to that of those that do not bind to the cells.

[0120] Finally, the MHC / antigen specificity of the increased dextramers is revealed by identifying the DNA tags by Q-PCR using DNA tag-specific probes or by sequencing the DNA tags.

[0121] 1. HLA-A * 0201 - Generation of three different DNA-tagged dextramers with peptide (pMHC) complexes. a. pMHC complexes are generated as follows and conjugated to dextran with unique DNA tags to identify individual pMHC complexes. i. Influenza (HLA-A * 0201 / GILGFVFTL / MP / lnfluenza), CMV(HLA-A *0201 / NLVPMVATV / pp65 / CMV) and negative (HLA * Preparation of DNA-tagged dextramers with 0201 / ALIAPVHAV / Neg.Control). 1. Prepare a 160 nanomolar (nM) dextramer stock and dilute the test oligo stock to 500 nM. Mix 10 microliters (μL) of the 160 nM dextramer stock with 10 μL of the 500 nM test oligo stock. Incubate at room temperature for 10 minutes. Mix with 1.5 μg of pMHC complex of desired specificity. Adjust the volume to 50 μL with a neutral pH buffer such as PBS or Tris (pH 7.4) and store at 4°C. This will yield DNA-tagged dextramers with approximately 3 oligo tags and 12 pMHC complexes per dextramer. a.Dex-Oligo-03 = Test Oligo-03 and HLA-A * Dextramers with 0201 / NLVPMVATV / pp65 / CMV. b.Dex-Oligo-04 = Test Oligo-04 and HLA-A * 0201 / GILGFVFTL / MP / Influenza and dextramer. c.Dex-Oligo-05 = Test Oligo-05 and HLA-A * Dextramers with 0201 / ALIAPVHAV / Neg.Control.

[0122] 2. Preparation of cell samples for antigen-specific T cell screening. Suitable cell samples for identifying antigen-specific T cells include lymphoid cell preparations, such as peripheral blood mononuclear cells (PBMCs) or anticoagulated blood preparations. Such cell sample preparations are prepared by standard techniques known to those skilled in the art. b. Place a range of 1E7 lymphoid cells (from PBMCs or EDTA-anticoagulated blood) into a 12 x 75 mm polystyrene test tube. c. Add 2 ml of PBS (pH 7.4) containing 5% fetal bovine serum. Centrifuge at 300 × g for 5 minutes. Remove the supernatant and resuspend the cells in a total volume of 2.5 ml of PBS (pH 7.4) containing 5% fetal bovine serum.

[0123] 3. Preparation and modification of a library of DNA-tagged dextramers (obtained in 1) with three MHC / peptide specificities. a. Mix 5 μl of 10 μM biotin with 10 μl each of Dex-Oligo-03, Dex-Oligo-04, and Dex-Oligo-05.

[0124] 4. A lymphoid cell sample was mixed with a library of DNA-tagged MHC dextramers. a. Mix 2.5 ml of 1E7 lymphoid cells (obtained in 2b) with 30 μL of the DNA-tagged dextramer library (obtained in 3a). b. Incubate at room temperature for 30 minutes. c. Centrifuge at 300 x g for 5 minutes and remove the supernatant. d. Resuspend the pellet in 2.5 ml of PBS (pH 7.4) containing 5% fetal bovine serum. Centrifuge at 300 x g for 5 minutes and remove the supernatant. e. Resuspend the pellet in 2.5 ml of PBS (pH 7.4) containing 5% fetal bovine serum.

[0125] 5. Capture of total CD8+ antigen-specific T cells by magnetic cell sorting according to Miltenyi Biotec's Whole Blood CD8 MicroBead protocol, catalog number 130-090.878. a. Add 100 μL of Whole Blood CD8 MicroBeads (Miltenyi Biotec, Catalog No. 130-090.878) to the lymphoid cells resuspended in 4e. Stir for 15 minutes at room temperature to capture CD8+ T cells. b. Place the Whole Blood Column in the magnetic field of the appropriate MACS Separator. See the Whole Blood Column Kit datasheet for details. c. Prepare the column by rinsing with 3 ml of separation buffer (autoMACS Running Buffer or PBS (pH 7.4) containing 5% fetal bovine serum). d. Add the magnetically labeled cell suspension (4e) to the prepared Whole Blood Column. Collect the flow-through containing unlabeled cells. e. Wash the Whole Blood Column with 3 x 3 ml of separation buffer (autoMACS Running Buffer or PBS (pH 7.4) containing 5% fetal bovine serum). f. Remove the Whole Blood Column from the separator and place a new collection tube in its place. g. Capture CD8+ T cells by pipetting 5 mL of Whole Blood Column Elution Buffer or 5% fetal bovine serum in PBS (pH 7.4) onto the Whole Blood Column. Immediately flush out the magnetically labeled cells by firmly depressing the plunger into the column. h. Centrifuge at 300 x g for 5 minutes and remove the supernatant. Resuspend the collected CD8+ cells in 50 μL and store at -20°C until further analysis.

[0126] 6. Identification of dextramers that significantly bound to antigen-specific T cells in lymphoid cell samples. a. The relative abundance of antigen-specific T cells in lymphoid cell samples is revealed by quantifying the ratio of DNA oligo tags in the input (3a) and captured fractions (5h) by sequencing or QPCR using DNA tag-specific probes LNA-3, LNA-4, and LNA-5. i. Quantification of the ratio of DNA oligo tags in the input (3a) and captured fractions (5h) by QPCR using DNA tag-specific probes LNA-3, LNA-4 and LNA-5. 1.a. DNA-tagged Dextramers Library Input (3a) b. DNA-tagged dextramer library output (5h) c. Standard curves of Test Oligo-03, Test Oligo-04, and Test Oligo-05 from 10 to 1E8, respectively. Make 25 µL of qPCR reaction. 2. Mix 12.5 μl of JumpStart Taq ReadyMix (Sigma-Aldrich, product code D7440) with 0.125 μl of 100 μM primers each of forward-01 and reverse-01, 0.625 μl of 10 μM probes LNA-3, LNA-4, or LNA-5, 0.025 μl of reference dye (Sigma-Aldrich, product code R4526), ​​and 12.5 μl of either the DNA-tagged dextramer library input (3a), the DNA-tagged dextramer library output (5h), or 10-1E8 standard curves of test oligo-03, test oligo-04, and test oligo-05, respectively. 3. Perform a two-step QPCR temperature profile: cycle 1 = 95 °C for 5 min, cycles 2-40 = 95 °C for 30 s and 60 °C for 1 min. 4. Estimate the relative abundance of T cells with antigen specificity for one of the three MHC dextramers by plotting the QPCR cycle time (Ct) values ​​of the DNA-tagged dextramer library input (3a) and DNA-tagged dextramer library output (5h) against a plot of the QPCR standard curves for Test Oligo-03, Test Oligo-04, and Test Oligo-05, respectively. ii. Quantification of the ratio of DNA oligo tags in the input (3a) and captured fractions (5h) by ultra-deep sequencing. 1.a. DNA-tagged Dextramers Library Input (3a) b. DNA-tagged dextramer library output (5h) Prepare 25 μL of PCR reaction. 2. Using any standard PCR master mix, combine the PCR reaction with 1.25 μL of 10 μM each of forward-01 and reverse-01 primers and 12.5 μL of either the DNA-tagged dextramer library input (3a) or the DNA-tagged dextramer library output (5h). Fill to 25 μL with purified water. For example, use Promega's 2x PCR Master Mix, which contains Taq DNA polymerase, dNTPs, MgCl2, and reaction buffer. 3. Ultra-deep sequencing of the above PCR products can be provided by a number of suppliers, such as Eurofins Genomics, GATC Biotech or Beckman Coulter Genomics, using well-established next generation sequencing technologies, such as high-throughput sequencing technologies for PCR amplicon sequencing, including Roche 454, Ion Torrent, Illumina technologies, etc. 4. PCR amplicon analysis of the relative abundance of DNA-tagged dextramers library input (3a) and DNA-tagged dextramers library output (5h) reveals the relative abundance of T cells with antigen specificity for one of the three MHC dextramers.

[0127] 7. Expected results and comments a. The relative abundance and ratio of DNA oligo tags in the library input of DNA-tagged dextramers (3a), as estimated by QPCR or sequencing, is expected to be influenced primarily by three parameters: i) the ratio in which the DNA oligo tags were provided during the production of the DNA-tagged dextramers (1.ai1), ii) the method of mixing the library input (3a), and iii) the efficiency with which individual DNA oligo tags are amplified in the PCR reaction. i. In one example, the relative ratios of DNA oligo tags in the input of the library of DNA-tagged dextramers generated in 3a and measured by QPCR or sequencing may be 1-10 fold greater than each other. b. The relative abundance and ratio of DNA oligo tags in the output of a library of DNA-tagged dextramers (5h), as estimated by QPCR or sequencing, is expected to be influenced by three other parameters in addition to the three listed in 7a, namely, i) the number of antigen-specific T cells with specificity for one of the three MHC-peptide combinations, ii) the affinity of the T cell receptor of a given T cell for a given MHC-peptide complex, and finally, iii) the efficiency of separating the antigen-specific T cells and their bound DNA-tagged MHC-dextramers from unbound DNA-tagged MHC-dextramers by washing and cell capture. i. In one example, the relative ratio of DNA oligo tags in the output of a library of DNA-tagged dextramers generated in 5 hours and measured by QPCR or sequencing will be greater than 10-fold higher in DNA oligo tags bound to MHC dextramers bearing MHC-peptide complexes in which antigen-specific T cells are present in a lymphoid cell sample. 1. HLA-A * 0201 / NLVPMVATV / pp65 / CMV and HLA-A * 0201 / GILGFVFTL / MP / lnfluenza antigen-specific T cells, and HLA-A * In lymphoid cell samples from influenza-positive, CMV-positive HLA-A0201 donors who did not have antigen-specific T cells against O201 / ALIAPVHAV / Neg.Control, test oligo-03 (Dex-Oligo-03 = test oligo-03 and HLA-A * Dextramer containing 0201 / NLVPMVATV / pp65 / CMV), test oligo-04 (Dex-Oligo-04 = test oligo-04 and HLA-A * Dextramer containing HLA-A1 / GILGFVFTL / MP / Influenza) and test oligo-05 (Dex-Oligo-05 = test oligo-05 and HLA-A *The relative ratio of test oligo-03 and test oligo-04 (dextramers with 0201 / ALIAPVHAV / Neg.Control) is expected to be greater than 10-fold greater than test oligo-05. That is, when provided in equal amounts in the DNA-tagged dextramers library input (3a), test oligo-03 and test oligo-04 are expected to be found in greater than 10-fold greater abundance or frequency than test oligo-05 when measured by sequencing or QPCR in the DNA-tagged dextramers library output (5h).

[0128] Example 3 In this example, peripheral blood mononuclear cells (PBMCs) were obtained by converting blood from a CMV-positive, HIV-negative donor, and the backbone was a dextran conjugate (Immudex's Dextramer backbone) with streptavidin and a fluorescent dye.

[0129] The MHC molecules were peptide-MHC (pMHC) complexes presenting either CMV-derived (positive antigen) or HIV-derived (negative antigen) peptide antigens. The MHC molecules were modified by biotinylation to generate a biotin capture tag on the MHC molecule. The MHC molecules were purified by HPLC and quality controlled for the formation of functional pMHC multimers that stained control T cell populations.

[0130] The oligonucleotide labels were synthesized by DNA Technology A / S (Denmark). The labels were synthetically modified with a terminal biotin capture tag. The labels were combinatorial oligonucleotide labels obtained by annealing oligonucleotide A (modified with biotin) with a partially complementary oligonucleotide label B, followed by enzymatic extension of oligos A and B with DNA polymerase to create a complete double-stranded label. MHC molecules were synthesized by attaching MHC molecules in the form of biotinylated pMHC and labels in the form of biotin-modified oligonucleotides onto a streptavidin-modified dextran backbone. The MHC molecules further contained a modification in the form of a fluorescent dye (5b). Two distinct MHC molecules were generated, each containing a different pMHC and encoded by a corresponding individual oligonucleotide label.

[0131] A fixed amount of sample PBMC (1b) was incubated with a fixed amount of mixed MHC molecules (5) under conditions that allow binding of the MHC molecules to T cells in the sample (6c).

[0132] After several rounds of washing of the PBMCs by initial cell sedimentation by centrifugation and resuspension in wash buffer, cell-bound MHC molecules were separated from non-cell-bound MHC molecules by fluorescence-activated cell sorting (FACS) of fluorescent dye-labeled cells (7). T cells capable of efficiently binding MHC molecules fluoresce due to the fluorescent dye contained within the MHC molecules, while T cells that cannot bind MHC molecules do not. FACS sorting enriches for fluorescent cells, and therefore for T cell-bound MHC molecules in the PBMC sample.

[0133] FACS-isolated cells were subjected to quantitative PCR analysis of oligonucleotide labels associated with MHC molecules bound to the isolated cells to reveal the identity of the MHC molecules bound to T cells present in the sample.

[0134] Thus, this experiment will reveal whether T cells expressing T cell receptors that recognize and bind to peptide-MHC molecules contained in the peptide-MHC multimer library are present in the blood.

[0135] 1. Sample Preparation. The cell samples used in this experiment were obtained by preparing PBMCs from blood drawn from donors who were CMV-positive and HIV-negative as determined by conventional MHC multimer staining. a. Sample acquisition: Blood was obtained from the Danish Blood Bank. b. Sample Modification: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation. The density gradient medium, Lymphoprep (Axis-Shield), consists of carbohydrate polymers and iodine-rich compounds, promoting the separation of individual blood components. The blood sample was diluted 1:1 with RPMI (RPMI 1640, GlutaMAX, 25 mM Hepes; Gibco Life Technologies) and carefully layered on top of Lymphoprep. After centrifugation at 490 g for 30 minutes, PBMCs and platelets were collected from the interphase of cells. The isolated buffy coat (BC) cells were washed twice with RPMI and frozen at -150°C in fetal calf serum (FCS; Gibco Life Technologies) containing 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich). The BCs used in this example are listed in Table 6 along with their respective virus specificities. These virus specificities were determined using a conventional MHC multimer staining protocol.

[0136] 2. Preparation of the backbone: The backbone is a dextran molecule conjugated with streptavidin and a fluorescent dye. Streptavidin serves as a binding site for biotinylated oligonucleotides and biotinylated pMHC complexes (MHC molecules). The fluorescent dye allows for the separation of cells bound to MHC molecules from cells not bound to MHC molecules. In this example, the backbone was a 1000-2000 KDa linear and branched dextran molecule to which streptavidin (5-10 per backbone) and PE-form fluorescent dye (2-20 per backbone) were covalently attached. The backbone was essentially a dextramer backbone as described by Immudex. In this example, the backbone is also referred to as an SA conjugate.

[0137] 3. Preparation of MHC molecules: The MHC molecules used in this example were two different class I MHC-peptide complexes. MHC heavy chains (HLA-A0201 and HLA-B0702) and B2M were expressed in E. coli as previously described (Hadrup et al., 2009) and refolded with two peptide antigens, respectively. Individual specificities (peptide-MHC molecule, allele, and peptide combinations) were generated as follows: a. Synthesis: The MHC molecule in this example is a UV-conditional 9-residue peptide ligand (p * Specific pMHC monomers were generated by UV exchange of selected HLA-I monomers with the conditional ligand. Exposure of the conditional ligand to UV light (366 nm) results in cleavage, leaving the binding groove empty. The empty MHC-I molecule is unstable, and the complex rapidly disintegrates unless rescued by replacement with another peptide that matches its HLA type. Thus, excess of the desired HLA ligand and pMHC-I monomers can be used to bind MHC-I molecules. * Specific pMHC monomers were generated by mixing with MHC monomers. * MHC monomers were refolded, biotinylated, and purified as previously described ( Hadrup et al., 2009 ). i. HIV-derived peptides ILKEPVHGV derived from the HIV polymerase antigen and TPRVTGGGAM, a CMV-derived peptide derived from the pp65TPR antigen (Pepscan Presto, The Netherlands) were diluted with phosphate-buffered saline (DPBS; Lonza) and mixed to a final concentration of 100 μg / ml: 200 μM (HLA-A02: ILKEPVHGV and HLA-B07: TPRVTGGGAM). The mixture was exposed to 366 nm UV light (UV cabinet; CAMAG) for 1 hour and optionally stored at 4°C for up to 24 hours. b. Modifications: No further modifications were performed. c. Purification: A panel of MHC molecules was transferred to an Eppendorf tube and centrifuged at 5000 g for 5 minutes before being added to the cells to sediment any MHC molecules remaining in solution.

[0138] 4. Label Preparation: In this example, two different oligonucleotides of the same length but with different sequences were prepared. Each oligonucleotide bound to, and thus encoded, a specific pMHC. The oligonucleotides were biotinylated to facilitate binding to a dextran-streptavidin conjugate backbone. a. Synthesis: Labels were DNA oligonucleotides purchased from DNA Technology (Denmark) and delivered as lyophilized powders. A 100 μM label stock dilution was made in nuclease-free water and stored at -20°C. i. The label used, termed the 2OS labeling system, was developed to increase the complexity of a limited number of oligonucleotide sequences through a combinatorial labeling strategy, enabling a larger number of unique labels to be generated from a more limited number of label precursors. In this strategy, termed 2OS, two partially complementary oligonucleotide sequences (A oligo and B oligo) were annealed and subsequently extended to generate new unique oligonucleotide sequences, which were then used as DNA oligonucleotide labels. For example, by combining 22 unique, all partially complementary oligonucleotide sequences (A label precursors) with 55 other unique oligonucleotide sequences (B label precursors), a combinatorial library of 1,210 unique (Ax + By) labels (e.g., 100 of Table 9) was obtained. 1. Partially complementary A and B oligonucleotides were annealed to yield two combined A+B oligonucleotide labels (A1+B1 yielded A1B1, and A2+B2 yielded A2B2). The A and B oligos were mixed as described in Table 3, heated to 65°C for 2 minutes, and gradually cooled to below 35°C over 15–30 minutes. The annealed A and B oligos were then extended as described in Table 3. The components of the extension reaction were mixed immediately before use. After mixing, the reaction was left at room temperature for 5 minutes to allow the annealed oligonucleotides to extend. The reagents used for annealing (left) and extending (right) the partially complementary oligonucleotides are listed in Table 3. Reagents in italics are from the Sequenase Version 2.0 DNA Sequencing Kit (Affymetrix, Inc., #70770). b. Modification: All labels were diluted to working concentration (640 nM) in nuclease-free water containing 0.1% Tween. c. Purification: No further purification of the label was performed.

[0139] 5. Preparation of MHC molecules: MHC molecules were formed by combining MHC molecules (pMHC) and labels (oligonucleotides) with a backbone (dextran-streptavidin-fluorescent dye conjugate) to ensure that a given pMHC bound to a given oligonucleotide. a. Synthesis: For preparation of MHC molecules, the backbone was labeled with a label in the form of a biotinylated AxBx oligo, followed by addition of pMHC. i. MHC molecules were prepared by adding a 2x excess of label over the backbone (label:backbone = 2:1) and incubated at 4°C for at least 30 minutes. After label attachment, the backbone was optionally stored at 4°C for up to 24 hours. Before combining the MHC molecules and pMHC monomers, they were centrifuged at 3300g for 5 minutes. Conjugated streptavidin (SA) and SA conjugates (dextramer backbone, Immudex) with fluorescent dyes (PE) were aliquoted onto a plate according to Table 1. MHC molecules were added to the aliquoted SA conjugates, avoiding precipitation, and incubated at room temperature for 30 minutes. After complex formation, D-biotin (Avidity Bio200) was added with 0.02% NaN2 in PBS to the final concentration of pMHC monomers listed in Table 1, and incubated at 4°C for at least 30 minutes or up to 24 hours. Assembled MHC molecules were stored at 4°C for up to 4 weeks. Two sets of two MHC molecules were generated. Each set with two specificities was individually labeled. The labels were reversed between the two sets as described below. 1.1×CMV-specific pMHC was combined with 2OS-A1B1, and 1×HIV-specific pMHC was combined with 2OS-A2B2. 2.1×CMV-specific pMHC was combined with 2OS-A2B2, and 1×HIV-specific pMHC was combined with 2OS-A1B1. b. Modifications: No further modifications were performed. c. Purification: Before adding the MHC molecules to the sample, they were centrifuged at 3300 g for 5 minutes to sediment any MHC molecules remaining in solution.

[0140] 6. Incubation of sample and MHC molecules: The cell sample and MHC molecules were mixed in one container to allow the MHC molecules to bind to the T cells they recognize. a. Sample amount: 1×10E6 to 2×10E6 cells of BC type were used. b. Amount of MHC molecules: according to Table 1. Per incubation, 1 μg / ml of each MHC molecule (peptide-MHC molecule) was required as calculated. c. Conditions: BCs were thawed in 10 ml of RPMI containing 10% fetal bovine serum (FBS) at 37°C, centrifuged at 490 g for 5 minutes, and washed twice with 10 ml of RPMI containing 10% FBS. All subsequent cell washes refer to centrifugation at 490 g for 5 minutes followed by removal of the supernatant. 2 x 10E6 cells were washed with 200 μl of barcode buffer (PBS / 0.5% BSA / 2 mM EDTA / 100 μg / ml herring DNA) and resuspended in this buffer at approximately 20 μl per staining run. Before incubation with MHC molecules, cells were incubated with 50 nM dasatinib at 37°C for 30 minutes (Lissina et al., 2009). Before adding MHC molecules to cells, the cells were centrifuged at 3300 g for 5 minutes. Five MHC molecules (per pMHC) were required at 1 μg / ml each per incubation. After adding the MHC molecules, the cells were incubated for 15 minutes at 37°C. The antibody mixture described in Table 2 was added along with 0.1 μl of a near-IR viability dye (Invitrogen, L10119) that stains free amines. Antibody staining was essentially the same as conventional MHC multimer staining. The cells were incubated for 30 minutes at 4°C. The cells were then washed twice with 200 μl of barcode buffer and incubated overnight at 4°C in 200 μl of 1% paraformaldehyde in phosphate-buffered saline (DPBS; Lonza).

[0141] 7. Enrichment of MHC molecules with desired characteristics: In this example, MHC molecules were enriched by using flow cytometry, more specifically, fluorescence-activated cell sorting (FACS). MHC molecules contain fluorescent dyes. Therefore, cells that bind to MHC molecules emit fluorescence, and by applying a FACS sorter, they can be separated from cells that do not bind to MHC molecules and therefore do not emit fluorescence. As a result, MHC molecules bound to cells are enriched. a. Application: Cells were sorted using a BD FACSAria equipped with three lasers (488 nm blue, 633 nm red, and 405 violet). Flow cytometry data analysis was performed using BD FACSDiva software version 6.1.2. The following gating strategy was applied: Lymphocytes were identified on an FSC / SSC plot. Additional gating on single cells (FSC-A / FSC-H), live cells (near-IR viability dye negative), and CD4, CD14, CD16, CD19, CD40 negative (FITC) / CD8 positive cells (PerCP) was used to define the CD8 T cell population (Table 2). Cells that bound MHC molecules were defined within the PerCP positive population. b. Washing: After being ready for acquisition by flow cytometry, cells were washed twice with barcode buffer. Optionally, cells were fixed with 1% paraformaldehyde overnight at 4°C and washed twice with barcode buffer. Fixed cells were stored at 4°C for up to 1 week. c. Isolation: Optionally, cells were harvested up to one week after fixation with 1% paraformaldehyde. Multimer-positive cells were sorted by FACS as described in 7a and placed in tubes containing 200 μl of barcode buffer, pre-saturated with 2% BSA solution for 2 hours to overnight to increase the stability of oligonucleotides associated with the sorted cells. Sorted fluorochrome (PE)-positive cells were centrifuged at 5000 g for 5 minutes to remove all excess buffer. Cells were stored at -80°C.

[0142] 8. Identification of enriched MHC molecules: By identifying the label (in this example, an oligonucleotide label), we were able to identify pMHC molecules bound to cells. Therefore, we analyzed the oligonucleotides contained in the MHC molecules recovered with the cells by quantitative PCR using a label-specific Q-PCR probe. This allowed us to identify pMHC molecules bound to cells in the cell sample. a. Labels from the sorted cells were analyzed by QPCR as described in Table 4. QPCR was performed using the following kit: Brilliant II QRT_PCR Low ROX Master Mix Kit (Agilent Technologies, No. 600837). The temperature profile is described in Table 5. PCR was performed using a thermal cycler: Mx3000P qPCR system (Agilent Technologies).

[0143] Results and Conclusions of Example 3 After fractionation and qPCR, the Ct values ​​obtained confirmed that the label was successfully recovered and concentrated only when bound to the CMV epitope, but no label was detected when bound to the HIV epitope (Figure 7).

[0144] Thus, it was confirmed that 2OS label was recovered after cell interaction, sorting, and qPCR only if T cells recognizing a given pMHC molecule were present in the sample.

[0145] Figure 7: Detection of B7 CMV pp65 TPR specificity in negative control barcode-labeled pMHC dextramers. In 1, a unique 2OS barcode was conjugated to the positive control reagent, and in 2, another unique 2OS barcode was conjugated to the positive control reagent. A: Representative dot plot showing the PE-positive population after staining with CMV pMHC multimers and HIV pMHC multimers bearing distinct 2OS barcodes. B: Ct values ​​obtained from multiplex qPCR of sorted PE-pMHC-dextramers positive cells. Cells were stained with 1 and 2, respectively. During staining, the reagent conjugated with the positive control (CMV) 2OS barcode and the reagent conjugated with the negative control (HIV) 2OS barcode were present, but the negative control (HIV) barcode-labeled pMHC dextramers were clearly washed away. Results from two individual experiments are shown as separate bars. Approximately 200 cells were subjected to separate PCRs. QPCR was performed in duplicate and Ct values ​​are shown as the mean ± range of duplicates.

[0146] Example 4 This is an example in which sample (1) was obtained from the blood of a CMV-positive, HIV-negative donor by modifying (1b) peripheral blood mononuclear cells (PBMCs).

[0147] The backbone (2) was a dextran conjugate with streptavidin and a fluorescent dye (dextramer backbone from Immudex).

[0148] This example is essentially the same as Example 1, except that a 1000-fold excess of unlabeled MHC molecules was used with irrelevant MHC molecules. The MHC molecules used (3) were peptide-MHC (pMHC) complexes presenting either CMV-derived (positive antigen) or HIV-derived (negative antigen) peptide antigens, or pMHC complexes presenting irrelevant peptide antigens. The MHC molecules were modified by biotinylation to produce a biotin capture tag on the MHC molecule (3b). The MHC molecules were purified by HPLC (2c). The label (4) was an oligonucleotide. The oligonucleotide was synthesized by DNA Technology A / S (Denmark) (4a). The label was synthetically modified with a terminal biotin capture tag (4b).

[0149] MHC molecules (5) were synthesized by attaching MHC molecules in the form of biotinylated pMHC and labels in the form of biotin-modified oligonucleotides onto a streptavidin-modified dextran backbone (5a). The MHC molecules further contained modifications in the form of fluorescent dyes (5b). Three different MHC molecules were generated, where two of these individual MHC molecules, including pMHC for CMV and pMHC for HIV, were encoded with corresponding individual oligonucleotide labels. The MHC molecule with the unrelated MHC molecule was not encoded with an oligonucleotide label. A fixed amount of sample PBMCs (1b) was incubated with a fixed amount of mixed MHC molecules (5) in a 1:1 ratio under conditions that allowed binding of the MHC molecules to T cells in the sample (6c), and also with 1000x unlabeled pMHC. * An MHC tag backbone was included.

[0150] After initial rounds of centrifugation to sediment the cells and resuspension in wash buffer, PBMCs were washed several times and then subjected to fluorescence-activated cell sorting (FACS) of fluorescently labeled cells to separate cell-bound MHC molecules from non-cell-bound MHC molecules (7). T cells capable of efficiently binding MHC molecules fluoresce due to the fluorescent dye contained within the MHC molecules, whereas T cells unable to bind MHC molecules do not. FACS sorting enriches for fluorescent cells and, therefore, for T cell-bound MHC molecules in the PBMC sample.

[0151] FACS-isolated cells were subjected to quantitative PCR analysis of oligonucleotide labels associated with MHC molecules bound to the isolated cells to reveal the identity of the MHC molecules bound to T cells present in the sample.

[0152] Thus, this experiment demonstrated the peptide-MHC specificity of the T cell receptors of T cells present in blood samples and demonstrated the feasibility of enriching T cells specific for CMV antigens (positive) over T cells specific for HIV antigens (negative) and over T cells specific for an excess of MHC molecules presenting irrelevant peptide antigens.

[0153] 1. Sample Preparation. The cell samples used in this experiment were obtained by preparing PBMCs from blood drawn from donors who were CMV-positive and HIV-negative as determined by conventional MHC multimer staining. a. Sample acquisition: Blood was obtained from the Danish Blood Bank. b. Sample Modification: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation. The density gradient medium, Lymphoprep (Axis-Shield), consists of carbohydrate polymers and iodine-rich compounds, promoting the separation of individual blood components. The blood sample was diluted 1:1 with RPMI (RPMI 1640, GlutaMAX, 25 mM Hepes; Gibco Life Technologies) and carefully layered on top of Lymphoprep. After centrifugation at 490 g for 30 minutes, PBMCs and platelets were collected from the interphase of cells. The isolated buffy coat (BC) cells were washed twice with RPMI and frozen at -150°C in fetal calf serum (FCS; Gibco Life Technologies) containing 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich). The BCs used in this example are listed in Table 6 along with their respective virus specificities. These virus specificities were determined using a conventional MHC multimer staining protocol.

[0154] 2. Backbone Preparation: The backbone used in this example is a dextran molecule conjugated with streptavidin and a fluorescent dye. Streptavidin serves as a binding site for biotinylated oligonucleotides (labels) and biotinylated pMHC complexes (MHC molecules). The fluorescent dye allows for the separation of cells bound to MHC molecules from cells not bound to MHC molecules. In this example, the backbone was a 1000-2000 KDa linear and branched dextran molecule to which streptavidin (5-10 per backbone) and PE-form fluorescent dye (2-20 per backbone) were covalently attached. The backbone was essentially a dextramer backbone as described by Immudex. In this example, the backbone is also referred to as an SA conjugate.

[0155] 3. Preparation of MHC molecules: The MHC molecules used in this example were two different class I MHC-peptide complexes. MHC heavy chains (HLA-A02 and HLA-B07) and B2M were expressed in E. coli as previously described (Hadrup et al., 2009) and refolded with two peptide antigens, respectively. Individual specificities (allele and epitope combinations) were generated as follows: a. Synthesis: A from Experiment 1. i. Same as Experiment 1. b. Modifications: No further modifications were performed. c. Purification: Same as Experiment 1.

[0156] 4. Label Preparation: In this example, two different oligonucleotides of the same length but with different sequences were prepared. Each oligonucleotide binds to, and thus encodes, a specific pMHC. The oligonucleotides were biotinylated to facilitate binding to a dextran-streptavidin conjugate backbone. Synthesis: In this example, the labels were DNA oligonucleotides purchased from DNA Technology (Denmark) and delivered as lyophilized powders. A 100 μM label stock dilution was made in nuclease-free water and stored at -20°C. i. Same as Experiment 1. ii. Partially complementary A and B oligonucleotides were annealed to yield two combined A+B oligonucleotide labels (A1+B1 yielded A1B1, and A2+B2 yielded A2B2). The A and B oligos were mixed as described in Table 3, heated to 65°C for 2 minutes, and gradually cooled to below 35°C over 15–30 minutes. The annealed A and B oligos were then extended as described in Table 3. The components of the extension reaction were mixed immediately before use. After mixing, the reaction was left at room temperature for 5 minutes to allow the annealed oligonucleotides to extend. The reagents used for annealing (left) and extending (right) the partially complementary oligonucleotides are described in Table 3. Reagents in italics are from the Sequenase Version 2.0 DNA Sequencing Kit (Affymetrix, Inc., #70770). b. Modification: All labels were diluted to working concentration (640 nM) in nuclease-free water containing 0.1% Tween. c. Purification: No further purification of the label was performed. 5. Preparation of MHC molecules: MHC molecules were formed by combining MHC molecules (pMHC) and labels (oligonucleotides) with a backbone (dextran-streptavidin-fluorescent dye conjugate) to ensure that a given pMHC bound to a given oligonucleotide. a. Synthesis: For preparation of MHC molecules, the backbone was labeled with a label in the form of a biotinylated AxBx oligo, followed by addition of pMHC. i. MHC molecules were assembled by adding a 2x excess of label over the backbone (label:backbone = 2:1) and incubating at 4°C for 30 minutes. Before combining the MHC molecules and pMHC monomers, they were centrifuged at 3300g for 5 minutes. Conjugated streptavidin (SA) and SA conjugates (dextramer backbone, Immudex) with fluorescent dyes (PE) were aliquoted into tubes according to Table 1. MHC molecules were added to the aliquoted SA conjugates, avoiding precipitation, and incubated at room temperature for 30 minutes. After complex formation, D-biotin (Avidity Bio200) was added with 0.02% NaN2 in PBS to the final pMHC monomer concentration listed in Table 1, and the mixture was incubated at 4°C for 30 minutes. Assembled MHC molecules were stored at 4°C for up to 4 weeks. Two sets of two MHC molecules were prepared. Each set, with two specificities, was individually labeled. The labels were reversed between the two sets as described below. 1.iv. 1x CMV-specific pMHC was combined with 2OS-A1B1, and 1x HIV-specific pMHC was combined with 2OS-A2B2. 2.v. 1x CMV-specific pMHC was combined with 2OS-A2B2, and 1x HIV-specific pMHC was combined with 2OS-A1B1. b. Modifications: No further modifications were performed. c. Purification: Before adding the MHC molecules to the sample, they were centrifuged at 5000 g for 5 minutes to sediment any MHC molecules remaining in solution.

[0157] 6. Incubation of sample and MHC molecules: The cell sample and MHC molecules were mixed in one container to allow the MHC molecules to bind to the T cells they recognize. a. Sample amount: 1×10E6 to 2×10E6 cells of BC type were used. b. Amount of MHC molecules: according to Table 1. 5 μl of each MHC molecule was required per incubation (1 μg / ml for pMHC). c. Conditions: BCs were thawed in 10 ml of RPMI containing 10% fetal bovine serum (FBS) at 37°C, centrifuged at 1500 g for 5 minutes, and washed twice with 10 ml of RPMI containing 10% FBS. All subsequent cell washes refer to centrifugation at 490 g for 5 minutes followed by removal of the supernatant. 1 x 10E6 to 2 x 10E6 cells were washed with barcode buffer (PBS / 0.5% BSA / 2 mM EDTA / 100 μg / ml herring DNA) and resuspended in this buffer at approximately 20 μl per staining run. Before incubation with MHC molecules, cells were incubated with 50 nM dasatinib at 37°C for 30 minutes. Before adding MHC molecules to cells, the cells were centrifuged at 3300 g for 5 minutes. 5 μl of each MHC molecule (per pMHC) was required per incubation (1 μg / ml for pMHC). After adding the MHC molecules, the cells were incubated for 15 minutes at 37°C. The antibody mixtures listed in Table 2 were added along with 0.1 μl of a near-IR viability dye (Invitrogen, L10119) that stains free amines. The antibody staining was essentially the same as conventional MHC multimer staining. The cells were incubated for 30 minutes at 4°C.

[0158] 7. Enrichment of MHC molecules with desired characteristics: In this example, MHC molecules were enriched by using flow cytometry, more specifically, a fluorescence-activated cell sorter (FACS). MHC molecules contain fluorescent dyes. Therefore, cells that bind to MHC molecules emit fluorescence and can be separated by the FACS sorter from cells that do not bind to MHC molecules and therefore do not emit fluorescence. As a result, MHC molecules bound to cells are enriched. a. Application: Two different flow cytometers were used for acquisition: a BD FACSCanto II with three lasers (488 nm blue, 633 nm red, and 405 violet) and a BD LSR II with five lasers. Only four of the LSR II's lasers were used throughout this experiment (488 nm blue, 640 nm red, 355 nm UV, and 405 nm violet). Additionally, cells were sorted using a BD FACSAria and a FACSAria II with three lasers (488 nm blue, 633 nm red, and 405 violet). BD FACSDiva software version 6.1.2 was used for analysis of both flow cytometry data. The following gating strategy was applied: Initial gating for CD8+ cells was performed identically in both cases. Lymphocytes were identified on an FSC / SSC plot. Additional gating on single cells (FSC-A / FSC-H), live cells (near-IR viability dye negative) and dump channel negative / CD8 positive cells (FITC / PerCP) was used to define the CD8 T cell population. b. Washing: After being ready for flow cytometry acquisition, cells were washed twice with barcode buffer. Optionally, cells were fixed with 1% paraformaldehyde overnight at 4°C and washed twice with FACS buffer or barcode buffer. Fixed cells were stored at 4°C for up to 1 week. c. Isolation: Optionally, cells were harvested up to one week after fixation with 1% paraformaldehyde. Multimer-positive cells were sorted by FACS as described in 7a, pre-saturated with 2% BSA solution for 2 hours to overnight to increase the stability of oligonucleotides associated with the sorted cells, and placed in tubes containing 200 μl of barcode buffer. Sorted fluorochrome (PE)-positive cells were centrifuged at 5000 g for 5 minutes to remove all excess buffer. Cells were stored at -80°C.

[0159] 8. Identification of enriched MHC molecules: Cell-bound pMHC can be identified by identifying the label (in this example, an oligonucleotide label). Therefore, the oligonucleotides contained in the MHC molecules collected with the cells were analyzed by quantitative PCR using a label-specific Q-PCR probe. This allowed the identification of cell-bound pMHC in the cell sample. a. Labels derived from the sorted cells were analyzed by QPCR as in Experiment 1.

[0160] Results and Conclusions of Example 4 After fractionation and qPCR, the Ct values ​​obtained confirmed that the label was successfully recovered and concentrated only when bound to the CMV epitope, but no label was detected when bound to the HIV epitope (Figure 8).

[0161] Only when combined with the positive control reagent was the 2OS label recovered after cell interaction, sorting, and qPCR.

[0162] Figure 8 Detection of CMV specificity in negative control barcode-labeled pMHC dextramers. In 1, a unique barcode was conjugated to the positive control reagent, and in 2, another unique barcode was conjugated to the positive control reagent. A spare barcode for each experiment was conjugated to the HIV negative control reagent. In addition, there are 998x unlabeled negative control reagents in both 1 and 2.

[0163] (A) Ct values ​​obtained from multiplex qPCR of sorted PE-pMHC-dextramer-positive cells. Cells were stained with 1 and 2, respectively. During staining, reagents conjugated with a positive control (CMV) barcode and reagents conjugated with a negative control (HIV) barcode were present, but the negative control (HIV) barcode-labeled pMHC-dextramer was clearly washed away. Approximately 575 cells were analyzed in each separate qPCR. (B) The number of barcodes conjugated per cell estimated based on the obtained Ct values. Although the same number of cells was present in each qPCR, some differences in the Ct values ​​shown in (B) are evident. However, when these values ​​are normalized to the specific probe, they are at the same level. QPCR was performed in duplicate, and the mean ± range of duplicates is shown here.

[0164] Example 5 This is an example in which blood was modified to obtain (1b) peripheral blood mononuclear cells (PBMCs) as sample (1).

[0165] The backbone (2) was a dextran conjugate with streptavidin and a fluorescent dye (dextramer backbone from Immudex).

[0166] The MHC molecule (3) is a peptide-MHC (pMHC) complex that presents a peptide antigen of 8–10 amino acids. The MHC molecule was modified by biotinylation to generate a biotin capture tag on the MHC molecule (3b). The MHC molecule was purified by HPLC (2c). The label (4) was an oligonucleotide. The oligonucleotide label was synthesized by DNA Technology A / S (Denmark) (4a) and synthetically modified with a terminal biotin capture tag (4b). In some examples, the oligonucleotide label was further modified by annealing with a partially complementary oligonucleotide label to form a combined oligonucleotide label.

[0167] MHC molecules (5) were synthesized by attaching MHC molecules in the form of biotinylated pMHC and labels in the form of biotin-modified oligonucleotides onto a streptavidin-modified dextran backbone (Dextramer backbone, Immudex, Denmark) (5a). The MHC molecules further contained modifications in the form of fluorescent dyes (5b). A library consisting of 110 different MHC molecules, each containing a different pMHC, was generated, with each MHC molecule encoded by a corresponding individual oligonucleotide label.

[0168] A fixed amount of sample PBMCs (1b) was incubated with a fixed amount of the library of MHC molecules (5) under conditions (e.g., incubation time, buffer, pH, and temperature) that allow binding of the MHC molecules to T cells in the sample (6c).

[0169] After initial rounds of centrifugation to sediment the cells and resuspension in wash buffer, PBMCs were washed several times and then subjected to fluorescence-activated cell sorting (FACS) of fluorescently labeled cells to separate cell-bound MHC molecules from non-cell-bound MHC molecules (7). T cells capable of efficiently binding MHC molecules fluoresce due to the fluorescent dye contained within the MHC molecules, whereas T cells unable to bind MHC molecules do not. FACS sorting enriches for fluorescent cells and, therefore, for T cell-bound MHC molecules in the PBMC sample.

[0170] FACS-isolated cells were subjected to PCR amplification of oligonucleotide tags associated with cell-associated MHC molecules, and the identities of the MHC molecules associated with T cells present in the sample were revealed by sequencing the individual DNA fragments generated by the PCR reaction.

[0171] Thus, this experiment revealed the peptide-MHC specificity of the T cell receptors of T cells present in the blood sample.

[0172] 1. Sample Preparation. The cell samples used in this experiment were obtained by mixing blood drawn from two different donors, BC260 and 171 (Table 6). To obtain increasing B0702 CMV pp65 TPR responses in B0702-negative donor samples, five-fold dilutions of BC260 in 171 were performed, i.e., 100%, 20%, 5%, 1%, 0.2%, 0.04%, 0.008%, 0.0016%, and 0.00032% BC260, corresponding to theoretical frequencies of B0702 CMV pp65 TPR-specific cells of 5%, 1%, 0.2%, 0.04%, 0.0125%, and 0.0025%. Therefore, the sensitivity of this method and the validity of the results obtained in this experiment could be evaluated at the end of the experiment by comparing them with data obtained simultaneously using another method on the same cells. a. Sample acquisition: Blood was obtained from the Danish Blood Bank. b. Sample Modification: i. Same as Experiment 1. ii. The above two blood samples were mixed.

[0173] 2. Preparation of the main chain: Same as experiment 1.

[0174] 3. Preparation of MHC molecules: The MHC molecules used in this example were class I MHC-peptide complexes. Individual specificities (allele and epitope combinations) were generated as described in Experiment 1. Here, a library of 110 different peptide MHC molecules corresponding to Table 10 was used. a. Synthesis: As described in Experiment 1. i. Both MHC heavy chain and B2M were expressed in E. coli as previously described (Hadrup et al., 2009). ii.p * MHC monomers were refolded and purified as previously described ( Hadrup et al., 2009 ). b. Modification:p * The UV-conditional peptide ligand was exchanged with the peptide antigen of interest to obtain specific peptide-MHC monomers. i. Peptides (Pepscan Presto) were diluted in phosphate-buffered saline (DPBS; Lonza) and mixed in individual wells of a 384-well plate to a final concentration of 100 μg / ml:200 μM (monomer:peptide). A maximum volume of 70 μl was prepared for each well format. The mixture was exposed to 366 nm UV light (UV cabinet; CAMAG) for 1 hour and optionally stored at 4°C for up to 24 hours. c. Purification: Same as experiment 2.

[0175] 4. Label Preparation: In this example, 110 different oligonucleotides of the same length but different sequences were generated. Each oligonucleotide bound to, and thus encoded, a specific pMHC. The oligonucleotides were biotinylated to facilitate binding to a dextran-streptavidin conjugate backbone. a. Synthesis: In this example, the labels were DNA oligonucleotides purchased from DNA Technology (Denmark) and delivered as lyophilized powders. 100 μM label stock dilutions were made in nuclease-free water and stored at -20°C. Two DNA oligonucleotide labels were used, designated 1OS and 2OS, respectively. The i.120 1OS label was ordered from DNA Technology as a single-stranded DNA oligonucleotide with a 5' biotinylated modification. The label was diluted to a working concentration (640 nM) in nuclease-free water containing 0.1% Tween. See Tables 9 and 10 for the sequence of the 1OS label. The ii.2OS labeling system was developed to increase the complexity of a limited number of oligonucleotide sequences through a combinatorial labeling strategy, enabling a larger number of unique labels to be generated from a more limited number of label precursors. This strategy, termed 2OS, involved the annealing and subsequent extension of two partially complementary oligonucleotide sequences to generate new unique oligonucleotide sequences that were then used as DNA oligonucleotide labels (Tables 9 and 10). For example, a combinatorial library of 1,200 distinct (Ax + By) labels was generated by combining 20 unique, all partially complementary, oligonucleotide sequences (A-label precursors) with 60 additional unique oligonucleotide sequences (B-label precursors). 1. Partially complementary A and B oligonucleotides were annealed to yield a combined A+B oligonucleotide label. The A and B oligos were mixed as described in Table 3, heated to 65°C for 2 minutes, and gradually cooled to below 35°C over 15-30 minutes. The annealed A and B oligos were then extended as described in Table 3.4. The components of the extension reaction were mixed immediately before use. After mixing, the reaction was left at room temperature for 5 minutes to allow the annealed oligonucleotides to extend. The reagents used for annealing (left) and extending (right) the partially complementary oligonucleotides are described in Table 3. Reagents shown in italics are from the Sequenase Version 2.0 DNA Sequencing Kit (Affymetrix, Inc., #70770). b. Modification: Both labels were diluted to a working concentration (640 nM) in nuclease-free water containing 0.1% Tween. The extended oligonucleotide sequence 2OS label was then treated in the same manner as the 1OS label.

[0176] 5. Preparation of MHC molecules: MHC molecules were formed by combining MHC molecules (pMHC) and labels (oligonucleotides) with a backbone (backbone, dextran-streptavidin-fluorochrome conjugate) ensuring that a given pMHC binds to a given oligonucleotide while maintaining a 1:1 relationship between pMHC and oligonucleotide. a. Synthesis: For the preparation of MHC molecules, the backbone was labelled in the form of a biotinylated oligonucleotide before adding pMHC. i. MHC molecules were prepared by adding a 2x excess of label over the backbone (label:backbone = 2:1) unless otherwise noted, and incubated at 4°C for 30 minutes. Whenever a new batch of backbone and / or label was used, the label-backbone (backbone) binding was determined after the titration procedure. Before combining MHC molecules and pMHC monomers, they were centrifuged at 3300 g for 5 minutes. Conjugated streptavidin (SA) and SA conjugates (dextramer backbone, Immudex) with fluorescent dyes (PE) were aliquoted into a new 96-well plate suitable for peptide exchange reaction setup. Differences in the method for assembling PE. MHC molecules were added to the aliquoted SA conjugates, avoiding precipitation, and incubated at room temperature for 30 minutes. After complex formation, D-biotin (Avidity Bio200) was added together with a 0.02% NaN2 solution in PBS and incubated at 4°C for 30 minutes. Assembled MHC molecules were stored at 4°C for up to 4 weeks. b. Modification: If the total volume of the panel of combined MHC molecules exceeded 100 μl per incubation with the sample, the volume was reduced. i. A Vivaspin 500 (Sartorius) size-exclusion spin column with a 300 kDa cutoff was saturated with 500 μl of 2% BSA / PBS and centrifuged at 5000 g until the volume passed through. The column was then washed twice by adding 500 μl of PBS and centrifuged at 5000 g until the column was nearly empty. The panel of combined MHC molecules was added to the spin column and centrifuged at 5000 g at 4°C until the desired volume was present on the column (approximately 80 μl per sample incubation). c. Purification: A panel of MHC molecules was transferred to an Eppendorf tube and centrifuged at 5000 g for 5 minutes before being added to the cells to sediment any MHC molecules remaining in solution.

[0177] 6. Incubation of sample and MHC molecules: The cell sample and MHC molecules were mixed in one container to allow the MHC molecules to bind to the T cells they recognize. a. Sample quantity: 2 x 10E6 cells of BC morphology. B amount of MHC molecules c. Conditions: All cell washes consisted of centrifugation at 490g for 5 minutes followed by removal of the supernatant. 2 x 10E6 cells were transferred to individual wells of a 96-well plate, washed with barcode buffer (PBS / 0.5% BSA / 2mM EDTA / 100µg / ml herring DNA), and resuspended in this buffer at approximately 20µl per staining. For MHC molecule incubation, cells were incubated with 50nM dasatinib at 37°C for 30 minutes (Lissina et al., 2009). Before adding MHC molecules to the cells, the cells were centrifuged at 3300g for 5 minutes. 3µl of each MHC molecule was required per incubation (1µg / ml for pMHC). After adding the MHC molecules, the cells were incubated at 37°C for 15 minutes. The antibody mixture listed in Table 2 was added along with 0.1µl of a near-IR viability dye (Invitrogen, L10119) that stains free amines. Antibody staining was essentially the same as conventional MHC multimer staining. Cells were incubated at 4°C for 30 minutes, and then unbound MHC molecules or antibodies were washed away. Cells were then fixed by adding 50 μl of 1% paraformaldehyde.

[0178] 7. Enrichment of MHC molecules with desired characteristics: In this example, MHC molecules were enriched by using flow cytometry, more specifically, a fluorescence-activated cell sorter (FACS). MHC molecules contain fluorescent dyes. Therefore, cells that bind to MHC molecules emit fluorescence and can be separated by the FACS sorter from cells that do not bind to MHC molecules and therefore do not emit fluorescence. As a result, MHC molecules bound to cells are enriched. a. Application: Two different flow cytometers were used for acquisition throughout this experiment: a BD FACSCanto II with three lasers (488 nm blue, 633 nm red, and 405 violet) and a BD LSR II cytometer with five lasers. Only four of the LSR II's lasers were used throughout this experiment (488 nm blue, 640 nm red, 355 nm UV, and 405 nm violet). Additionally, cells were sorted using a BD FACSAria and FACSAria II with three lasers (488 nm blue, 633 nm red, and 405 violet). BD FACSDiva software version 6.1.2 was used for analysis of all flow cytometry data. i. The following gating strategy was used: Initial gating for CD8+ cells was performed identically in all cases. Lymphocytes were identified on an FSC / SSC plot. Additional gating on single cells (FSC-A / FSC-H), live cells (near-IR viability dye negative), and dump channel negative / CD8+ cells (FITC / PerCP) was used to define the CD8+ T cell population. b. Washing: After being ready for flow cytometry acquisition, cells were washed twice with barcode buffer. Optionally, cells were fixed with 1% paraformaldehyde overnight at 4°C and washed twice with FACS buffer or barcode buffer. Fixed cells were stored at 4°C for up to 1 week. c. Isolation: Optionally, cells were harvested up to one week after fixation with 1% paraformaldehyde. Multimer-positive cells were sorted into tubes containing 200 μl of barcode buffer pre-saturated with 2% BSA for 2 hours to overnight to increase the stability of oligonucleotides associated with the sorted cells. Sorted multimer-positive cells were centrifuged at 5000 g for 5 minutes to remove all excess buffer. Cells were stored at -80°C. i. Gates were set to define positive events with a single conjugated fluorochrome, ie, PE or APC. ii. The potency of pMHC dextramers was assessed based on mean fluorescence intensity (MFI) or staining index (SI), which is a measure of population separation that takes into account the possible influence on the negative population (background) and the background range.

[0179] 8. Identification of enriched MHC molecules: By identifying the label (in this example, the oligonucleotide label), pMHC bound to the cells can be identified. Therefore, the oligonucleotides contained in the MHC molecules collected with the cells were sequenced. This allowed the identification of pMHC bound to the cells in the cell sample. a. Prior to sequencing, the tags from the sorted cells were amplified by PCR. See Table 4 for the PCR composition. PCR was performed using the following kit: Taq PCR Master Mix Kit (Qiagen, No. 201443). The temperature profile is shown in Table 5. PCR was performed in a thermal cycler: GeneAmp, PCR System 9700 (Applied Biosystem). PCR products were visualized after gel electrophoresis using a Bio-Rad Gel Doc EZ Imager. i. The forward and reverse primers contained adapters for the sequencing reaction (A and P1 keys, respectively, compatible with Ion Torren sequencing (Life Technologies)). ii. Additionally, the forward primers carried sample identification barcodes (Table 8). Primers with specific sample identification sequences were used to amplify the labels and associated MHC molecules on sorted cells from individual samples (Table 8). This facilitated the distribution of sequence reads from each single sample. Furthermore, the input of the enriched MHC molecule panel (before mixing with cells) was assigned a sample identification barcode by PCR (referred to as the panel input). Sequencing of the panel input would allow for normalization of the analyzed sequence output. iii. Positive sequence reads were compared to the sequence read from the 5' end sample barcode identity through the pMHC barcode identity. Read counts were normalized by the total number of reads mapped to the same sample barcode identity and the number of reads from the panel input. Deconvolution into MHC molecules DNA oligonucleotide labels were sequenced on a b.314 Ion Torrent chip (GeneDx). Adapters were introduced via primers during PCR (see Table 8 for adapter sequences). i. A sequence database was created containing all possible combinations of 15 sample identification barcodes and 358 pMHC barcodes (118 1OS + 240 2OS), as well as primer and annealing sequences for both the 1OS and 2OS systems. This pooled data yielded 5,370 sequences that could be predicted from a single sequencing run. Each sequencing read was then used to search the database for alignments using the nucleotide BLAST algorithm, with a match reward of 1, a mismatch reward of -2, and a gap cost of 2 for both gap initiation and gap extension. In this way, sequencing errors, whether miscalled or inserted / deleted, were penalized equally compared to the actual sequence. ii. Alignments were discarded based on the following criteria: 1. E-value > 1e-12; alignment length is insufficient (1OS and 2OS systems must exceed 60 or 102 bases, respectively). 2. The target sequence has two or more start positions, i.e., the alignment contains four or fewer bases out of the six bases in the unique portion of the sample identification barcode. 3. If multiple alignments were still found for any sequencing read, only the alignment with the highest percent identity was retained. Finally, the number of reads that mapped to each barcode in the database was counted. iii. Identification of overrepresented barcodes: Relative read counts were calculated by normalizing each read to the total read counts mapped to the same sample identity barcode. Relative read counts were then used to calculate the fold change per barcode compared to a control sample barcode input (a barcode-labeled detection molecule panel not mixed with cells). Significantly overrepresented barcodes were identified using a two-sample test comparing the equality of raw read count proportions between sample and control barcode inputs, and p-values ​​were corrected for multiple testing using the Benjamini-Hochberg FDR method.

[0180] Results of Example 5: This example demonstrates the feasibility of detecting antigen-responsive T cells in large mixtures of different pMHC multimers (MHC molecules). We demonstrate that the sensitivity of barcode-labeled MHC multimers is such that they can detect at least 0.00032% of specific T cells among CD8+ T cells. This correlates accurately with previously described (low-throughput) methods.

[0181] Figure 9 shows a schematic representation of the number of unique 1OS barcode reads mapped to seven different samples. HLA-B7-negative BCs were spiked with 5% B7 CMV pp65 TPR response (barcode 88) at 5-fold dilutions to generate seven samples (5%, 1%, 0.2%, 0.04%, 0.008%, 0.0016%, and 0.00032%). These BCs contained a population of A11 EBV-EBNA4-specific T cells (corresponding to barcode 4). The samples were stained with the same panel containing 110 pMHC-dextramers variously barcoded with 1OS. Bars indicate the total number of reads normalized to the input panel in each sample. Experiments were performed in duplicate; averages are shown here.

[0182] Figure 10 shows a schematic representation of the number of unique 2OS barcoded reads mapped to seven different samples. HLA-B7-negative BCs were spiked with 5% B7 CMV pp65 TPR response (barcode A3B18) at 5-fold dilutions to generate seven samples (5%, 1%, 0.2%, 0.04%, 0.008%, 0.0016%, and 0.00032%). These BCs contained a population of A11 EBV-EBNA4-specific T cells (corresponding to barcode A1B4). The samples were stained with the same panel containing 110 pMHC-dextramers, each barcoded in 2OS. The bars indicate the total number of reads in each sample, normalized to the input panel. Experiments were performed in duplicate. Averages are shown.

[0183] Example 6: Example 6 is carried out exactly like Example 5, except that different samples are used, in which antigen-responsive T cells are detected in five different donor blood samples.

[0184] Results of Example 6: This example demonstrates the feasibility of detecting multiple distinct specificities in different donor samples using DNA-barcoded MHC multimers. The data obtained demonstrate the feasibility of high-throughput screening of T cell reactivity in multiple donors to assess immune reactivity associated with disease manifestations, vaccination, infection, etc.

[0185] Figure 11 shows a schematic representation of the number of unique 1OS barcoded reads that mapped to six different samples. Six BCs were stained with the same panel containing 110 pMHC dextramers variably barcoded with 1OS. The bar graphs show the total number of reads normalized to the input panel in each sample (p<0.05). Each pie chart shows the number of significant (p<0.01) reads that mapped to that sample.

[0186] Figure 12 shows a schematic representation of the number of unique 2OS barcoded reads mapped to six different samples. Six BCs were stained with the same panel containing 110 pMHC-dextramers variably barcoded with 2OS. The bar graph shows the total number of reads normalized to the input panel in each sample (p<0.05).

[0187] table: [Table 1]

[0188] [Table 2]

[0189] [Table 3]

[0190] [Table 4]

[0191] [Table 5] TIFF2025128229000009.tif30166

[0192] [Table 6]

[0193] [Table 7] TIFF2025128229000012.tif117162

[0194] [Table 8] TIFF2025128229000014.tif204166TIFF2025128229000015.tif25166

[0195]

Table 9

[0196]

Table 10

[0197]

Table 11

[0198]

Table 12

[0199] (References) 1. Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell Jl, et al. Phenotypic analysis of antigen-specific T lymphocytes. Science. 1996;274:94-6. 2. Davis MM, Bjorkman PJ. T-cell antigen receptor genes and T-cell recognition. Nature. 1988;334:395-402. 3.Robins HS,Campregher P V,Srivastava SK,Wacher A,Turtle CJ,Kahsai O,et al.Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells.Blood.2009;14:4099-107. 4.Hadrup SR,Bakker AH,Shu CJ,Andersen RS,van VJ,Hombrink P,et al.Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers.Nature Methods.2009;6:520-6. 5.Andersen RS,Kvistborg P,Morch TF,Pedersen NW,Lyngaa R,Bakker AH,et al.Parallel detection of antigen-specific T-cell responses by combinatorial encoding of MHC multimers.NatProtoc.2012 6.Newell EW,Sigal N,Nair N,Kidd Ba,Greenberg HB,Davis MM.Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization.Nat Biotechnol.2013;1-9. 7.Soen Y,Chen DS,Kraft DL,Davis MM,Brown PO.Detection and characterization of cellular immune responses using peptide-MHC microarrays.PLoSBiol.2003;1:429-38. 8.Stone JD,Demkowicz Jr.WE,Stern LJ.HLA-restricted epitope identification and detection of functional T cell responses by using MHC-peptide and costimulatory microarrays.ProcNatlAcadSciUSA.2005;102:3744-9. 9.Newell EW,Davis MM.Beyond model antigens:high-dimensional methods for the analysis of antigen-specific T cells.Nat Biotechnol.2014;32. 10.Dossinger G,Bunse M,Bet J,Albrecht J,Paszkiewicz PJ,WeiBbrich B,et al.MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy.PLoS One.2013;8:e61384. 11.Cha E,Klinger,Hou Y,Cummings C,Ribas A,Faham M,et al.Improved Survival with T Cell Clonotype Stability After Anti-CTLA-4 Treatment in Cancer Patients.Sci Transl Med.2014;6:238ra70. 12.Robert L,Tsoi J,Wang X,Emerson RO,Hornet B,Chodon T,et al.CTLA4 blockade broadens the peripheral T cell receptor repertoire.Clin Cancer Res.2014 13.Morgan RA,Dudley ME,Wunderlich JR,Hughes MS,Yang JC,Sherry RM,et al.Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes.Science.2006. 14.Pannetier C,Even J,Kourilsky P.T-cell repertoire diversity and clonal expansions in normal and clinical samples.ImmunolToday.1995;16:176-81. 15.Cameron BJ,Gerry AB,Dukes J,Harper J V,Kannan V,Bianchi FC,et al.Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells.Sci Transl Med.2013;5:197ra103. 16.Linette GP,Stadtmauer Ea,Maus M V,Rapoport AP,Levine BL,Emery L,et al.Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma.Blood.2013;122:863-71.

Claims

1. A multimeric major histocompatibility complex (MHC), a) at least eight given MHC-peptide molecules linked to a backbone molecule; at least one nucleic acid molecule linked to the backbone molecule; wherein the at least one nucleic acid molecule comprises a 5' first primer region, a barcode region, and a 3' second primer region; the barcode region serves as a label specific to the at least eight given MHC-peptide molecules; and / or b) two or more given MHC-peptide molecules linked to a dextran backbone molecule; at least one nucleic acid molecule linked to said dextran backbone molecule; wherein the at least one nucleic acid molecule comprises a 5' first primer region, a barcode region, and a 3' second primer region; the barcode region serves as a label specific to the two or more given MHC-peptide molecules; and / or c) two or more given MHC-peptide molecules linked to a backbone molecule; at least one nucleic acid molecule linked to the backbone molecule; wherein the at least one nucleic acid molecule comprises a 5' first primer region, a barcode region, and a 3' second primer region, and the at least one nucleic acid molecule is linked to the backbone via a streptavidin-biotin bond and / or a biotin-avidin bond; A multimeric MHC, wherein the barcode region serves as a specific label for the two or more given MHC-peptide molecules.

2. 2. The multimeric MHC of claim 1, wherein the backbone molecule is selected from the group consisting of polysaccharides, including glucans such as dextran, streptavidin, and streptamer multimers.

3. 2. The multimeric MHC of claim 1, wherein the backbone molecule is dextran.

4. 4. The multimeric MHC complex according to claim 1, wherein the two or more MHC-peptide molecules are bound to the backbone via a streptavidin-biotin bond and / or biotin-avidin, and / or via an MHC heavy chain and / or via a light chain (B2M).

5. The multimeric MHC according to claims b) and c) of claim 1, wherein said multimeric MHC consists of at least 4 given MHC-peptide molecules, such as at least 8, for example at least 10, for example 2-30, for example 2-20, for example 2-10, for example 4-10, or for example 15-30 given MHC-peptide molecules.

6. The multimeric MHC according to claim 1 a), wherein the multimeric MHC consists of at least 10 given MHC-peptide molecules, such as 15 to 30 given MHC-peptide molecules.

7. 7. The multimeric MHC of claim 1, wherein the backbone molecule is dextran and the multimeric MHC consists of at least eight given MHC-peptide molecules.

8. 8. The multimeric MHC according to any one of claims 1 to 7, wherein said at least one nucleic acid molecule has a length of 20 to 200 nucleotides, such as 20 to 150, such as 20 to 100, such as 30 to 100, such as 30 to 80, such as 30 to 50 nucleotides, and / or said at least one nucleic acid molecule comprises or consists of DNA, RNA and / or artificial nucleotides such as PNA or LNA.

9. 2. The multimeric MHC of claim 1, wherein the at least one nucleic acid molecule is linked to the backbone molecule via a streptavidin-biotin bond and / or a biotin-avidin bond.

10. The multimeric MHC according to any one of claims 1 to 9, wherein the MHC is selected from the group comprising class I MHC, class II MHC, CD1, and other MHC-like molecules.

11. 11. The multimeric MHC of any one of claims 1 to 10, wherein the backbone further comprises one or more selectable labels, such as one or more labels selected from the group consisting of a fluorescent label, a His tag, and a metal ion tag.

12. The multimeric MHC of any one of claims 1 to 11, wherein the backbone further comprises a fluorescent label.

13. 13. A composition comprising different subsets of multimeric major histocompatibility complexes (multimeric MHC) according to any one of claims 1 to 12, wherein each subset of multimeric MHC has a unique barcode region specific for a given MHC-peptide molecule of each subset.

14. 14. The composition of claim 13, comprising at least 10 different multimeric MHC subsets, such as at least 100, such as at least 500, at least 1000, at least 5000, such as 10-100000, such as 10-50000, such as 10-1000, or such as in the range of 50-500 multimeric MHC subsets.

15. The composition of any one of claims 13 to 14, wherein the primer region of the nucleic acid molecule is identical for each subset of multimeric MHC.

16. 1. A method for detecting antigen-responsive cells in a sample, comprising: i) providing one or more multimeric MHCs according to any one of claims 1 to 12 or a composition according to any one of claims 13 to 14; ii) contacting said multimeric MHC with said sample; iii) detecting binding of said multimeric MHC to said antigen-responsive cells, thereby detecting cells responsive to antigens present on said multimeric MHC, wherein said binding a. amplifying the barcode region of the nucleic acid molecule linked to the multimeric MHC by PCR; b. Sequencing the amplified barcode region; to be detected by, to detect A method comprising:

17. 17. The method of claim 16, wherein in step iii)a), the barcode region of the nucleic acid molecule is amplified using one primer set.

18. 18. The method of any one of claims 16 to 17, wherein the sample is selected from the group consisting of a blood sample, such as a peripheral blood sample, a blood-derived sample, a tissue sample, or a body fluid, such as cerebrospinal fluid or saliva.

19. 19. The method of any one of claims 16 to 18, wherein the method further comprises one or more steps of sorting or selecting one or more populations of antigen-responsive cells prior to amplification, such as one or more steps of single cell sorting prior to amplification.

20. 20. The method of claim 19, wherein sorting or selecting the cells comprises a method selected from the group consisting of flow cytometry such as FACS, magnetic bead selection, size exclusion, gradient centrifugation, column adsorption and gel filtration.

21. a) detecting binding of the antigen-responsive cells comprises comparing the measured value with a reference level, such as a negative control and / or total response level; b) the frequency of specific T cells is calculated by comparing the frequency of barcode sequence reads with the number of sorted T cells; or 21. The method of any one of claims 16 to 20, wherein c) the relative contribution of multiple different peptide-MHC complexes bound to a single T cell is determined by the number of barcode reads of a given sequence.

22. The method of any one of claims 16 to 21, wherein sequencing the amplified barcode region comprises deep sequencing, high-throughput sequencing or next-generation sequencing.

23. 23. The method of any one of claims 19 to 22, wherein the antigen specificity of a given sorted single cell is determined in combination with one or more additional single cell characteristics, such as single cell TCR sequencing.

24. The method of any one of claims 16 to 23, wherein the affinity and binding motif of a given TCR is determined.

25. 25. The method of any one of claims 16 to 24, wherein the method further comprises one or more steps of removing unbound MHC multimers in the sample that have been contacted with the multimeric MHC prior to amplification, such as by washing and / or centrifugation.

26. The method of any one of claims 16 to 25, wherein the method is for detecting a plurality of different antigen-responsive cells in a sample, such as in a single sample.

27. The method of any one of claims 16 to 26, wherein the antigen-responsive cells are T cells.

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