Methods for assessing antigen-specific T cell responses

Abstract

The present disclosure provides methods and kits for determining antigen-specific T cell responses in a subject based on the use of a whole blood sample rather than isolated, enriched, and / or extracted peripheral blood mononuclear cells. The use of a whole blood sample to measure antigen-specific T cell responses in a subject streamlines the method by reducing sample preparation and consumption. These methods for determining antigen-specific T cell responses in a subject include contacting a whole blood sample from the subject with at least one antigen to form a mixture, contacting the whole blood sample with at least one staining reagent, and analyzing the mixture for antigen-specific T cells.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application was filed as a PCT international patent application on November 13, 2023, and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 383,412, filed November 11, 2022, the entire disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] (Introduction) The development of specific adaptive immune responses is crucial for controlling and eliminating infections, including viral infections. T cells can orchestrate and execute immune responses to various antigens to acquire a highly diverse set of functional properties, which provide the basis for immune defense. Therefore, analyzing and understanding specific T cell responses is important for better understanding protective immunity in many infectious diseases.

[0003] The ability to analyze and characterize enhanced cellular immunity is a key milestone for vaccine and treatment development across many fields, including virology, oncology, and immunology. The presence of antigen-specific T cells can be assessed in vitro based on their ability to proliferate, express activation markers, and / or produce cytokines and transcription factors in the presence of the antigen of interest. Among available methods, the enzyme-linked immunospot (ELISpot) assay is a technique for determining the frequency of antigen-specific immune cells that secrete cytokines upon recall antigen stimulation. The IFN-γ ELISpot technique has become the most common approach in clinical settings to assess both T-CD4+ and T-CD8+ antigen-specific responses. Based on the use of isolated, enriched, and / or extracted peripheral blood mononuclear cells (PBMCs), this technique allows for the detection of low frequencies of antigen-specific T cells but does not allow for the differentiation of CD4+- and CD8+-driven responses.

[0004] Another method for measuring and characterizing specific T cell responses is intracellular cytokine staining (ICS). When combined with multiparameter flow cytometry, ICS allows for the simultaneous detection of various markers (both extracellular and intracellular) resulting in the identification of responding cell subsets based on cell phenotyping with a functional readout of cytokine production. Typically, ICS allows for the exploration of the polarization of the cellular response by analyzing cytokines (e.g., IL-2, TNF, and IFN-γ). Preparation of isolated, enriched, and / or extracted PBMCs is often recognized as a critical prerequisite for ICS and is therefore often the first step in the ICS workflow. Preparation of PBMCs is time-consuming and relies on technically competent individuals who are often already busy in diagnostic laboratories. As a direct result, ICS is rarely considered in routine clinical practice.

[0005] Classical approaches to determining antigen-specific T cell responses in subjects use isolated, enriched, and / or extracted PBMCs. As discussed, examples of these approaches include ELISpot and ICS. Both ELISpot and ICS require the extraction of PBMCs from whole blood before determining antigen-specific T cell responses. Current techniques (including ELISpot and ICS) suffer from a lack of resolution, a lack of sensitivity, and / or labor-intensive and tedious workflows. Further deficiencies with current techniques include the fact that many current techniques only examine a single activation marker. These deficiencies with current techniques are exacerbated by the low frequency of antigen-specific T cells (often as low as 1 cell per 100,000 T cells). Summary of the Invention [Means for solving the problem]

[0006] (Brief summary of the invention) Methods for assessing antigen-specific T cell responses in a subject The present disclosure provides a method and kit for determining antigen-specific T cell responses in a subject, based on the use of whole blood samples rather than isolated, enriched, and / or extracted PBMCs.The use of whole blood samples to measure antigen-specific T cell responses in a subject streamlines the method by simplifying sample preparation and analysis, reducing sample volume, and reducing sample consumption.The methods and kits disclosed herein can provide a more realistic physiological environment for stimulating antigen-specific T cells without loss.The methods and kits can further simplify sample preparation and analysis through the use of dry reagent technology and / or the use of intact antigens rather than pre-processed peptides.

[0007] In its broadest aspect, the present disclosure provides methods and kits for determining antigen-specific T cell responses in a subject, the methods comprising: contacting a whole blood sample from the subject with at least one antigen to form a mixture; contacting the mixture with at least one staining reagent, where the contacting of the whole blood sample with the at least one staining reagent occurs before, simultaneously with, or after the contacting of the whole blood sample with the at least one antigen to form a mixture, or any combination thereof; and analyzing the mixture for T cells responsive to the antigen. In embodiments, the analyzing step is by flow cytometry. In embodiments, the whole blood sample is about 10 μL to about 10 mL.

[0008] In certain embodiments, the at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and / or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, or any combination thereof. In certain embodiments, at least one reagent is added to the mixture formed by contacting the whole blood sample with the at least one antigen.

[0009] In certain embodiments, the disclosed methods further comprise incubating the mixture formed by contacting the whole blood sample with at least one antigen prior to contacting the whole blood with at least one staining reagent, and / or after contacting the whole blood sample with at least one antigen prior to contacting the whole blood with at least one staining reagent. In certain embodiments, incubating the mixture comprises incubation at about 37° C. for about 30 minutes to about 24 hours.

[0010] The method of the present disclosure may also include, after incubating the mixture, obtaining a cell concentrate from the mixture, wherein obtaining a cell concentrate from the mixture includes one or more of the following steps: adding a fixation reagent to the mixture, adding a lysis reagent to the mixture, adding a permeabilization reagent to the mixture, staining the mixture with a staining reagent, and washing and concentrating the mixture to obtain the cell concentrate.

[0011] In certain embodiments, the staining reagent is a dry staining reagent, and the staining reagent may be provided in at least one pre-filled reagent container. In embodiments, the at least one dry reagent contained in the pre-filled dry reagent container comprises a monoclonal antibody reagent that allows for identification of extracellular and / or intracellular T cell markers.

[0012] In certain embodiments, the at least one antigen is a dry reagent, and the at least one antigen may be provided in at least one pre-filled reagent container. In some embodiments, the at least one dry antigen reagent contained in the pre-filled dry reagent container further comprises at least one dry staining reagent.

[0013] Some examples of extracellular T cell markers include CD2, CD3, CD4, CD8, CD11a, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, and CD161 (KLRB1). These include, but are not limited to, CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, OX40, and / or any combination thereof. Some examples of intracellular T cell markers include, but are not limited to, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-γ, TGFβ (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1), RORC (RORy), STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-α, and / or any combination thereof.

[0014] In certain embodiments, analyzing the mixture includes analyzing the presence of CD4+ T cell activation markers and / or CD8+ T cell activation markers, and / or the presence of intracellular cytokines or transcription factors. In certain embodiments, analyzing the mixture includes analyzing the presence of markers indicative of Th1-specific T cells, Th2-specific T cells, Th17-specific T cells, or Treg-specific T cells that respond to allergens during immunotherapy. Activation markers for CD4+ T cells may include, but are not limited to, CD69, CD154, CD137, and CD107a. Activation markers for CD8+ T cells may include, but are not limited to, CD69, CD154, CD137, and CD107a.

[0015] In certain embodiments, the at least one antigen comprises at least one virus-specific antigen, which may include, but is not limited to, a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS-CoV, MERS, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), monkeypox (MP), human immunodeficiency virus (HIV), or human papillomavirus (HPV). The full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 may include, but is not limited to, spike protein (S), spike protein subunit S1 (S1), spike protein subunit S2 (S2), spike protein (S+), spike protein RBD domain, nucleocapsid protein, membrane protein, envelope protein, and / or any combination thereof.

[0016] In certain embodiments, the at least one antigen may comprise at least one bacteria-specific antigen, at least one parasite-specific antigen, and / or at least one allergen-specific antigen. In certain embodiments, the at least one antigen is one or more proteins, variants, or fusion proteins isolated from bacterial lysates derived from infection with at least one bacterial strain. In certain embodiments, the at least one antigen is derived from tuberculosis, L. monocytogenes, E. coli, or Borrelia bacterium. In some embodiments, the at least one antigen is derived from one or more pollen or environmental allergens. A wide range of allergenic pollen species, including grasses, weeds, and trees, is known and available through literature. In some embodiments, the at least one antigen is derived from one or more food allergens. A wide range of allergenic food species, including dairy products, wheat, and nuts, is known and available through literature.

[0017] In certain embodiments, the method excludes the steps of peripheral blood mononuclear cell (PBMC) isolation, enrichment, and / or extraction.

[0018] In certain embodiments, the method for determining antigen-specific T cell response in a subject is used to analyze the antigen-specific T cell response of a subject before and / or after vaccination.In certain embodiments, the method for determining antigen-specific T cell response in a subject is used to monitor the immune status of a subject after a viral infection.In certain embodiments, the method for determining antigen-specific T cell response in a subject is used to monitor the immune status of a subject before a viral infection.

[0019] In certain embodiments, the present disclosure is directed to a kit for determining an antigen-specific T cell response in a subject, the kit comprising at least one antigen and at least one staining reagent. In embodiments, the at least one antigen and the at least one staining reagent are compatible with a whole blood sample of about 10 μL to about 10 mL. In certain embodiments, the at least one antigen is a dry reagent, and the at least one antigen may be provided in at least one pre-filled reagent container. In some embodiments, the at least one dry antigen reagent is contained within a pre-filled dry reagent container. In other embodiments, the at least one staining reagent is a dry staining reagent, and the at least one staining reagent may be provided in at least one pre-filled reagent container. In other embodiments, the pre-filled dry reagent container may comprise at least one dry staining reagent and at least one dry antigen reagent.

[0020] In some embodiments, kits can be prepared for determining antigen-specific T cell responses based on a cell type of interest or for specific uses (e.g., predicting response to therapeutic agents, diagnosing and prognosing various diseases or conditions). In some embodiments, the kits of the present disclosure can also be used to identify antigen-specific T cell responses to determine the effectiveness of treatments for cancer, allergies, autoimmunity, bacterial infections, viral infections, and / or parasitic infections. The kits of the present disclosure can also be used to identify antigen-specific T cell responses to determine immunity to cancer, allergies, bacterial infections, viral infections, and / or parasitic infections, and / or immunity resulting from cancer, allergies, bacterial infections, viral infections, and / or parasitic infections. In some embodiments, the kits of the present disclosure can also be used to identify antigen-specific T cell responses to determine a subject's recovery from treatments for cancer, allergies, bacterial infections, viral infections, and / or parasitic infections.

[0021] In certain embodiments, kits for determining antigen-specific T cell responses may include staining reagents specific for detecting extracellular markers of T cell responses. In certain embodiments, kits for determining antigen-specific T cell responses may include staining reagents specific for intracellular markers of T cell responses, including cytokine-specific markers and transcription factor-specific markers. Staining reagents include extracellular T cell markers (CD2, CD3, CD4, CD8, CD11a, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1)). Examples of antibodies that may be included include antibody reagents for identification of CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, OX40, and / or any combination thereof. Staining reagents may also include antibody reagents for identification of intracellular T cell markers (including, but not limited to, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-γ, TGFβ (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1), RORC (RORy), STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-α, or any combination thereof).

[0022] In certain embodiments, a kit for determining antigen-specific T cell responses may include at least one pre-filled reagent container, a lysis reagent, a permeabilization reagent, a fixation reagent, or any combination thereof. In certain embodiments, the at least one antigen or the at least one staining reagent is a dry reagent. In some embodiments, the at least one dry reagent is provided in at least one pre-filled reagent container.

[0023] In certain embodiments, a kit for determining antigen-specific T cell responses may include at least one virus-specific antigen. The virus-specific antigen may be, but is not limited to, a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS-CoV, MERS, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), monkeypox (MP), or human papillomavirus (HPV). The virus-specific antigen may be, but is not limited to, a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, including spike protein (S), spike protein subunit S1 (S1), spike protein subunit S2 (S2), spike protein (S+), spike protein RBD domain, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof.

[0024] In certain embodiments, the kit for determining an antigen-specific T cell response may comprise at least one parasite-specific antigen, at least one allergen-specific antigen, or at least one bacteria-specific antigen. In certain embodiments, the kit for determining an antigen-specific T cell response may be used to analyze a subject's antigen-specific T cell response before and / or after vaccination, or to monitor a subject's immune status before or after a viral infection event.

[0025] In certain embodiments, kits for determining antigen-specific T cell responses may include staining reagents specific for characterizing the phenotype of various T cell subsets (including, but not limited to, naive T cells, memory T cells, regulatory T cells, helper T cells, cytotoxic T cells, and / or natural killer T cells).

[0026] In certain embodiments, the disclosed kits include a combination of a T cell activation marker for an extracellular marker and a T cell activation marker for an intracellular marker. The disclosed kits may also include one or more additional vials, tubes, inhibitors, modulators, therapeutic agents, fixatives, buffers, physical devices, and software for performing analysis of the cells (e.g., by flow cytometry).

[0027] In one aspect, the present disclosure provides a method for determining an antiviral immune response to SARS-CoV-2 in a subject, the method comprising: contacting a whole blood sample from the subject with at least one antigen to form a mixture; and contacting the whole blood sample with at least one staining reagent, wherein the contacting of the whole blood sample with the at least one staining reagent is performed prior to, simultaneously with, or after the contacting of the whole blood sample with the at least one antigen to form a mixture, or any combination thereof. In an embodiment, the mixture is analyzed by flow cytometry. In an embodiment, the whole blood sample has a volume of about 10 μL to about 10 mL, or any volume between about 10 μL and about 10 mL. In a specific embodiment, the whole blood sample is greater than about 10 μL but less than about 1 mL. In certain embodiments, the whole blood sample is between about 200 μL and about 1 ml.

[0028] In some embodiments, the at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length proteins and / or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, or any combination thereof. In some embodiments, at least one reagent is added to the mixture formed by contacting the whole blood sample with at least one antigen. In certain embodiments, analyzing the mixture comprises identifying a T cell response to contacting the whole blood sample with at least one antigen.

[0029] In certain embodiments, the disclosed methods further comprise incubating the mixture formed by contacting the whole blood sample with at least one antigen prior to contacting the whole blood with at least one staining reagent, and / or after contacting the whole blood sample with at least one antigen prior to contacting the whole blood with at least one staining reagent. In certain embodiments, incubating the mixture comprises incubation at about 37° C. for about 30 minutes to about 24 hours.

[0030] The method of the present disclosure may also include, after incubating the mixture, obtaining a cell concentrate from the mixture, wherein obtaining a cell concentrate from the mixture includes one or more of the following steps: adding a fixation reagent to the mixture, adding a lysis reagent to the mixture, adding a permeabilization reagent to the mixture, staining the mixture with a staining reagent, and washing and concentrating the mixture to obtain the cell concentrate.

[0031] In certain embodiments, the staining reagent is a dry staining reagent, and the staining reagent may be provided in at least one pre-filled reagent container. In embodiments, the at least one dry reagent contained in the pre-filled dry reagent container comprises a monoclonal antibody reagent that allows for identification of extracellular and / or intracellular T cell markers.

[0032] Some examples of extracellular T cell markers include CD2, CD3, CD4, CD8, CD11a, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, and CD161 (KLRB1). These include, but are not limited to, CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, OX40, and / or any combination thereof. Some examples of intracellular T cell markers include, but are not limited to, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-γ, TGFβ (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1), RORC (RORy), STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-α, and / or any combination thereof.

[0033] In certain embodiments, analyzing the mixture includes analyzing the presence of CD4+ T cell activation markers and / or CD8+ T cell activation markers, and / or the presence of intracellular cytokines and / or transcription factors. CD4+ T cell activation markers may include, but are not limited to, CD69, CD154, CD137, and CD107a. CD8+ T cell activation markers may include, but are not limited to, CD69, CD154, CD137, and CD107a.

[0034] In certain embodiments, the at least one antigen comprises at least one virus-specific antigen. The at least one virus-specific antigen may include, but is not limited to, a full-length protein, a protein derivative, a peptide, or a peptide pool derived from SARS-CoV-2. The full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 may include, but is not limited to, the spike protein (S), the spike protein subunit S1 (S1), the spike protein subunit S2 (S2), the spike protein (S+), the spike protein RBD domain, the nucleocapsid protein, the membrane protein, the envelope protein, and / or any combination thereof.

[0035] In certain embodiments, the method excludes the steps of peripheral blood mononuclear cell (PBMC) isolation, enrichment, and / or extraction.

[0036] In certain embodiments, the method for determining an antiviral immune response to SARS-CoV-2 in a subject is used to analyze the subject's antigen-specific T cell response before and / or after vaccination. In certain embodiments, the method for determining an antiviral immune response to SARS-CoV-2 in a subject is used to monitor the subject's immune status after an event of viral infection. In certain embodiments, the method for determining an antiviral immune response to SARS-CoV-2 in a subject is used to monitor the subject's immune status before an event of viral infection. [Brief explanation of the drawings]

[0037] [Figure 1A] FIG. 1 shows exemplary workflows for the extracellular staining workflow (FIG. 1A and FIG. 1B), intracellular staining workflow I (FIG. 1C), and intracellular staining workflow II (FIG. 1D), which are discussed in Examples 1-6. [Figure 1B] FIG. 1 shows exemplary workflows for the extracellular staining workflow (FIG. 1A and FIG. 1B), intracellular staining workflow I (FIG. 1C), and intracellular staining workflow II (FIG. 1D), which are discussed in Examples 1-6. [Figure 1C] FIG. 1 shows exemplary workflows for the extracellular staining workflow (FIG. 1A and FIG. 1B), intracellular staining workflow I (FIG. 1C), and intracellular staining workflow II (FIG. 1D), which are discussed in Examples 1-6. [Figure 1D] FIG. 1 shows exemplary workflows for the extracellular staining workflow (FIG. 1A and FIG. 1B), intracellular staining workflow I (FIG. 1C), and intracellular staining workflow II (FIG. 1D), which are discussed in Examples 1-6.

[0038] [Figure 2A]Figure 2 shows data and information obtained from two blood donor samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 2A-2B show flow cytometry gating for the two donor samples. Figures 2C-2D show flow cytometry analysis of CD4+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. Figures 2E-2F show flow cytometry analysis of CD8+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. [Figure 2B] Figure 2 shows data and information obtained from two blood donor samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 2A-2B show flow cytometry gating for the two donor samples. Figures 2C-2D show flow cytometry analysis of CD4+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. Figures 2E-2F show flow cytometry analysis of CD8+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. [Figure 2C]Figure 2 shows data and information obtained from two blood donor samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 2A-2B show flow cytometry gating for the two donor samples. Figures 2C-2D show flow cytometry analysis of CD4+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. Figures 2E-2F show flow cytometry analysis of CD8+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. [Figure 2D] Figure 2 shows data and information obtained from two blood donor samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 2A-2B show flow cytometry gating for the two donor samples. Figures 2C-2D show flow cytometry analysis of CD4+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. Figures 2E-2F show flow cytometry analysis of CD8+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. [Figure 2E]Figure 2 shows data and information obtained from two blood donor samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 2A-2B show flow cytometry gating for the two donor samples. Figures 2C-2D show flow cytometry analysis of CD4+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. Figures 2E-2F show flow cytometry analysis of CD8+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. [Figure 2F] Figure 2 shows data and information obtained from two blood donor samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 2A-2B show flow cytometry gating for the two donor samples. Figures 2C-2D show flow cytometry analysis of CD4+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX. Figures 2E-2F show flow cytometry analysis of CD8+ T cells from the two donor samples using a negative control (Neg), one activation condition for cytomegalovirus (CMV), and one activation condition for CEFX.

[0039] [Figure 3A]Figure 3 shows data and information obtained from whole blood and PBMC samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 3A-3B show flow cytometry gating for the two samples. Figures 3C-3D show flow cytometry analysis of CD4+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3E-3F show flow cytometry analysis of CD8+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3G-3H show flow cytometry analysis of NK cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3I-3J show flow cytometry analysis of NKT cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. [Figure 3B]Figure 3 shows data and information obtained from whole blood and PBMC samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 3A-3B show flow cytometry gating for the two samples. Figures 3C-3D show flow cytometry analysis of CD4+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3E-3F show flow cytometry analysis of CD8+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3G-3H show flow cytometry analysis of NK cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3I-3J show flow cytometry analysis of NKT cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. [Figure 3C]Figure 3 shows data and information obtained from whole blood and PBMC samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 3A-3B show flow cytometry gating for the two samples. Figures 3C-3D show flow cytometry analysis of CD4+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3E-3F show flow cytometry analysis of CD8+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3G-3H show flow cytometry analysis of NK cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3I-3J show flow cytometry analysis of NKT cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. [Figure 3D]Figure 3 shows data and information obtained from whole blood and PBMC samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 3A-3B show flow cytometry gating for the two samples. Figures 3C-3D show flow cytometry analysis of CD4+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3E-3F show flow cytometry analysis of CD8+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3G-3H show flow cytometry analysis of NK cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3I-3J show flow cytometry analysis of NKT cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. [Figure 3E]Figure 3 shows data and information obtained from whole blood and PBMC samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 3A-3B show flow cytometry gating for the two samples. Figures 3C-3D show flow cytometry analysis of CD4+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3E-3F show flow cytometry analysis of CD8+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3G-3H show flow cytometry analysis of NK cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3I-3J show flow cytometry analysis of NKT cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. [Figure 3F]Figure 3 shows data and information obtained from whole blood and PBMC samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 3A-3B show flow cytometry gating for the two samples. Figures 3C-3D show flow cytometry analysis of CD4+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3E-3F show flow cytometry analysis of CD8+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3G-3H show flow cytometry analysis of NK cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3I-3J show flow cytometry analysis of NKT cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. [Figure 3G]Figure 3 shows data and information obtained from whole blood and PBMC samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 3A-3B show flow cytometry gating for the two samples. Figures 3C-3D show flow cytometry analysis of CD4+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3E-3F show flow cytometry analysis of CD8+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3G-3H show flow cytometry analysis of NK cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3I-3J show flow cytometry analysis of NKT cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. [Figure 3H]Figure 3 shows data and information obtained from whole blood and PBMC samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 3A-3B show flow cytometry gating for the two samples. Figures 3C-3D show flow cytometry analysis of CD4+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3E-3F show flow cytometry analysis of CD8+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3G-3H show flow cytometry analysis of NK cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3I-3J show flow cytometry analysis of NKT cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. [Figure 3I]Figure 3 shows data and information obtained from whole blood and PBMC samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 3A-3B show flow cytometry gating for the two samples. Figures 3C-3D show flow cytometry analysis of CD4+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3E-3F show flow cytometry analysis of CD8+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3G-3H show flow cytometry analysis of NK cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3I-3J show flow cytometry analysis of NKT cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. [Figure 3J]Figure 3 shows data and information obtained from whole blood and PBMC samples using an embodiment of the extracellular staining workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137. Figures 3A-3B show flow cytometry gating for the two samples. Figures 3C-3D show flow cytometry analysis of CD4+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3E-3F show flow cytometry analysis of CD8+ T cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3G-3H show flow cytometry analysis of NK cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike. Figures 3I-3J show flow cytometry analysis of NKT cells from the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for spike.

[0040] [Figure 4A]Figure 4 shows data and information obtained from a whole blood sample using an embodiment of the extracellular staining workflow to generate antigen-specific T cells for intact protein versus peptide. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and / or CD154. Figure 4A shows flow cytometry gating of the whole blood sample. Figure 4B shows flow cytometry analysis of CD4+ T cells from the whole blood sample using a negative control (Neg), one activation condition for spiked JPT, one activation condition for intact spike (7.2 pmol concentration), one activation condition for intact spike (14.4 pmol concentration), and one activation condition for intact spike (28.8 pmol concentration). Figure 4C shows flow cytometry analysis of CD8+ T cells from whole blood samples using a negative control (Neg), one activation condition for spiked JPT, one activation condition for intact spike (7.2 pmol concentration), one activation condition for intact spike (14.4 pmol concentration), and one activation condition for intact spike (28.8 pmol concentration). [Figure 4B]Figure 4 shows data and information obtained from a whole blood sample using an embodiment of the extracellular staining workflow to generate antigen-specific T cells for intact protein versus peptide. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and / or CD154. Figure 4A shows flow cytometry gating of the whole blood sample. Figure 4B shows flow cytometry analysis of CD4+ T cells from the whole blood sample using a negative control (Neg), one activation condition for spiked JPT, one activation condition for intact spike (7.2 pmol concentration), one activation condition for intact spike (14.4 pmol concentration), and one activation condition for intact spike (28.8 pmol concentration). Figure 4C shows flow cytometry analysis of CD8+ T cells from whole blood samples using a negative control (Neg), one activation condition for spiked JPT, one activation condition for intact spike (7.2 pmol concentration), one activation condition for intact spike (14.4 pmol concentration), and one activation condition for intact spike (28.8 pmol concentration). [Figure 4C]Figure 4 shows data and information obtained from a whole blood sample using an embodiment of the extracellular staining workflow to generate antigen-specific T cells for intact protein versus peptide. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and / or CD154. Figure 4A shows flow cytometry gating of the whole blood sample. Figure 4B shows flow cytometry analysis of CD4+ T cells from the whole blood sample using a negative control (Neg), one activation condition for spiked JPT, one activation condition for intact spike (7.2 pmol concentration), one activation condition for intact spike (14.4 pmol concentration), and one activation condition for intact spike (28.8 pmol concentration). Figure 4C shows flow cytometry analysis of CD8+ T cells from whole blood samples using a negative control (Neg), one activation condition for spiked JPT, one activation condition for intact spike (7.2 pmol concentration), one activation condition for intact spike (14.4 pmol concentration), and one activation condition for intact spike (28.8 pmol concentration).

[0041] [Figure 5A]Figure 5 shows data and information obtained from a whole blood sample using an embodiment of an extracellular staining workflow to generate antigen-specific T cells for intact cells using antigen in either a dry or liquid format. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and / or CD154. Figure 5A shows flow cytometry gating of the whole blood sample. Figure 5B shows flow cytometry analysis of CD4+ T cells from the whole blood sample using a negative control (Neg), one activation condition for liquid spike (Liq), one activation condition for dry spike (Dry), one activation condition for liquid EMN (Liq), and one activation condition for dry EMN (Dry). Figure 5C shows flow cytometry analysis of CD84+ T cells in whole blood samples using a negative control (Neg), one activation condition for liquid spike (Liq), one activation condition for dry spike (Dry), one activation condition for liquid EMN (Liq), and one activation condition for dry EMN (Dry). [Figure 5B]Figure 5 shows data and information obtained from a whole blood sample using an embodiment of an extracellular staining workflow to generate antigen-specific T cells for intact cells using antigen in either a dry or liquid format. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and / or CD154. Figure 5A shows flow cytometry gating of the whole blood sample. Figure 5B shows flow cytometry analysis of CD4+ T cells from the whole blood sample using a negative control (Neg), one activation condition for liquid spike (Liq), one activation condition for dry spike (Dry), one activation condition for liquid EMN (Liq), and one activation condition for dry EMN (Dry). Figure 5C shows flow cytometry analysis of CD84+ T cells in whole blood samples using a negative control (Neg), one activation condition for liquid spike (Liq), one activation condition for dry spike (Dry), one activation condition for liquid EMN (Liq), and one activation condition for dry EMN (Dry). [Figure 5C]Figure 5 shows data and information obtained from a whole blood sample using an embodiment of an extracellular staining workflow to generate antigen-specific T cells for intact cells using antigen in either a dry or liquid format. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and / or CD154. Figure 5A shows flow cytometry gating of the whole blood sample. Figure 5B shows flow cytometry analysis of CD4+ T cells from the whole blood sample using a negative control (Neg), one activation condition for liquid spike (Liq), one activation condition for dry spike (Dry), one activation condition for liquid EMN (Liq), and one activation condition for dry EMN (Dry). Figure 5C shows flow cytometry analysis of CD84+ T cells in whole blood samples using a negative control (Neg), one activation condition for liquid spike (Liq), one activation condition for dry spike (Dry), one activation condition for liquid EMN (Liq), and one activation condition for dry EMN (Dry).

[0042] [Figure 6A]Figure 6 shows data and information obtained from whole blood samples using an embodiment of the intracellular staining workflow using antigens in either a dry or liquid format. The cells were stained for CD3, CD4, CD8, IFNγ, CD45, CD154, IL-4, TNFα, and / or IL-17. Figure 6A shows flow cytometry analysis of CD4+ T cells from a sample from a patient with rheumatoid arthritis (RA) using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dried antigen (Dry CMV), one activation condition for Dry CEFX dried antigen, and one activation condition for Dry PMA / ionomycin dried antigen (PMA / Iono). FIG. 6B shows flow cytometry analysis of CD8+ T cells in samples from patients with rheumatoid arthritis (RA) using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono). FIG. 6C shows flow cytometry analysis of CD4+ T cells from samples from healthy patients using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono).Figure 6D shows flow cytometry analysis of CD8+ T cells from samples from healthy patients using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono). [Figure 6B]Figure 6 shows data and information obtained from whole blood samples using an embodiment of the intracellular staining workflow using antigens in either a dry or liquid format. The cells were stained for CD3, CD4, CD8, IFNγ, CD45, CD154, IL-4, TNFα, and / or IL-17. Figure 6A shows flow cytometry analysis of CD4+ T cells from a sample from a patient with rheumatoid arthritis (RA) using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dried antigen (Dry CMV), one activation condition for Dry CEFX dried antigen, and one activation condition for Dry PMA / ionomycin dried antigen (PMA / Iono). FIG. 6B shows flow cytometry analysis of CD8+ T cells in samples from patients with rheumatoid arthritis (RA) using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono). FIG. 6C shows flow cytometry analysis of CD4+ T cells from samples from healthy patients using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono).Figure 6D shows flow cytometry analysis of CD8+ T cells from samples from healthy patients using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono). [Figure 6C]Figure 6 shows data and information obtained from whole blood samples using an embodiment of the intracellular staining workflow using antigens in either a dry or liquid format. The cells were stained for CD3, CD4, CD8, IFNγ, CD45, CD154, IL-4, TNFα, and / or IL-17. Figure 6A shows flow cytometry analysis of CD4+ T cells from a sample from a patient with rheumatoid arthritis (RA) using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dried antigen (Dry CMV), one activation condition for Dry CEFX dried antigen, and one activation condition for Dry PMA / ionomycin dried antigen (PMA / Iono). FIG. 6B shows flow cytometry analysis of CD8+ T cells in samples from patients with rheumatoid arthritis (RA) using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono). FIG. 6C shows flow cytometry analysis of CD4+ T cells from samples from healthy patients using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono).Figure 6D shows flow cytometry analysis of CD8+ T cells from samples from healthy patients using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono). [Figure 6D]Figure 6 shows data and information obtained from whole blood samples using an embodiment of the intracellular staining workflow using antigens in either a dry or liquid format. The cells were stained for CD3, CD4, CD8, IFNγ, CD45, CD154, IL-4, TNFα, and / or IL-17. Figure 6A shows flow cytometry analysis of CD4+ T cells from a sample from a patient with rheumatoid arthritis (RA) using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dried antigen (Dry CMV), one activation condition for Dry CEFX dried antigen, and one activation condition for Dry PMA / ionomycin dried antigen (PMA / Iono). FIG. 6B shows flow cytometry analysis of CD8+ T cells in samples from patients with rheumatoid arthritis (RA) using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono). FIG. 6C shows flow cytometry analysis of CD4+ T cells from samples from healthy patients using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono).Figure 6D shows flow cytometry analysis of CD8+ T cells from samples from healthy patients using a negative control (CTRL), one activation condition for P8A liquid antigen, one activation condition for P8B liquid antigen, one activation condition for PAD2 liquid antigen, one activation condition for PAD4 liquid antigen, one activation condition for cytomegalovirus dry antigen (Dry CMV), one activation condition for Dry CEFX dry antigen, and one activation condition for Dry PMA / ionomycin dry antigen (PMA / Iono).

[0043] [Figure 7A]Figure 7 shows data and information obtained from two blood donor samples using an embodiment of the intracellular staining workflow II. The cells were stained for CD3, CD4, CD8, IFNγ, CD19, CD45, CD154, IL-4, IL-10, TNFα, and / or IL-17. Figure 7A shows flow cytometry analysis of CD4+ T cells from the samples using a negative control (NEG), one activation condition for the SARS-CoV-2 spike glycoprotein antigen, one activation condition for the global CMV antigen, one activation condition for the CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for the PMA / ionomycin antigen (PMA / Iono). Figure 7B shows flow cytometry analysis of CD8+ T cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7C shows flow cytometry analysis of CD19+ B cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7D shows flow cytometry analysis of NK cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for CMV global antigen, one activation condition for CMV pp65 antigen, one activation condition for allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono).Figures 7E-7H show significant differences in aggregate mean marker expression across multiple whole blood samples caused by activator type: Figure 7E shows significant differences for CD4+ cells, Figure 7F shows CD8+ cells, Figure 7G shows CD19+ B cells, and Figure 7H shows NK cells. [Figure 7B]Figure 7 shows data and information obtained from two blood donor samples using an embodiment of the intracellular staining workflow II. The cells were stained for CD3, CD4, CD8, IFNγ, CD19, CD45, CD154, IL-4, IL-10, TNFα, and / or IL-17. Figure 7A shows flow cytometry analysis of CD4+ T cells from the samples using a negative control (NEG), one activation condition for the SARS-CoV-2 spike glycoprotein antigen, one activation condition for the global CMV antigen, one activation condition for the CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for the PMA / ionomycin antigen (PMA / Iono). Figure 7B shows flow cytometry analysis of CD8+ T cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7C shows flow cytometry analysis of CD19+ B cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7D shows flow cytometry analysis of NK cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for CMV global antigen, one activation condition for CMV pp65 antigen, one activation condition for allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono).Figures 7E-7H show significant differences in aggregate mean marker expression across multiple whole blood samples caused by activator type: Figure 7E shows significant differences for CD4+ cells, Figure 7F shows CD8+ cells, Figure 7G shows CD19+ B cells, and Figure 7H shows NK cells. [Figure 7C]Figure 7 shows data and information obtained from two blood donor samples using an embodiment of the intracellular staining workflow II. The cells were stained for CD3, CD4, CD8, IFNγ, CD19, CD45, CD154, IL-4, IL-10, TNFα, and / or IL-17. Figure 7A shows flow cytometry analysis of CD4+ T cells from the samples using a negative control (NEG), one activation condition for the SARS-CoV-2 spike glycoprotein antigen, one activation condition for the global CMV antigen, one activation condition for the CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for the PMA / ionomycin antigen (PMA / Iono). Figure 7B shows flow cytometry analysis of CD8+ T cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7C shows flow cytometry analysis of CD19+ B cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7D shows flow cytometry analysis of NK cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for CMV global antigen, one activation condition for CMV pp65 antigen, one activation condition for allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono).Figures 7E-7H show significant differences in aggregate mean marker expression across multiple whole blood samples caused by activator type: Figure 7E shows significant differences for CD4+ cells, Figure 7F shows CD8+ cells, Figure 7G shows CD19+ B cells, and Figure 7H shows NK cells. [Figure 7D]Figure 7 shows data and information obtained from two blood donor samples using an embodiment of the intracellular staining workflow II. The cells were stained for CD3, CD4, CD8, IFNγ, CD19, CD45, CD154, IL-4, IL-10, TNFα, and / or IL-17. Figure 7A shows flow cytometry analysis of CD4+ T cells from the samples using a negative control (NEG), one activation condition for the SARS-CoV-2 spike glycoprotein antigen, one activation condition for the global CMV antigen, one activation condition for the CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for the PMA / ionomycin antigen (PMA / Iono). Figure 7B shows flow cytometry analysis of CD8+ T cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7C shows flow cytometry analysis of CD19+ B cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7D shows flow cytometry analysis of NK cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for CMV global antigen, one activation condition for CMV pp65 antigen, one activation condition for allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono).Figures 7E-7H show significant differences in aggregate mean marker expression across multiple whole blood samples caused by activator type: Figure 7E shows significant differences for CD4+ cells, Figure 7F shows CD8+ cells, Figure 7G shows CD19+ B cells, and Figure 7H shows NK cells. [Figure 7E]Figure 7 shows data and information obtained from two blood donor samples using an embodiment of the intracellular staining workflow II. The cells were stained for CD3, CD4, CD8, IFNγ, CD19, CD45, CD154, IL-4, IL-10, TNFα, and / or IL-17. Figure 7A shows flow cytometry analysis of CD4+ T cells from the samples using a negative control (NEG), one activation condition for the SARS-CoV-2 spike glycoprotein antigen, one activation condition for the global CMV antigen, one activation condition for the CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for the PMA / ionomycin antigen (PMA / Iono). Figure 7B shows flow cytometry analysis of CD8+ T cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7C shows flow cytometry analysis of CD19+ B cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7D shows flow cytometry analysis of NK cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for CMV global antigen, one activation condition for CMV pp65 antigen, one activation condition for allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono).Figures 7E-7H show significant differences in aggregate mean marker expression across multiple whole blood samples caused by activator type: Figure 7E shows significant differences for CD4+ cells, Figure 7F shows CD8+ cells, Figure 7G shows CD19+ B cells, and Figure 7H shows NK cells. [Figure 7F]Figure 7 shows data and information obtained from two blood donor samples using an embodiment of the intracellular staining workflow II. The cells were stained for CD3, CD4, CD8, IFNγ, CD19, CD45, CD154, IL-4, IL-10, TNFα, and / or IL-17. Figure 7A shows flow cytometry analysis of CD4+ T cells from the samples using a negative control (NEG), one activation condition for the SARS-CoV-2 spike glycoprotein antigen, one activation condition for the global CMV antigen, one activation condition for the CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for the PMA / ionomycin antigen (PMA / Iono). Figure 7B shows flow cytometry analysis of CD8+ T cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7C shows flow cytometry analysis of CD19+ B cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7D shows flow cytometry analysis of NK cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for CMV global antigen, one activation condition for CMV pp65 antigen, one activation condition for allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono).Figures 7E-7H show significant differences in aggregate mean marker expression across multiple whole blood samples caused by activator type: Figure 7E shows significant differences for CD4+ cells, Figure 7F shows CD8+ cells, Figure 7G shows CD19+ B cells, and Figure 7H shows NK cells. [Figure 7G]Figure 7 shows data and information obtained from two blood donor samples using an embodiment of the intracellular staining workflow II. The cells were stained for CD3, CD4, CD8, IFNγ, CD19, CD45, CD154, IL-4, IL-10, TNFα, and / or IL-17. Figure 7A shows flow cytometry analysis of CD4+ T cells from the samples using a negative control (NEG), one activation condition for the SARS-CoV-2 spike glycoprotein antigen, one activation condition for the global CMV antigen, one activation condition for the CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for the PMA / ionomycin antigen (PMA / Iono). Figure 7B shows flow cytometry analysis of CD8+ T cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7C shows flow cytometry analysis of CD19+ B cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7D shows flow cytometry analysis of NK cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for CMV global antigen, one activation condition for CMV pp65 antigen, one activation condition for allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono).Figures 7E-7H show significant differences in aggregate mean marker expression across multiple whole blood samples caused by activator type: Figure 7E shows significant differences for CD4+ cells, Figure 7F shows CD8+ cells, Figure 7G shows CD19+ B cells, and Figure 7H shows NK cells. [Figure 7H]Figure 7 shows data and information obtained from two blood donor samples using an embodiment of the intracellular staining workflow II. The cells were stained for CD3, CD4, CD8, IFNγ, CD19, CD45, CD154, IL-4, IL-10, TNFα, and / or IL-17. Figure 7A shows flow cytometry analysis of CD4+ T cells from the samples using a negative control (NEG), one activation condition for the SARS-CoV-2 spike glycoprotein antigen, one activation condition for the global CMV antigen, one activation condition for the CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for the PMA / ionomycin antigen (PMA / Iono). Figure 7B shows flow cytometry analysis of CD8+ T cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7C shows flow cytometry analysis of CD19+ B cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for global CMV antigen, one activation condition for CMV pp65 antigen, one activation condition for an allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono). Figure 7D shows flow cytometry analysis of NK cells from samples using a negative control (NEG), one activation condition for SARS-CoV-2 spike glycoprotein antigen, one activation condition for CMV global antigen, one activation condition for CMV pp65 antigen, one activation condition for allergen mix, and one activation condition for PMA / ionomycin antigen (PMA / Iono).Figures 7E-7H show significant differences in aggregate mean marker expression across multiple whole blood samples caused by activator type: Figure 7E shows significant differences for CD4+ cells, Figure 7F shows CD8+ cells, Figure 7G shows CD19+ B cells, and Figure 7H shows NK cells.

[0044] [Figure 8A] Figure 8 shows data and information obtained from three blood donor samples using an embodiment of the intracellular staining workflow II. The whole blood cells were stained for CD3, CD4, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137 and were either unactivated, activated with spiked JPT, or activated with CEFX. Figure 8A shows flow cytometry gating for the three donor samples. Figure 8B shows flow cytometry analysis of CD4+ T cells from the three whole blood samples using a negative control (Neg), one activation condition with spiked JPT, and one activation condition with CEFX. Figure 8C shows flow cytometry analysis of CD4+ T cells from the three whole blood samples using a negative control (Neg), one activation condition with spiked JPT, and one activation condition with CEFX. [Figure 8B]Figure 8 shows data and information obtained from three blood donor samples using an embodiment of the intracellular staining workflow II. The whole blood cells were stained for CD3, CD4, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137 and were either unactivated, activated with spiked JPT, or activated with CEFX. Figure 8A shows flow cytometry gating for the three donor samples. Figure 8B shows flow cytometry analysis of CD4+ T cells from the three whole blood samples using a negative control (Neg), one activation condition with spiked JPT, and one activation condition with CEFX. Figure 8C shows flow cytometry analysis of CD4+ T cells from the three whole blood samples using a negative control (Neg), one activation condition with spiked JPT, and one activation condition with CEFX. [Figure 8C] Figure 8 shows data and information obtained from three blood donor samples using an embodiment of the intracellular staining workflow II. The whole blood cells were stained for CD3, CD4, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137 and were either unactivated, activated with spiked JPT, or activated with CEFX. Figure 8A shows flow cytometry gating for the three donor samples. Figure 8B shows flow cytometry analysis of CD4+ T cells from the three whole blood samples using a negative control (Neg), one activation condition with spiked JPT, and one activation condition with CEFX. Figure 8C shows flow cytometry analysis of CD4+ T cells from the three whole blood samples using a negative control (Neg), one activation condition with spiked JPT, and one activation condition with CEFX. DETAILED DESCRIPTION OF THE INVENTION

[0045] (Detailed explanation) While the concepts of the present disclosure have been shown and described in detail in the drawings and specification herein, it is understood that the results in the drawings and their description are to be considered as examples and not as limiting in character; it is understood that only exemplary embodiments have been shown and described, and that all changes and modifications that come within the scope of this disclosure are desired to be protected.

[0046] Unless otherwise defined, scientific and technical nomenclature has the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0047] It will be appreciated by those skilled in the art that other suitable modifications and adaptations to the methods described herein will be readily apparent from the disclosure description contained herein in light of the information known to those skilled in the art, and may be made without departing from the scope of the disclosure or any of its embodiments. Having now described the disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included herein for illustrative purposes only and are not intended to be limitations of the disclosure.

[0048] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0049] (definition) As used herein, "g" refers to grams; "L" refers to liters; and "mg" refers to milligrams (10 -3 "mL" or "cc" indicates milliliters (10 -3 liters). 1 "μL" is 1 microliter (10 -6 The unit of temperature used herein is degrees Celsius (°C).

[0050] The term "about" is used in conjunction with a numerical value to include normal variations in measurement expected by one of ordinary skill in the art, and is understood to have the same meaning as "approximately" and include the typical tolerance of error for the stated value (e.g., ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%). Whether modified by the term "about," the claims include the equivalent of the quantity.

[0051] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a method" includes having two or more methods that are either the same as each other or different from each other. It should also be noted that the term "or" is commonly used to include "and / or" within its meaning unless the context clearly dictates otherwise. As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0052] For the sake of brevity and simplicity, any range of values ​​given herein contemplates all values ​​within that range and should be interpreted as support for any claim reciting any subrange having endpoints that are real values ​​within the range specified. As a hypothetical illustrative example, the disclosure of a range of 1 to 5 herein should be interpreted as support for a claim to any of the following ranges: 1-5; 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3-4; and 4-5.

[0053] The term "substantially" is used herein to indicate the inherent degree of uncertainty that may result from any quantitative comparison, value, measurement, or other representation. The term "substantially" is also used herein to indicate the extent to which a quantitative representation may vary from the stated recitation without resulting in a change in the basic functionality of the subject matter under consideration.

[0054] As used herein, the terms "comprise," "comprises," and "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0055] The term "antigen," as used herein, is defined as any substance or molecule (e.g., a polypeptide) capable of eliciting a specific immune response. An antigen may be, be derived from, or be immunologically cross-reactive with an infectious agent, epitope, biomolecule, cell, or tissue associated with infectious disease, cancer, autoimmune disease, allergy, or any other condition in which stimulation of an antigen-specific immune response is involved or is desirable or beneficial. An antigen may be a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and / or partial proteins, a protein derivative, a single epitope, a partial proteome, an unprocessed extract, a mixture of polypeptides and other biomolecules, or any combination thereof.

[0056] As used herein, the term "extracellular T cell marker" includes any marker or molecule characteristic of the plasma membrane of a T cell, or any marker or molecule that is partially or completely exposed on the outer surface of that plasma membrane, and can be accessed without modulating cell permeability. As used herein, extracellular T cell markers include extracellular T cell markers expressed on resting cells, activated cells, responding cells, and / or diseased cells, and as used herein, extracellular T cell markers include CD2, CD3, CD4, CD8, CD11a, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), and the like. ), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1), CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, OX40, and / or any combination thereof.

[0057] As used herein, the term "intracellular T cell marker" includes any marker or molecule located within a T-0 cell, including, but not limited to, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26, IFN-γ, TGFβ (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1), RORC (RORy), STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-α, and / or any combination thereof. Intracellular T cell markers may include cytokines and transcription factors. Intracellular and extracellular T cell markers can be detected in the same cell. Detection of some intracellular T cell markers can require disruption of the integrity of the cell membrane or other manipulation (e.g., preparation and / or treatment of the cell with one or more permeabilization reagents described herein).

[0058] The term "full-length protein" as used herein includes any amino acid sequence that represents the complete amino acid sequence of a given nucleotide sequence. Those skilled in the art will understand the term "full-length protein" as used herein. GenBank, the sequence database of the National Institutes of Health (登録商標) can be used as a reference to identify the full-length protein sequence, which is available at https: / / www.ncbi.nlm.nih.gov / genbank / .

[0059] As used herein, the term "peptide pool" includes a combination of mixed peptides of the same or different lengths (often from about 2 amino acids to about 50 amino acid residues). The peptides within the peptide pool may include synthetic peptides and / or naturally occurring peptides. The peptide pool may contain overlapping peptides derived from an antigen. Peptide pools are standards for stimulation of antigen-specific T cells in functional T cell assays (e.g., ELISpot and ICS) and may be used with the present disclosure.

[0060] As used herein, the term "protein derivative" includes nonisomorphous derivative proteins.

[0061] As used herein, the term "dry reagent" includes reagents that are provided in dry rather than liquid form, which often do not require refrigeration and are more stable.

[0062] As used herein, the term "dry staining reagent" includes a staining reagent that allows target identification (e.g., a monoclonal antibody that allows T cells to be identified based on their interaction with a specific target marker). Dry staining reagents are provided in a dry form rather than a liquid form, and often do not require refrigeration and are more stable. An example of a dry staining reagent is the DURA Innovations reagent available from Beckman Coulter (https: / / www.beckman.com / resources / technologies / dura-innovations).

[0063] The term "allergen mix" as used herein includes allergenic raw extracts, recombinant allergens or purified allergens, whether in the form of native proteins, whole proteins, undigested or denatured proteins, whole denatured proteins or denatured proteins digested with enzymes, or synthetic peptides of allergenic proteins.

[0064] As used herein, the term "spike" includes purified or recombinant SARS-CoV-2 proteins, or synthetic peptides (either as individual peptides or pools of several peptides).

[0065] As used herein, the term "spike JPT" includes synthetic peptide pools of entire or specific domains of SARS-CoV-2 proteins. An example of spike JPT is sold by JPT Technologies (JPT Peptide Technologies GmbH, Volmerstrasse, 12489 Berlin, Germany).

[0066] As used herein, the term "CEFX" includes a mixture of 176 synthetic peptide epitopes derived from a variety of viral and bacterial organisms. An example of CEFX is sold by JPT Technologies.

[0067] (General Description) The present disclosure overcomes the deficiencies of classical approaches to analyzing antigen-specific T cell responses by replacing the use of isolated, enriched, and / or extracted PBMCs with whole blood and intact antigens rather than pre-processed peptides. In one embodiment, a method for determining antigen-specific T cell responses in a subject avoids the isolation, enrichment, and / or extraction of PBMCs by contacting a whole blood sample from the subject with at least one antigen to form a mixture. The whole blood sample is contacted with at least one staining reagent (e.g., a dried staining reagent contained in a reagent container), which includes a monoclonal antibody reagent that allows for the identification of T cell markers. The mixture is incubated to obtain a cell concentrate from the mixture, which is then analyzed to identify T cell responses to the antigen. In an embodiment, the analysis is by flow cytometry.

[0068] The method and kit of the present disclosure can be applied in various fields, including determining antigen-specific T cell responses to infectious diseases, autoimmune disorders, cancer, allergies, and aging. Furthermore, the method and kit of the present disclosure can be used to determine the need for treatment (e.g., vaccine), the subject's response to treatment, and / or the need for further treatment or no need for further treatment.

[0069] Some embodiments of the disclosed methods overcome the limitations of prior methods through the simple use of intact or whole proteins as antigens. The production of synthetic peptides is labor-intensive, which can be a daunting task, especially when the goal is to encompass the entire protein sequence. Furthermore, the use of peptide pools can bias T cell responses toward T cell sets that do not naturally respond; for example, short peptides can bias responses toward CD8+ T cells. The use of unmodified, intact, or larger proteins can contain hundreds of overlapping 8- to 12-mer peptides for a single antigen. These methods also open up the possibility of testing antigen-specific T cell responses in a subject sample mixed with a potentially antigenic protein mixture, without any priori targeting one protein or another.

[0070] Disclosed herein are streamlined methods and kits that utilize whole blood, rather than isolated, extracted, or enriched PBMCs, to determine antigen-specific T cell responses in subjects, and are based on the use of extracellular and / or intracellular activation markers. These have been developed and further compared with more conventional techniques (e.g., ELISpot and flow cytometry-based ICS). The disclosed methods rely on the direct detection of rare antigen-specific lymphocytes by identifying membrane markers of T cell activation in whole blood samples (referred to as extracellular staining protocols) or by identifying intracellular markers of T cell activation in whole blood samples (intracellular staining protocols I and II). These simple streamlined methods have been developed that can be used in a wide variety of situations, including clinical settings. These simple, streamlined methods are capable of individual stratification capabilities; for example, using the methods of the present disclosure, a combination of extracellular markers (CD154, CD137, and CD107a) demonstrated greater orthogonality than cytokines commonly considered in ICS (IFN-γ, TNF-α, and IL-2).

[0071] The present disclosure provides methods and kits for determining antigen-specific T cell responses in a subject, the methods comprising: contacting a whole blood sample from the subject with at least one antigen to form a mixture; contacting the whole blood sample with at least one staining reagent, wherein the contacting of the whole blood sample with the at least one staining reagent occurs before, simultaneously with, or after the contacting of the whole blood sample with the at least one antigen to form a mixture; and analyzing the mixture for T cells responsive to the antigen contact.

[0072] In embodiments, the subject is a mammal, a domestic pet (e.g., a dog or cat), or a human. In certain embodiments, the subject is screened for antigen-specific T cell responses to determine the presence of cancer, allergies, autoimmunity, bacterial infection, viral infection, and / or parasitic infection. The methods of the present disclosure can also be used to identify antigen-specific T cell responses to determine the effectiveness of treatment for cancer, allergies, autoimmunity, bacterial infection, viral infection, and / or parasitic infection. The methods of the present disclosure can also be used to identify antigen-specific T cell responses to determine immunity against cancer, allergies, bacterial infection, viral infection, and / or parasitic infection, and / or immunity derived from cancer, allergies, bacterial infection, viral infection, and / or parasitic infection. The methods of the present disclosure can also be used to identify antigen-specific T cell responses to determine the recovery of a subject from treatment for cancer, allergies, bacterial infection, viral infection, and / or parasitic infection.

[0073] In certain embodiments, the method for determining antigen-specific T cell response in a subject is used to analyze the antigen-specific T cell response of a subject before treatment and / or vaccination.The method of the present disclosure can also be used to determine antigen-specific T cell response in a subject, and used to analyze the antigen-specific T cell response of a subject after treatment and / or vaccination.The method of the present disclosure can also be used to determine antigen-specific T cell response in a subject, and used to monitor the immune status of a subject after infection (including viral or bacterial infection).The method of the present disclosure can also be used to determine antigen-specific T cell response in a subject, and used to monitor the immune status of a subject before infection (including bacterial or viral infection).

[0074] In certain embodiments, the methods of the present disclosure are used to determine the presence of infection before infection and / or to determine the antiviral immune response to SARS-CoV-2 in a subject after infection. The methods of the present disclosure can also be used to determine the presence of vaccination, after vaccination, or some combination thereof to determine the antiviral immune response to SARS-CoV-2 in a subject.

[0075] In certain embodiments, the methods of the present disclosure can be used to determine the presence of antigen-specific T cells and to determine anti-tumor immune responses in a subject before and after telomerase-based vaccination.

[0076] In certain embodiments, the methods of the present disclosure may be used to determine the allergen-specific immune response in a subject before, during, and after receiving allergen immunotherapy.

[0077] In certain embodiments, the whole blood sample is about 10 μL to about 10 mL. In certain embodiments, the whole blood sample is about 10 μL to about 2 mL, about 10 μL to about 1 mL, 10 μL to about 750 μL, about 50 μL to about 500 μL, or about 100 μL to about 400 μL. In certain embodiments, the whole blood sample is greater than 0, less than about 2 mL, less than about 1 mL, less than about 750 μL, less than about 500 μL, less than about 300 μL, less than about 250 μL, less than about 100 μL, or less than about 50 μL. In certain embodiments, the whole blood sample is greater than about 500 μL, greater than about 1 mL, greater than about 2 mL, greater than about 3 mL, or greater than about 4 mL.

[0078] In certain embodiments, the method for determining antigen-specific T cell responses involves at least one antigen. In certain embodiments, the at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and / or partial proteins, a protein derivative, a single epitope, a partial proteome, an unprocessed extract, a mixture of polypeptides and other biomolecules, or any combination thereof. The peptide pool may include peptides derived from CEFX or CMV, or any other commercially available pool. The antigens (including whole proteins, peptides, peptide pools, and protein derivatives) may be commercially available, naturally occurring, synthetically engineered, or some combination thereof. In certain embodiments, the at least one antigen comprises an intact protein, as opposed to a processed protein. In certain embodiments, the at least one antigen comprises a single epitope. In certain embodiments, the at least one antigen comprises a partial proteome. In certain embodiments, the at least one antigen comprises a whole proteome, including hundreds of intact proteins. In such embodiments, the at least one antigen may comprise a crude extract (e.g., of an allergen). In certain embodiments, the at least one antigen is an allergen extract, which comprises a homogenous mixture of polypeptides and other biomolecules.

[0079] The number of antigens added to the whole blood mixture is not limited by the parameters of the disclosed methods and kits. In certain embodiments, the number of antigens in a single tube, well, sample, or mixture is between 1 and 20, between 1 and 15, between 1 and 10, or between 3 and 8. In certain embodiments, the number of antigens is greater than 2, greater than 3, greater than 5, greater than 7, greater than 9, or greater than 15. In certain embodiments, the number of antigens is at least 1 but less than 20, less than 15, less than 10, or less than 5.

[0080] In certain embodiments, at least one reagent is added to the mixture formed by contacting the whole blood sample with at least one antigen. The at least one reagent may include one or more reagents commonly used for flow cytometry, cell sample preparation, ELISpot, or ICS and known to those skilled in the art. For example, the at least one reagent may include phosphate-buffered saline, water, acid, base, pH-adjusting reagent, modulator, therapeutic agent, fixative, pH-stabilizing reagent, lysis buffer, wash buffer, and / or some combination thereof. The at least one reagent may include commercially available reagents, such as DURAclone flow cytometry reagent (Beckman Coulter) or Optilyse C (Beckman Coulter). Possible reagents may include, but are not limited to, permeabilization reagents, fixatives, and / or lysis reagents commonly used for sample preparation for flow cytometry.

[0081] In certain embodiments, analyzing the mixture by flow cytometry includes identifying a T cell response to contacting the whole blood sample with at least one antigen. Analyzing the mixture by flow cytometry can include one or more techniques, assays, algorithms, formulas, programs, or any combination thereof, known to those skilled in the art. For example, analyzing the mixture by flow cytometry can include using Kaluza Analysis Software version 2.1 (available from Beckman Coulter). Analyzing the mixture by flow cytometry can include, but is not limited to, analyzing the presence or absence of markers of various cell compartments to maximize stratification and thoroughly evaluate one or more phenotypes present in the blood sample. This can include responding or non-responding cells, immature or mature cells, memory cells, or cells with more specialized phenotypes (e.g., CD4 cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, Treg cells, NKT cells, CD8 cells, Tc1 cells, Tc2 cells, Tc9 cells, T EFF cell, T CM cell, T SCM cell, T RM cell, T EM These may include compartments clearly defined as γδ T cells, γδ T cells, and / or the presence of intracellular cytokines and transcription factors.

[0082] In certain embodiments, the present disclosure further includes incubating the mixture formed by contacting the whole blood sample with at least one antigen before contacting the whole blood with at least one staining reagent and / or after contacting the whole blood sample with at least one antigen before contacting the whole blood with at least one staining reagent. There may be zero incubation steps, one incubation step, at least two incubation steps, or more than three incubation steps. The incubation step(s) may include agitation or mixing of the mixture by shaking, rocking, stirring, vortexing, and / or some combination thereof.

[0083] In certain embodiments, a staining reagent (e.g., an antibody) is added to the mixture before the addition of the antigen, after the addition of the antigen, simultaneously with the addition of the antigen, or any combination thereof. For example, in certain embodiments, at least one staining reagent is added both before and after antigen stimulation.

[0084] In certain embodiments, the step of incubating the mixture comprises incubating at about 37°C for about 30 minutes to about 24 hours. In embodiments, incubation of the mixture may be performed at room temperature, about 37°C, about 4°C, or some combination thereof. In certain embodiments, incubation of the mixture may be for about 30 seconds to about 24 hours, about 5 minutes to about 24 hours, about 10 minutes to about 12 hours, or about 10 minutes to about 30 minutes. In certain embodiments, the incubation is for more than 0 seconds but less than about 24 hours, less than about 16 hours, less than about 12 hours, less than about 6 hours, less than about 2 hours, less than about 1 hour, less than about 30 minutes, less than about 15 minutes, or less than about 10 minutes. In certain embodiments, the incubation comprises more than about 5 minutes, more than about 10 minutes, more than about 30 minutes, more than about 1 hour, more than about 12 hours, or more than about 16 hours.

[0085] The disclosed method may also include, after incubating the mixture, obtaining a cell concentrate from the mixture, the step of obtaining a cell concentrate from the mixture comprising one or more of the following steps: adding a fixation reagent to the mixture, adding a lysis reagent to the mixture, adding a permeabilization reagent to the mixture, staining the mixture with a staining reagent, and washing and concentrating the mixture to obtain the cell concentrate. The disclosed method may include one or more of these steps in any order. Exemplary lysis reagents include, but are not limited to, OptiLyse (Beckman Coulter) and VersaLyse (Beckman Coulter). Exemplary permeabilization and fixation reagents include, but are not limited to, Intraprep Permeabilization Reagent (Beckman Coulter) and PerFix Reagent (Beckman Coulter). Exemplary staining reagents include, but are not limited to, monoclonal antibody reagents that enable identification of extracellular and / or intracellular T cell markers. For example, conjugated antibodies against CD3 (APC-Alexa750, clone UCHT1), CD4 (APC, clone 13B8.2), CD8 (Alexa700, clone B9.11), CD154 (PE, clone TRAP-1), and CD69 (FITC, clone FN50) (obtained from Beckman Coulter), as well as conjugated antibodies against TNF-α (PC7, clone Mab11), IL-2 (BV605, clone MQ1-17H12), and IFN-γ (BV650, clone 4S.B3) (obtained from BioLegend) to detect intracellular cytokines.

[0086] In certain embodiments, the staining reagent is a dry staining reagent, and the staining reagent may be provided in at least one pre-filled reagent container. The pre-filled reagent container may include a single-well plate, a multi-well plate (e.g., a 96-well plate), a test tube, or an Eppendorf tube. The dry staining reagent may be free from the reagent container, attached to the bottom of the reagent container, attached to the side of the reagent container, or distributed throughout the reagent container. In embodiments, the at least one dry reagent contained in a pre-filled dry reagent container comprises a monoclonal antibody reagent (including the monoclonal antibody reagents identified herein) that enables identification of extracellular and / or intracellular T cell markers.

[0087] Some examples of extracellular T cell markers include CD2, CD3, CD4, CD8, CD11a, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, and CD161 (KLRB1). These include, but are not limited to, CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, OX40, and / or any combination thereof. Some examples of intracellular T cell markers include, but are not limited to, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-γ, TGFβ (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1), RORC (RORy), STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-α, and / or any combination thereof.

[0088] In certain embodiments, the at least one antigen comprises at least one virus-specific antigen, which may include, but is not limited to, a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS-CoV, MERS, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), monkeypox (MP), or human papillomavirus (HPV). The full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 may include, but is not limited to, spike protein (S), spike protein subunit S1 (S1), spike protein subunit S2 (S2), spike protein (S+), spike protein RBD domain, nucleocapsid protein, membrane protein, envelope protein, and / or any combination thereof.

[0089] In certain embodiments, the at least one antigen may comprise at least one bacteria-specific antigen, at least one parasite-specific antigen, and / or at least one allergen-specific antigen.

[0090] In certain embodiments, the method excludes the steps of isolating, enriching, and / or extracting PBMCs.

[0091] In one aspect, the present disclosure provides a method for determining an antiviral immune response to SARS-CoV-2 in a subject, the method comprising: contacting a whole blood sample from the subject with at least one antigen to form a mixture; contacting the whole blood sample with at least one staining reagent, the contacting step being performed prior to, simultaneously with, or after the contacting step; and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In some embodiments, the whole blood sample is about 10 μL to about 10 mL.

[0092] In one aspect, the present disclosure provides a method for determining a T cell response to a CPI in a subject, the method comprising: contacting a whole blood sample from the subject with at least one antigen to form a mixture; contacting the whole blood sample with at least one staining reagent, the contacting step being performed before, simultaneously with, or after the contacting step; and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In some embodiments, the whole blood sample is about 10 μL to about 10 mL.

[0093] In one aspect, the present disclosure provides a method for determining specific T cell responses in a subject receiving a telomerase-based vaccination as part of tumor treatment, the method comprising: contacting a whole blood sample from the subject with at least one antigen to form a mixture; contacting the whole blood sample with at least one staining reagent, the contacting step being performed prior to, simultaneously with, or after the contacting step; and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In some embodiments, the whole blood sample is about 10 μL to about 10 mL.

[0094] In one aspect, the present disclosure provides a method for determining allergen-specific T cell responses in a subject undergoing allergen immunotherapy (AIT), the method comprising the steps of: contacting a whole blood sample from the subject with at least one antigen to form a mixture; contacting the whole blood sample with at least one staining reagent, the step of contacting the whole blood sample with at least one staining reagent occurring prior to, simultaneously with, or after the step of contacting the whole blood sample with at least one antigen to form a mixture; and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In some embodiments, the whole blood sample is about 10 μL to about 10 mL.

[0095] In one aspect, the present disclosure provides a method for determining T cell responses in a subject with autoimmunity against peptidyl arginyl deiminase (PAD), the method comprising the steps of: contacting a whole blood sample from the subject with at least one antigen to form a mixture; contacting the whole blood sample with at least one staining reagent, the step of contacting the whole blood sample with at least one staining reagent occurring prior to, simultaneously with, or after the step of contacting the whole blood sample with at least one antigen to form a mixture; and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In some embodiments, the whole blood sample is about 10 μL to about 10 mL.

[0096] In one aspect, the present disclosure provides a method for determining a T cell response to CMV in a subject, the method comprising: contacting a whole blood sample from the subject with at least one antigen to form a mixture; contacting the whole blood sample with at least one staining reagent, where the contacting of the whole blood sample with the at least one staining reagent occurs before, simultaneously with, or after the contacting of the whole blood sample with the at least one antigen to form a mixture; and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In some embodiments, the whole blood sample is about 10 μL to about 10 mL. [Example]

[0097] (Example) The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the disclosed methods and compositions, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0098] Figure 1 shows the workflow required for each of the assays considered herein. While both the IFN-γ ELISpot assay and the ICS assay require initial PBMC preparation, the assays disclosed herein rely on direct incubation of whole blood and antigenic composition(s). Working with whole blood instead of isolated, enriched, or extracted PBMCs offers many advantages, including a reduced blood sample volume required, reduced technical time required to complete the assay, reduced assay difficulty, and increased assay robustness.

[0099] Example 1 (Extracellular Workflow Protocol) The disclosed method allows for the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound markers of T cell activation in whole blood samples.

[0100] In this example (using the method shown in Figure 1A), 250 mL of whole blood was incubated with antigen (CEFX or CMV) and CD107a- and CD154-specific antibodies for 16-20 hours at 37°C to stain for extracellular T cell markers. At the end of the incubation, the samples were stained with the remaining antibodies for 30 minutes at room temperature. 1.25 mL Optilyse C was then added and incubated for 10 minutes. The samples were then centrifuged at 300 g, the supernatant removed, and the pellet reconstituted in 250 mL of PBS for analysis by flow cytometry.

[0101] As shown in Figures 2A-2F, the whole blood samples were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137 and activated with CMV or CEFX. Figures 2A-2B show flow cytometry gating of two donor samples. Figures 2B-2F show flow cytometry analysis of T cells obtained according to the disclosed method. Analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2.1 (Beckman Coulter). Populations of interest were manually gated.

[0102] Example 2 (Extracellular Workflow Protocol: Whole Blood vs. PBMC) The disclosed method allows for the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound markers of T cell activation in whole blood samples.

[0103] In this example (using the method shown in Figures 1A and 1B), to stain for extracellular T cell markers, 250 mL of whole blood or isolated PBMCs were incubated with antigen (CEFX or spiked) and CD107a- and CD154-specific antibodies for 16-20 hours at 37°C. At the end of the incubation, a 30-minute staining step with the remaining antibodies was performed at RT. Next, 1.25 mL of Optilyse C was added and incubated for 10 minutes. Samples were then centrifuged at 300 g, the supernatant removed, and the pellet reconstituted in 250 mL of PBS for flow cytometric analysis.

[0104] As shown in Figures 3A-3J, the whole blood or PBMC samples were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137 and activated with CMV or CEFX. Figures 3A-3B show flow cytometry gating of the samples. Figures 3B-3J show flow cytometry analysis of T cells, NK cells, and NKT cells obtained according to the disclosed methods. Analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2.1 (Beckman Coulter). Populations of interest were manually gated.

[0105] Example 3 (Extracellular workflow protocol: Intact spike protein vs. spike protein peptides at various concentrations) The disclosed method allows for the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound markers of T cell activation in whole blood samples that respond to intact proteins versus peptides.

[0106] In this example (using the method shown in Figures 1A and 1B), 250 mL of whole blood was incubated with antigen (spiked JPT or intact spike at concentrations between 7.2 pmol and 28.8 pmol) and CD107a- and CD154-specific antibodies for 16 to 20 hours at 37°C to stain for extracellular T cell markers. At the end of the incubation, a 30-minute staining step with the remaining antibodies was performed at RT. Next, 1.25 mL of Optilyse C was added and incubated for 10 minutes. Samples were then centrifuged at 300 g, the supernatant removed, and the pellet reconstituted in 250 mL of PBS for flow cytometric analysis.

[0107] As shown in Figures 4A-4C, the whole blood samples were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and / or CD154 and activated with spiked JPT or various concentrations of intact spike. Figure 4A shows the flow cytometry gating of the samples. Figures 4B-4C show flow cytometry analysis of T cells obtained according to the disclosed methods. Analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2.1 (Beckman Coulter). Populations of interest were manually gated.

[0108] Example 4 (Extracellular Workflow Protocol: Liquid vs. Dry Antigens) The disclosed method allows for the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound markers of T cell activation in whole blood samples that respond to intact proteins versus peptides.

[0109] In this example (using the method shown in Figures 1A and 1B), to stain for extracellular T cell markers, 250 mL of whole blood or isolated PBMCs were incubated with antigen (spiked JPT or intact spike at concentrations between 7.2 pmol and 28.8 pmol) and CD107a- and CD154-specific antibodies for 16 to 20 hours at 37°C. At the end of the incubation, a 30-minute staining step with the remaining antibodies was performed at RT. Next, 1.25 mL of Optilyse C was added and incubated for 10 minutes. Samples were then centrifuged at 300 g, the supernatant removed, and the pellet reconstituted in 250 mL of PBS for flow cytometric analysis.

[0110] As shown in Figures 5A-5C, the whole blood samples were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and / or CD154 and activated with liquid or dry spikes or liquid or dry EMN. Figure 5A shows flow cytometry gating of the samples. Figures 5B-5C show flow cytometry analysis of T cells obtained according to the disclosed methods. Analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2.1 (Beckman Coulter). Populations of interest were manually gated.

[0111] Example 5 Intracellular Workflow I Protocol: Liquid vs. Dry Antigens The disclosed method allows for the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound and intracellular markers of T cell activation in whole blood samples using liquid versus dried antigens.

[0112] In this example (using the method shown in Figure 1C), 250 μL of whole blood was incubated with brefeldin (dried or liquid) and antigen (dried or liquid) for 5 hours to stain intracellular T cell markers. Next, 1.25 mL of Intraprep Permeabilization Reagent 1 (R1) (Beckman Coulter) was added to each sample, followed by a 15-minute incubation. Next, 1.5 mL of Intraprep Permeabilization Reagent 2 (R2) was added to each sample, followed by a 10-minute incubation. The samples were then centrifuged at 500 g for 5 minutes, and the supernatant was removed. Additional reconstituted staining marker was added to the cell pellet (if dried, first reconstituted in 1.5 mL), followed by a 45-minute incubation. Next, 3 mL of 1× IntraPrep Permeabilization Reagent 3 (R3) was added to the stained cells, the samples were centrifuged at 500 g for 5 minutes, and the supernatant was removed. 250 mL of 1× R3 was then added to reconstitute the cell pellet. The samples were analyzed by flow cytometry.

[0113] As shown in Figures 6A-6D, the whole blood samples were stained for CD3, CD4, CD8, IFNγ, CD45, CD154, IL-4, TNFα, and / or IL-17 and activated with liquid or dried antigens (liquid: P8A, P8B, PAD2, PAD4; dried: CMV, CEFX, PMA / Iono). Figure 6A shows flow cytometry gating of the samples. Figures 6B-6D show flow cytometry analysis of T cells obtained according to the disclosed method. Analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2.1 (Beckman Coulter). Populations of interest were manually gated.

[0114] Example 6 (Intracellular Workflow II Protocol) The disclosed method allows for the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound and intracellular markers of T cell activation in whole blood samples.

[0115] In this example, to stain intracellular T cell markers (using the method shown in Figure ID), 250 μL of whole blood was incubated with brefeldin (dried or liquid) and antigen (dried or liquid) for 3 to 5 hours. Extracellular staining was then performed. Each sample was then incubated with 400 μL of OptiC lysis solution for 10 minutes, followed by the addition of 1.25 mL of Intraprep Permeabilization Reagent 1 (R1) (Beckman Coulter), followed by an additional 30-minute incubation. Each sample was then washed, followed by the addition of 1.5 mL of Intraprep Permeabilization Reagent 2 (R2), followed by a 10-minute incubation. Reconstituted additional staining markers were added to the cell pellet (if dry, they were first reconstituted in 1.5 mL), followed by a 45-minute incubation. Next, 3 mL of 1× IntraPrep Permeabilization Reagent 3 (R3) was added to the stained cells, the samples were centrifuged at 500 g for 5 minutes, and the supernatant was removed. 250 mL of 1× R3 was then added to reconstitute the cell pellet. The samples were analyzed by flow cytometry.

[0116] As shown in Figures 7A-7D, the whole blood samples were stained for CD3, CD4, CD8, IFNγ, CD19, CD45, CD154, IL-4, IL-10, TNFα, and / or IL-17 and activated with SARS-CoV-2 spike glycoprotein, CMV global, CMV pp65, allergen mix, or PMA / ionomycin. Figures 7A-7D show flow cytometry analysis of T cells, B cells, and NK cells obtained according to the disclosed methods. Figures 7E-7H show significant mean differences in marker expression between multiple whole blood samples. Analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2.1 (Beckman Coulter). Populations of interest were manually gated.

[0117] Example 7 (Various whole blood volumes) The disclosed method enables the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound markers of T cell activation in whole blood samples of various volumes. (Using the method shown in Figure 1B) In this example, 250 μL, 500 μL, or 1000 μL of whole blood from three separate patients was incubated with antigen (spiked JPT or CEFX, or no antigen as a negative control) and CD107a- and CD154-specific antibodies for 20 hours at 37°C to stain for extracellular T cell markers. At the end of the incubation, the samples were stained with the remaining antibodies (CD8, CD3, CD137, CCR7, CD69, CD56, and CD45RA) for 20 minutes at room temperature. Optilyse C (1.5 mL, 3 mL, or 6 mL, depending on the original whole blood volume) was then added and incubated at room temperature for 10 minutes. Samples were then centrifuged at 250 g, the supernatant removed, and the pellet reconstituted in PBS (1.5 mL, 3 mL, or 6 mL depending on the original whole blood volume) for analysis by flow cytometry.

[0118] The whole blood samples were stained for CD3, CD4, CD45RA, CD8, CD154, CD69, CD107a, and / or CD137 and were either unactivated, activated with spiked JPT, or activated with CEFX, as shown in Figures 8A-8C. Figure 8A shows flow cytometry gating of the three donor samples (500 μL whole blood samples are shown in Figure 8A; however, no significant differences were observed between 250 μL, 500 μL, or 1000 μL whole blood). Figures 8B-8C show flow cytometry analysis of CD4+ T cells (Figure 8B) and CD8+ T cells (Figure 8C) from the three whole blood samples obtained according to the disclosed methods (500 μL whole blood samples are shown in Figures 8A-8C; however, no significant differences were observed between 250 μL, 500 μL, or 1000 μL whole blood). Analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2.1 (Beckman Coulter). Populations of interest were manually gated.

[0119] The following numbered paragraphs define further exemplary aspects and features of the present disclosure: Item 1. A method for determining an antigen-specific T cell response in a subject, comprising: a) contacting a whole blood sample from the subject with at least one antigen to form a mixture; b) contacting the whole blood sample with at least one staining reagent, wherein contacting the whole blood sample with at least one staining reagent occurs before step (a), simultaneously with step (a), after step (a), or any combination thereof; and c) analyzing the mixture to identify T cells that respond to the antigen challenge. A method comprising: Item 2. The method according to Item 1, wherein the at least one antigen is a T cell activator. Item 3. The method according to any one of Items 1 and 2, wherein the mixture is analyzed by flow cytometry. Item 4. The method of any one of Items 1 to 3, wherein the at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length proteins and / or partial proteins, a protein derivative, a single epitope, a partial proteome, an unprocessed extract, a mixture of polypeptides and other biomolecules, or any combination thereof. Item 5. Adding at least one reagent to the mixture of step (b) Item 5. The method according to any one of items 1 to 4, further comprising: Item 6. Incubating the mixture after step (a), before step (b), after step (b), or any combination thereof. Item 6. The method according to any one of Items 1 to 5, further comprising: Item 7. After incubating the mixture, obtaining a cell concentrate from the mixture. Item 7. The method according to any one of items 1 to 6, further comprising: Item 8. The step of obtaining a cell concentrate from the mixture, a) adding a lysis reagent to the mixture; b) adding a permeabilization reagent to the mixture; c) staining the mixture with a staining reagent; and d) washing and concentrating the mixture to obtain the cell concentrate. Item 8. The method according to any one of Items 1 to 7, comprising: Item 9. The method according to any one of Items 1 to 8, wherein the step of obtaining a cell concentrate from the mixture further comprises: a) adding a fixation reagent to the mixture. Item 10. The method according to any one of Items 1 to 9, wherein the at least one staining reagent and / or the at least one antigen is a dry reagent. Item 11. The method according to any one of Items 1 to 10, wherein the dry reagent is provided in at least one pre-filled reagent container. Item 12. The method according to any one of Items 1 to 11, wherein the dry reagent contained in the pre-filled dry reagent container comprises a monoclonal antibody reagent that enables identification of an extracellular T cell marker. Item 13. The staining reagent includes a monoclonal antibody reagent for identifying extracellular T cell markers, and the extracellular T cell markers include CD2, CD3, CD4, CD8, CD11a, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, and CD161 (KLRB1). 13. The method of any one of items 1 to 12, comprising CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, OX40, or any combination thereof. Item 14. The method according to any one of Items 1 to 13, wherein the at least one dry reagent contained in the pre-filled dry reagent container comprises a monoclonal antibody reagent that enables identification of an intracellular T cell marker. Item 15. The method of any one of Items 1 to 14, wherein the staining reagent comprises a monoclonal antibody reagent for identifying an intracellular T cell marker, and the intracellular T cell marker comprises IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26, IFN-γ, TGFβ (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1), RORC (RORy), STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-α, or any combination thereof. Item 16. The method according to any one of Items 1 to 15, wherein the step of incubating the mixture comprises incubation at about 37°C for about 30 minutes to about 24 hours. Item 17. The method according to any one of Items 1 to 16, wherein the volume of the whole blood sample is about 10 μL to about 10 mL. Item 18. The method according to any one of Items 1 to 17, wherein the step of analyzing the mixture by flow cytometry comprises analyzing the presence of CD4+ T cell activation markers and / or CD8+ T cell activation markers, and / or the presence of intracellular cytokines and / or transcription factors. Item 19. The method according to any one of Items 1 to 18, wherein the activation markers for CD4+ T cells include CD69, CD154, CD137, and CD107a, and the activation markers for CD8+ T cells include CD69, CD154, CD137, and CD107a. Item 20. The method according to any one of Items 1 to 19, wherein the at least one antigen comprises at least one virus-specific antigen. Clause 21. The method of any one of clauses 1 to 20, wherein the at least one virus-specific antigen is a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS-CoV, MERS, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), monkeypox (MP), or human papillomavirus (HPV). Item 22. The method of any one of items 1 to 21, wherein the full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 comprises spike protein (S), spike protein subunit S1 (S1), spike protein subunit S2 (S2), spike protein (S+), spike protein RBD domain, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof. Item 23. The method of any one of Items 1 to 22, wherein the at least one antigen comprises at least one parasite-specific antigen, at least one allergen-specific antigen, or at least one bacteria-specific antigen. Item 24. The method according to any one of items 1 to 23, wherein the method excludes the steps of isolating, enriching, and / or extracting peripheral blood mononuclear cells (PBMCs). Clause 25. Use of the method according to any one of clauses 1 to 24 for analyzing antigen-specific T cell responses in a subject before and / or after vaccination. Clause 26. Use of the method of any one of clauses 1 to 24 to monitor the immune status of a subject before or after an event of viral infection. Item 27. A method for determining an antigen-specific T cell response in a subject, comprising: a) contacting a whole blood sample from the subject with at least one antigen to form a mixture; b) contacting the mixture with a staining reagent for CD107a and a staining reagent for CD154; c) incubating the mixture; d) contacting the mixture with at least one additional staining reagent; e) adding a lysis reagent to the mixture; f) washing and concentrating the mixture to obtain a cell concentrate; and g) analyzing the mixture to identify T cells that respond to the antigen contact. A method comprising: Item 28. A method for determining an antiviral immune response to SARS-CoV-2 in a subject, comprising: a) contacting a whole blood sample from the subject with at least one SARS-CoV-2 antigen to form a mixture; b) contacting the whole blood sample with at least one staining reagent, wherein contacting the whole blood sample with at least one staining reagent occurs before step (a), simultaneously with step (a), after step (a), or any combination thereof; and c) analyzing the mixture to identify T cells that respond to the SARS-CoV-2 antigen exposure. A method comprising: Item 29. The method according to any one of Items 27 to 28, wherein the mixture is analyzed by flow cytometry. Item 30. Adding at least one reagent to the mixture of step (b) 30. The method according to any one of items 27 to 29, further comprising: Item 31. Incubating the mixture after step (a), before step (b), after step (b), or any combination thereof. Item 31. The method according to any one of Items 27 to 30, further comprising: Item 32. After incubating the mixture, obtaining a cell concentrate from the mixture. Item 32. The method according to any one of Items 27 to 31, further comprising: Item 33. The step of obtaining a cell concentrate from the mixture, a) adding a lysis reagent to the mixture; b) adding a permeabilization reagent to the mixture; c) staining the mixture with a staining reagent; and d) washing and concentrating the mixture to obtain the cell concentrate. Item 33. The method according to any one of Items 27 to 32, comprising: Item 34. The method according to any one of Items 27 to 33, wherein the step of obtaining a cell concentrate from the mixture further comprises: a) adding a fixation reagent to the mixture. Item 35. The method according to any one of Items 27 to 34, wherein the at least one staining reagent and / or the at least one antigen is a dry reagent. Item 36. The method according to any one of Items 27 to 35, wherein the dry reagent is provided in at least one pre-filled reagent container. Item 37. The method of any one of Items 27 to 36, wherein the at least one dry reagent contained in the pre-filled dry reagent container comprises a monoclonal antibody reagent that enables identification of an extracellular T cell marker. Item 38. The extracellular T cell markers are CD2, CD3, CD4, CD8, CD11a, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, and CD161 (KLRB1). 38. The method of any one of paragraphs 27 to 37, comprising CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, OX40, and / or any combination thereof. Item 39. The method according to any one of Items 27 to 38, wherein the at least one dry reagent contained in the pre-filled dry reagent container comprises a monoclonal antibody reagent that enables identification of an intracellular T cell marker. Item 40. The method of any one of Items 27 to 39, wherein the staining reagent comprises a monoclonal antibody reagent for identifying an intracellular T cell marker, and the intracellular T cell marker comprises IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26, IFN-γ, TGFβ (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1), RORC (RORy), STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-α, or any combination thereof. Item 41. The method according to any one of Items 27 to 40, wherein the step of incubating the mixture comprises incubation at about 37°C for about 30 minutes to about 24 hours. Item 42. The method according to any one of Items 27 to 41, wherein the volume of the whole blood sample is about 10 μL to about 10 mL. Item 43. The method of any one of Items 27 to 42, wherein the step of analyzing the mixture by flow cytometry comprises analyzing the presence of CD4+ T cell activation markers and / or CD8+ T cell activation markers, and / or the presence of intracellular cytokines and / or transcription factors. Item 44. The method according to any one of Items 27 to 43, wherein the CD4+ T cell activation markers include CD69, CD154, CD137, and CD107a, and the CD8+ T cell activation markers include CD69, CD154, CD137, and CD107a. Clause 45. The method of any one of clauses 27 to 44, wherein the at least one antigen is a full-length protein derived from SARS-CoV-2; a processed protein derived from SARS-CoV-2; a peptide derived from SARS-CoV-2; a pool of peptides derived from SARS-CoV-2; a complex mixture of full-length and / or partial proteins derived from SARS-CoV-2; a protein derivative derived from SARS-CoV-2; a single epitope derived from SARS-CoV-2; a partial proteome derived from SARS-CoV-2; an unprocessed extract derived from SARS-CoV-2; or a mixture of polypeptides derived from SARS-CoV-2 and other biomolecules. Paragraph 46. The method of any one of paragraphs 27 to 45, wherein the full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 comprises spike protein (S), spike protein subunit S1 (S1), spike protein subunit S2 (S2), spike protein (S+), spike protein RBD domain, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof. Item 47. The method of any one of Items 27 to 46, wherein the method excludes the steps of isolating, enriching, and / or extracting peripheral blood mononuclear cells (PBMCs). Clause 48. Use of the method of any one of clauses 27 to 47 for analyzing antigen-specific T cell responses in a subject before and / or after vaccination. Clause 49. Use of the method of any one of clauses 27 to 47 to monitor the immune status of a subject following an event of viral infection. Clause 50. Use of the method of any one of clauses 27 to 47 to monitor the immune status of a subject prior to the event of viral infection. Item 51. At least one antigen; and At least one staining reagent A kit for analyzing antigen-specific T cell responses in a sample, comprising: Item 52. At least one pre-filled reagent container Item 52. The kit of Item 51, further comprising: Item 53. The kit according to any one of Items 51 to 52, wherein the at least one antigen or the at least one staining reagent is a dry reagent. Item 54. The kit according to any one of Items 51 to 53, wherein the at least one dry reagent is provided in at least one pre-filled reagent container. Item 55. The kit according to any one of Items 51 to 54, wherein the at least one antigen is a T cell activator. Clause 56. The kit of any one of clauses 51 to 55, wherein the at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and / or partial proteins, a protein derivative, a single epitope, a partial proteome, an unprocessed extract, a mixture of polypeptides and other biomolecules, or any combination thereof. Item 57.a) Lytic reagent; b) a permeabilization reagent; and c) Fixation reagent Item 57. The kit according to any one of Items 51 to 56, further comprising: Item 58. The kit according to any one of Items 51 to 57, wherein the dry reagent contained in the pre-filled dry reagent container comprises a monoclonal antibody reagent that enables identification of an extracellular T cell marker. Item 59. The staining reagent includes a monoclonal antibody reagent for identifying extracellular T cell markers, and the extracellular T cell markers are CD2, CD3, CD4, CD8, CD11a, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) 59. The kit of any one of paragraphs 51 to 58, comprising CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, OX40, or any combination thereof. Item 60. The kit according to any one of Items 51 to 59, wherein the at least one dry reagent contained in the pre-filled dry reagent container comprises a monoclonal antibody reagent that enables identification of an intracellular T cell marker. Item 61. The kit of any one of Items 51 to 60, wherein the staining reagent comprises a monoclonal antibody reagent for identifying an intracellular T cell marker, and the intracellular T cell marker comprises IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26, IFN-γ, TGFβ (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1), RORC (RORy), STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-α, or any combination thereof. Item 62. The kit according to any one of Items 51 to 61, wherein the at least one antigen comprises at least one virus-specific antigen. Clause 63. The kit of any one of clauses 51 to 62, wherein the at least one virus-specific antigen is a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS-CoV, MERS, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), monkeypox (MP), or human papillomavirus (HPV). Clause 64. The kit of any one of clauses 51 to 63, wherein the full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 comprises spike protein (S), spike protein subunit S1 (S1), spike protein subunit S2 (S2), spike protein (S+), spike protein RBD domain, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof. Clause 65. The kit of any one of clauses 51 to 64, wherein the at least one antigen comprises at least one parasite-specific antigen, at least one allergen-specific antigen, or at least one bacteria-specific antigen. Clause 66. Use of the kit according to any one of clauses 51 to 65 for analyzing antigen-specific T cell responses in a subject before and / or after vaccination. Clause 67. Use of the kit of any one of clauses 51 to 65 to monitor the immune status of a subject before or after an event of viral infection. Item 68. A method for determining an antigen-specific T cell response in a subject, comprising: a) contacting a whole blood sample from the subject with at least one antigen to form a mixture, wherein the volume of the whole blood sample is between about 10 μL and about 10 mL, and the at least one antigen is a T cell activator, and the at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length proteins and / or partial proteins, a protein derivative, a single epitope, a partial proteome, an unprocessed extract, a mixture of polypeptides and other biomolecules, or any combination thereof; b) contacting the whole blood sample with at least one staining reagent, the staining reagent comprising a monoclonal antibody reagent for identification of extracellular T cell markers, the extracellular T cell markers being CD2, CD3, CD4, CD8, CD11a, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) the step of contacting the whole blood sample with at least one staining reagent comprises CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, OX40, or any combination thereof, wherein the contacting step comprises incubation for about 30 minutes to about 24 hours, and the step of contacting the whole blood sample with at least one staining reagent is performed before step (a), simultaneously with step (a), after step (a), or any combination thereof; and c) analyzing the mixture by flow cytometry to identify T cells that respond to the antigen challenge. A method comprising: Item 69. Adding at least one reagent to the mixture of step (b) Item 69. The method of Item 68, further comprising: Item 70. Incubating the mixture after step (a), before step (b), after step (b), or any combination thereof. Item 69. The method according to any one of items 68 to 69, further comprising: Item 71. After incubating the mixture, a step of obtaining a cell concentrate from the mixture. Item 71. The method according to any one of items 68 to 70, further comprising: Item 72. The step of obtaining a cell concentrate from the mixture, a) adding a lysis reagent to the mixture; b) adding a permeabilization reagent to the mixture; c) staining the mixture with a staining reagent; and d) washing and concentrating the mixture to obtain the cell concentrate. Item 72. The method according to any one of items 68 to 71, comprising: Item 73. The method according to any one of Items 68 to 72, wherein the step of obtaining a cell concentrate from the mixture further comprises adding a fixation reagent to the mixture. Item 74. The method according to any one of Items 68 to 73, wherein the at least one staining reagent and / or the at least one antigen is a dry reagent. Item 75. The method of any one of Items 68 to 74, wherein the dry reagent is provided in at least one pre-filled reagent container. Item 76. The method of any one of Items 68 to 75, wherein the staining reagent comprises a monoclonal antibody reagent for identifying an intracellular T cell marker, and the intracellular T cell marker comprises IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26, IFN-γ, TGFβ (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1), RORC (RORy), STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-α, or any combination thereof. Item 77. The method of any one of Items 68 to 76, wherein the step of analyzing the mixture by flow cytometry comprises analyzing the presence of CD4+ T cell activation markers and / or CD8+ T cell activation markers, wherein the CD4+ T cell activation markers include CD69, CD154, CD137, and CD107a, and the CD8+ T cell activation markers include CD69, CD154, CD137, and CD107a. Clause 78. The method of any one of clauses 68 to 77, wherein the at least one antigen comprises at least one virus-specific antigen, and the at least one virus-specific antigen is a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS-CoV, MERS, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), monkeypox (MP), or human papillomavirus (HPV), spike protein (S), spike protein subunit S1 (S1), spike protein subunit S2 (S2), spike protein (S+), spike protein RBD domain, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof. Paragraph 79. The method of any one of paragraphs 68 to 79, wherein the method excludes the steps of peripheral blood mononuclear cell (PBMC) isolation, enrichment, and / or extraction. Clause 80. Use of the method of any one of clauses 68 to 79 to monitor the immune status of a subject before or after an event of viral infection. Paragraph 81. A method for determining an antigen-specific T cell response in a subject, comprising: a) contacting a whole blood sample from the subject with at least one antigen to form a mixture; b) contacting the mixture with a staining reagent for CD107a and a staining reagent for CD154; c) incubating the mixture; d) contacting the mixture with at least one additional staining reagent; e) adding a lysis reagent to the mixture; f) washing and concentrating the mixture to obtain a cell concentrate; and g) analyzing the mixture to identify T cells that respond to the antigen challenge. A method comprising: Paragraph 82. A method for determining an antiviral immune response to SARS-CoV-2 in a subject, comprising: a) contacting a whole blood sample from the subject with at least one SARS-CoV-2 antigen to form a mixture; b) contacting the whole blood sample with at least one staining reagent, wherein contacting the whole blood sample with at least one staining reagent occurs before step (a), simultaneously with step (a), after step (a), or any combination thereof; and c) analyzing the mixture to identify T cells that respond to the SARS-CoV-2 antigen challenge. A method comprising:

Claims

1. 1. A method for determining an antigen-specific T cell response in a subject, comprising: a) contacting a whole blood sample from the subject with at least one antigen to form a mixture, wherein the volume of the whole blood sample is from about 10 μL to about 10 mL, and the at least one antigen is an activator of T cells, and comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length proteins and / or partial proteins, a protein derivative, a single epitope, a partial proteome, an unprocessed extract, a mixture of polypeptides and other biomolecules, or any combination thereof; b) contacting the whole blood sample with at least one staining reagent, the staining reagent comprising a monoclonal antibody reagent for identification of extracellular T cell markers, the extracellular T cell markers being CD2, CD3, CD4, CD8, CD11a, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) and wherein the staining reagent comprises CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, OX40, or any combination thereof, wherein the contacting step comprises incubation for about 30 minutes to about 24 hours, and wherein contacting the whole blood sample with the at least one staining reagent occurs before step (a), simultaneously with step (a), after step (a), or any combination thereof; and c) analyzing the mixture by flow cytometry to identify T cells that respond to the antigen contact. A method comprising:

2. 10. The method of claim 1, further comprising the step of adding at least one reagent to the mixture of step (b).

3. 10. The method of claim 1, further comprising incubating the mixture after step (a), before step (b), after step (b), or any combination thereof.

4. The method of claim 3, further comprising obtaining a cell concentrate from the mixture after incubating the mixture.

5. Obtaining a cell concentrate from the mixture, a) adding a lysis reagent to the mixture; b) adding a permeabilization reagent to the mixture; c) staining the mixture with a staining reagent; and d) washing and concentrating the mixture to obtain the cell concentrate. The method of claim 4, comprising:

6. The method of claim 5 , wherein the step of obtaining a cell concentrate from the mixture further comprises adding a fixation reagent to the mixture.

7. The method of claim 1 , wherein the at least one staining reagent and / or the at least one antigen is a dry reagent.

8. The method of claim 7 , wherein the dry reagents are provided in at least one pre-filled reagent container.

9. 9. The method of claim 1 or 8, wherein the staining reagent comprises a monoclonal antibody reagent for identification of intracellular T cell markers, the intracellular T cell markers comprising IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26, IFN-γ, TGF-β (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1), RORC (RORy), STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-α, or any combination thereof.

10. 2. The method of claim 1, wherein analyzing the mixture by flow cytometry comprises analyzing the presence of activation markers for CD4+ T cells and / or CD8+ T cells, wherein the activation markers for CD4+ T cells include CD69, CD154, CD137, and CD107a, and the activation markers for CD8+ T cells include CD69, CD154, CD137, and CD107a.

11. 2. The method of claim 1, wherein the at least one antigen comprises at least one virus-specific antigen, wherein the at least one virus-specific antigen is a full-length protein, protein derivative, peptide, or peptide pool, spike protein (S), spike protein subunit S1 (S1), spike protein subunit S2 (S2), spike protein (S+), spike protein RBD domain, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof, derived from SARS-CoV-2, SARS-CoV, MERS, cytomegalovirus (CMV), respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), monkeypox (MP), or human papillomavirus (HPV).

12. 10. The method of claim 1, wherein the method excludes the steps of peripheral blood mononuclear cell (PBMC) isolation, enrichment, and / or extraction.

13. 10. Use of the method of claim 1 to monitor the immune status of a subject before or after an event of viral infection.

14. 1. A method for determining an antigen-specific T cell response in a subject, comprising: a) contacting a whole blood sample from the subject with at least one antigen to form a mixture; b) contacting the mixture with a staining reagent for CD107a and a staining reagent for CD154; c) incubating the mixture; d) contacting the mixture with at least one additional staining reagent; e) adding a lysis reagent to the mixture; f) washing and concentrating the mixture to obtain a cell concentrate; and g) analyzing the mixture to identify T cells that respond to the antigen contact. A method comprising:

15. 1. A method for determining an antiviral immune response to SARS-CoV-2 in a subject, comprising: a) contacting a whole blood sample from the subject with at least one SARS-CoV-2 antigen to form a mixture; b) contacting the whole blood sample with at least one staining reagent, wherein contacting the whole blood sample with at least one staining reagent occurs before step (a), simultaneously with step (a), after step (a), or any combination thereof; and c) analyzing the mixture to identify T cells that respond to the SARS-CoV-2 antigen challenge. A method comprising:

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