Manipulation and use of antigen-specific regulatory t cells

Abstract

To provide highly selective antigen-specific therapies that control autoimmune diseases and other inflammatory diseases by manipulating immune system cells.SOLUTION: Compositions and methods are provided for isolating, manipulating and using for therapeutic and other purposes, mammalian, MHC Class I restricted, antigen-specific regulatory T cells. The regulatory T cells can be characterized as CD8+ cells that specifically suppress responses of self-reactive and / or pathogenic CD4+ T cells by cytotoxic mechanisms including, but not limited to, perforin and other components of the perforin / granzyme apoptosis pathway, etc. The regulatory T cells are antigen-specific, but are not activated by the same antigen as the self-reactive and / or pathogenic CD4+ T cells. In humans, the regulatory T cells express inhibitory KIR proteins, e.g., one or more of KIR2DL2, KIR2DL3, and KIR3DL1.SELECTED DRAWING: None

Description

[Background technology]

[0001] There has been long-standing interest in manipulating cells of the immune system to achieve control of autoimmune and other inflammatory diseases. Traditional treatment methods have generally been non-antigen specific. For example, common immunosuppressants utilize drugs such as methylprednisolone, other steroids, methotrexate, cladribine, and cyclophosphamide. However, the global immunosuppression these therapies provide has significant undesirable side effects.

[0002] More selective modulation of the immune system utilizes agents such as cytokine blockers, e.g., anti-TNFα antibodies, soluble TNFα receptors, soluble IL-1 receptors (Anakinra), and anti-IL-6R antibodies (Tocilizumab), cell-targeted therapies (CTLA4-Ig [Abatacept]), B-cell-targeted therapies (anti-CD20 [Rituximab]), etc.

[0003] Alternatively, anti-inflammatory cytokines such as interferon beta (IFNβ)-1b (Betafenol / Betaseron) have also found use. However, although these therapies are more targeted, they still immunosuppress entire classes of responses rather than specifically immunosuppressing unwanted responses.

[0004] Thus, the promise of highly selective, antigen-specific therapy remains attractive but challenging. Such specificity could provide effective treatment of unwanted immune responses without involving all immune system populations or responses. However, despite known autoantigen targets, the strong correlation between many autoimmune diseases and specific major histocompatibility complex (MHC) class II alleles, and well-established T cell involvement, antigen-targeted therapy has not been readily translated into clinical use.

[0005] The development of therapies for this purpose is provided herein. Summary of the Invention

[0006] Compositions and methods are provided for isolating, manipulating, and using mammalian, MHC class I-restricted, antigen-specific regulatory T cells for therapeutic and other purposes. Regulatory T cells can be characterized as CD8+ cells that specifically suppress autoreactive and / or pathogenic CD4+ T cell responses through cytotoxic mechanisms, including, but not limited to, perforin and other components of the perforin / granzyme apoptosis pathway. Regulatory T cells are antigen-specific but are not activated by the same antigen as autoreactive and / or pathogenic CD4+ T cells. This novel regulatory CD8+ T cell subset exists in humans and has been demonstrated to suppress pathogenic CD4+ T cells arising from autoreactivity in autoimmune disorders or cross-reactivity to autoantigens in infectious diseases via cytotoxicity. Regulatory T cells have been shown to express cytotoxic molecules and trigger the programmed cell death of autoreactive CD4+ T cells. The ability of antigen-specific regulatory cells to selectively target specific activated CD4+ cells provides a means for the highly selective treatment of inflammatory diseases, e.g., autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, transplant rejection, atopic conditions, etc., and is therefore a highly desirable therapeutic approach.

[0007] As used herein, during the development of a T cell response to an initiating antigen, CD4 + There is an expansion of T cells, and their CD4 + Regulatory CD8 that can specifically suppress cells + The expansion of related T cell populations has been shown to be associated with the simultaneous expansion of T cells in peripheral blood, as well as in synovial tissue, the CNS, and autoimmune diseases. It is found in disease-related tissues such as the site of a tumor.

[0008] Initiating antigens include, but are not limited to, autoantigens involved in disease development, including known autoantigens such as myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), aquaporins, glutamic acid decarboxylase (GAD), insulin and proinsulin, matrix metalloproteinase-1 (MMP-1), type II collagen (COL II), thyroglobulin, proteolipid protein (PLP), myelin-associated glycoprotein (MAG), chondrocyte glycoprotein, heat shock proteins (HSPs), and citralinated proteins such as filaggrin. Initiating antigens may also include non-self antigens, including, for example, non-self antigens that induce an autoimmune response, antigens present on transplanted tissue, pathogen antigens, including viral antigens such as SARS-CoV-2 proteins, and bacterial antigens, including Borrelia.

[0009] Regulatory T cells are CD8 + Regulatory T cells are characterized by a phenotype that is CD44+, CD122+, and MHC class I-restricted. In humans, regulatory T cells express inhibitory KIR proteins, and in mice, they express the mouse counterpart, Ly49 protein. The specific KIR proteins expressed by the cells can include one or more of the inhibitory KIR proteins, such as KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, and KIR3DL2, and specifically can include one or more of KIR2DL2, KIR2DL3, and KIR3DL1. Regulatory T cells can also be characterized as CD44+, CD122+, and not Qa-1b-restricted. In multiple sclerosis, regulatory cells are characterized by CD8 + CD38 + HLA-DR + KIR + CD29 + Regulatory T cells can be brain-homing, having a phenotype of: Affinity agents specific for one or more of the cell surface markers can be used to detect and isolate regulatory T cells.

[0010] In some embodiments, methods are provided for treating undesirable T cell-mediated inflammatory conditions, which may include, but are not limited to, autoimmune diseases, transplant rejection, and the like. The treatment methods provide antigen-specific suppression of specific pathogenic CD4+ T cells through a cytotoxic mechanism. In some embodiments, an effective dose of regulatory T cells is provided to an individual. In some embodiments, the regulatory T cells are autologous to the individual being treated. In some embodiments, the regulatory T cells are allogeneic to the individual being treated. In other embodiments, an effective dose of a regulatory peptide is administered to the individual, and the regulatory peptide induces an antigen-specific regulatory response. The peptide can be administered in a suitable format, for example, complexed with antigen-presenting cells, as a multimer, as a free peptide, etc.

[0011] The treatment can be administered at the onset of disease symptoms. The treatment can be administered at the onset of disease relapse, at the peak of disease relapse, etc. The treatment can be administered in combination with an additional agent, e.g., a disease-modifying therapy, e.g., interferon beta, glatiramer acetate, teriflunomide, dimethyl fumarate, fingolimod, natalizumab, ocrelizumab, alemtuzumab, cladribine, mitoxantrone, etc. The additional agent can also be administered concurrently, cross-over, following, e.g., an antigen-specific therapy.

[0012] In certain embodiments, the treatment is directed to an autoimmune disease. In some embodiments, the autoimmune disease is a demyelinating disease, e.g., multiple sclerosis, neuromyelitis optica, etc. In some embodiments, the inflammatory condition is initiated by an autoantigen. In some embodiments, the initiating autoantigen is a peptide of a myelin-associated protein, e.g., MOG, MBP, MAG, etc. In other embodiments, the inflammatory condition is initiated by an infection, e.g., a viral infection.

[0013] For example, COVID-19 can cause serious clinical problems that are thought to be related to autoimmunity. KIRs correlate with disease severity and the development of vasculitis in patients + CD8+ There are elevated levels of T cells, suggesting that many of the complications of COVID-19 are the result of various types of autoimmunity.

[0014] In some embodiments, methods are provided for determining the antigen specificity of regulatory T cells, thereby providing identification of regulatory peptide sequences. In one such method, the T cell receptors (TCRs) of antigen-specific regulatory T cells, e.g., α / β TCR pairs, are expressed as soluble multimers, e.g., tetramers. The TCR multimers are utilized in binding assays against a library of diverse peptides in an MHC context, e.g., a yeast display system. The use of human MHC proteins associated with inflammatory diseases is particularly interesting. After one or more rounds of binding and selection, the enriched peptide sequences provide identification of the antigen specificity of regulatory T cells. The peptide antigens thus identified may be natural peptides or surrogate peptides that act to specifically activate the regulatory T cells of interest.

[0015] In a related embodiment, peptide antigens that specifically bind to and activate regulatory T cells are provided, and these peptides can be identified by the methods described herein. A characteristic of regulatory peptides is that they activate regulatory T cells to antigen- and MHC class I-restricted anti-T cell responses. In other words, regulatory T cells are activated by regulatory peptides, resulting in a state in which the regulatory T cells suppress autoreactive and / or pathogenic CD4+ T cell responses through a cytotoxic mechanism.

[0016] The regulatory peptide composition may be a pharmaceutical composition comprising a pharmaceutically acceptable excipient, wherein the peptide is in a form suitable for T cell activation. In certain such embodiments, a package is provided that includes an anti-CD49e agent, one or more second therapeutic compounds, and a package insert or label indicating that the anti-CD49e agent is to be administered in combination with the second compound to a patient for the treatment of a neurological inflammatory disease.

[0017] In some embodiments, an isolated population of antigen-specific, MHC class I-restricted CD8+ regulatory T cells is provided. In some embodiments, the regulatory T cells are activated in vivo after administration of an initiating antigen to an individual and isolated from a sample from the individual, e.g., peripheral blood, lymph nodes, etc. In some embodiments, the regulatory T cells are activated in an in vitro culture system after administration of the initiating antigen. In other embodiments, the T cells are activated in vitro by contact with a regulatory peptide, e.g., in co-culture with suitable antigen-presenting cells. In some embodiments, the regulatory T cells initially activated in vivo or in vitro are expanded in an in vitro culture system in a medium containing one or more suitable cytokines for expansion, e.g., including, but not limited to, IL-15, IL-1, IL-18, IL-33, etc.

[0018] In some embodiments, the regulatory T cells express an engineered T cell receptor with specificity for the regulatory peptide. In some embodiments, T cells are obtained from the individual to be treated and engineered in vitro to express a TCR specific for the regulatory peptide in an MHC class I context. In some embodiments, the T cells are obtained from the individual after an immune challenge, e.g., during an expansion period after vaccination. In other embodiments, the regulatory T cells are allogeneic with respect to the individual to be treated.

[0019] In some embodiments, methods are provided for the analysis of inflammation, including infectious diseases, including but not limited to viral infections such as SARS-CoV-2, in an individual, the methods comprising detecting the presence or changes in levels of regulatory CD8+ T cells, particularly CD8+KIR+ T cells. The methods can be performed, for example, by flow cytometry. -, mass cytometry, etc., in patient samples, or in patient samples over time, e.g., after therapeutic treatment, during disease onset, etc. + KIR +The method may include detecting the presence of regulatory T cells. The cells may be more specifically characterized by expression of specific KIR proteins, including, but not limited to, one or more of KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, and KIR3DL2, and may specifically include one or more of KIR2DL2, KIR2DL3, and KIR3DL1. The cells may be further characterized by expression of markers, including, but not limited to, CD38, HLA-DR, CD29, CD44, CD122, and the like. The cells may also be characterized for expression of TCRs associated with regulatory T cells, for example, by binding to multimeric peptide / MHC polypeptides. The presence of increased levels of regulatory T cells may indicate the presence of autoimmune complications in viral diseases, but may also be associated with improved disease prognosis. Depending on the prognosis, the patient may be treated with the therapeutic methods described herein or with conventional therapeutic methods. Assays may also evaluate patient samples for the presence of pathogenic CD4+ T cells, eg, expansion, γδ T cell expansion, etc., specific for autoantigens, etc. [Brief explanation of the drawings]

[0020] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, the dimensions of the various features have been arbitrarily expanded or reduced for clarity. The drawings include the following figures:

[0021] [Figure 1]Concurrent activation of all T cells after EAE immunization. As shown in a, C57BL / 6J mice were immunized for EAE induction, and cells from the blood, CNS, spleen, and draining LN were isolated and analyzed for total T cell frequency at different days post-immunization (PI). b–e show the total frequencies of CD4+, CD8+, and γδ+ T cells in the blood (b), CNS (c), LN (d), and spleen (e) on different days after immunization: D0 (non-immunized) (n = 5), D3 (n = 4), D5 (n = 4), D7 (n = 4), D10 (n = 4), D12 (n = 4), D15 (n = 5), D17 (n = 5), D19 (n = 5), D21 (n = 5), D23 (n = 5), and D30 (n = 3). Data are mean ± sem and represent two independent experiments. *P = 0.05, **P = 0.0097, ***P = 0.0008, ****P < 0.0001, one-way analysis of variance (ANOVA) followed by Dunnett's post hoc multiple comparison test. [Figure 2] CD4+, CD8+, and γδ+ T cells were clonally expanded after EAE. C57BL / 6J mice were immunized for EAE induction (a). On different days after immunization (D0 (non-immunized), D7, D10, D15, and D19), blood and CNS-infiltrating CD4+, CD8+, and γδ+ T cells were single-cell FACS-sorted based on activation markers (CD44 high, CD62L low), and their TCRs were sequenced (b). CFA, complete Freund's adjuvant; PTX, pertussis toxin. c, d are pie charts showing the clonal expansion of CD4+, CD8+, and γδ+ T cells in the blood (c) and CNS (d) on different days after immunization. Each pie chart is a summary of the number of TCR sequences from three individual mice pooled together per tissue per time point. The number of cells in which β or both γ and δ chains were successfully identified is indicated above each pie chart. For each TCR clone expressed by more than one cell (clonally expanded), the absolute number of cells expressing that clone is indicated by different color segments. Sequencing data are from one experiment consisting of three individual mice per time point. [Figure 3]Clonally expanded CD8 TCRs do not respond to myelin. Four clonally expanded CD4 TCRs (EAE1-CD4 (a), EAE2-CD4 (b), EAE3-CD4 (c), and EAE4-CD4 (d)) were expressed on SKWαβ- / - cells and stained with MOG35-55 and ovalbumin 327-337 (OVA327) I-AbpMHC tetramer. Nine clonally expanded CD8 TCRs (EAE1-CD8 to EAE9-CD8) were expressed on 58αβ- / - cells (e). Cells were stimulated for 12–16 h with a pool of myelin peptides from MOG, myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), SIINFEKL, myelin, or ovalbumin protein, as well as anti-CD3 and anti-CD28, and examined for expression of the activation marker CD69. Each peptide pool (PP1–PP7) consisted of variable-length peptides (8–12 nucleotides), and each peptide pool contained 50 peptides. 58αβ− / − cells expressing the OT-1 TCR were stimulated with either SIINFEKL peptide or ovalbumin protein. Data represent three independent experiments. [Figure 4] Clonal expanded EAE CD8 TCRs bind novel peptides. Tetramer staining of 9-nucleotide and 10-nucleotide H2-Db yeast-pMHC libraries with the 6218 TCR (a), EAE6-CD8 TCR (b), and EAE7-CD8 TCR (c) at the end of three rounds of selection is shown. Heat maps of amino acid preferences by position for the 6218 (left) (d), EAE6-CD8 (center) (e), and EAE7-CD8 (right) (f) TCRs after three rounds of selection are shown. The sequences of the top seven peptides after three rounds of selection for each TCR are shown below, along with their amino acid preferences. MHC anchor residues are colored red (P5, Asn; N) or blue (P9 / 10, Met, Ile, and Let; M / I / L). Each TCR was screened once on the yeast library. [Figure 5]CD8+ T cell-specific SP immunization abrogates EAE. Spleen and LN cells were isolated from non-immunized mice and mice 10 days after immunization, and the cells were enriched for CD4+ T cells specific for I-Ab MOG35-55 (a) or CD8+ T cells specific for SP (SMRPNHFFFL, ASRSNRYFWL, HDRVNWEYI, and YQPGNWEYI) (b). Representative dot plots of non-immunized and immunized mice are shown. Representative data are from two independent experiments. c) The frequency of MOG35-55-specific CD4+ T cells in wild-type (n = 4) and immunized (n = 5 mice) mice. Data are means ± sem. d) The frequency of SP-specific CD8+ T cells in wild-type (n = 4) and different immunization groups (n = 5 mice per group). Data are means ± sem. Figure 1(e) shows the EAE clinical scores (n=10) of C57BL / 6J mice immunized with MOG35-55 + CFA and PTX (n=10), or with an emulsion containing only MOG35-55 + CFA, PTX, and SP, or SP, CFA, and PTX (n=10). Figure 1(f) shows the EAE clinical scores (n=10) of C57BL / 6J mice immunized with MOG35-55 + CFA and PTX (n=10) and then challenged with SP, ICFA, and PTX 7 days after immunization. Figure 1(g) shows the EAE clinical scores (n=10) of C57BL / 6J mice immunized with SP, CFA, and PTX and then challenged with MOG35-55 + ICFA and PTX 7 days after immunization. Data are mean ± sem and represent two independent experiments. **P=0.0040, ****P<0.0001, regression analysis by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc multiple comparison test. [Figure 6]CD8+ T cell-specific SP immunization suppresses MOG35-55-specific CD4+ T cells. C57BL / 6J mice were immunized with an emulsion containing MOG35-55, CFA, and PTX, with or without SP (n=4) or without SP (n=5). Spleen and LN cells were isolated from unimmunized (n=4) and D10-immunized (n=5 mice per group) mice and enriched for MOG35-55I-AbpMHC-specific CD4+ T cells. (a) FACS dot plots from representative mice from different groups. (b) Frequency of MOG35-55-specific CD4+ T cells from each group. Data are mean ± sem and representative of two independent experiments. C57BL / 6J mice were immunized with (n = 2 mice) or without (n = 2 mice) SP as in a and b. Spleen and LN cells isolated from unimmunized (n = 2) and post-D10 immunization mice were enriched for CD4+, CD8+ T cells, or antigen-presenting cells (APCs). Dye-labeled CD4+ T cells from MOG-immunized mice were cocultured with APCs from MOG-immunized mice in the absence (c) or presence (d) of CD8+ T cells from mice immunized with MOG, MOG and SP, or SP alone. Cells were analyzed for proliferation 7 days after coculture. Representative data from two independent experiments are shown. In e, the frequency of CD8+ T cells with a control phenotype (CD44+CD122+Ly49+) is shown in wild-type (n = 4) and different immunization groups (n = 5 mice per group). Data are mean ± SEM and represent two independent experiments. *P = 0.0382; ***P = 0.001; one-way analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparison test. CD4+ T cells from MOG-immunized mice were cocultured without (f) or with (g) total CD8+ T cells, or with purified CD8+CD44+CD122+Ly49+ (Ly49+) (h) or CD8+CD44+CD122+Ly49- (Ly49-) (i) T cells from mice immunized with MOG and SP (n = 2 mice per group). Data represent two independent experiments. (j) Heatmap of gene expression in RNA-seq samples.Genes were selected based on being differentially expressed (log2-transformed fold change >0.75 and corrected P<0.005; and two-tailed Benjamini-Hochberg corrected P<0.005) as defined by DESeq2 in both Ly49+ vs. Ly49- and MOG vs. MOG+SP comparisons. Columns display samples. Rows and columns are ordered based on hierarchical clustering. Normalized gene expression values ​​are centered for each gene by subtracting the mean value of all samples from each sample value. Data represent two independent experiments. [Figure 7] This represents the massive clonal expansion of all T cells after EAE immunization. C57BL / 6J mice were immunized for EAE induction, and cells from the blood, draining LN, spleen, and CNS were isolated and stained with a cocktail of cell surface antibodies on different days after immunization (D0 (non-immunized), D3, D5, D7, D10, D12, D15, D17, D19, D21, D23, and D3). For a, infiltrating CD4+, CD8+, and γδ+ T cells were single-cell sorted on D0 (non-immunized), D7, D10, D15, and D19 after immunization. Cells were subjected to single-cell paired TCR sequencing (n = 3 mice per group / time point). In total, we sequenced 1,302 (CD4+), 1,660 (CD8+), and 1,451 (γδ+) paired TCR sequences. Figures b, c, and d show the mean percentage of clonal expansion of CD4+ (b), CD8+ (c), and γδ+ (d) T cells between non-immunized and immunized mice across all days and tissues combined. Data are mean ± sem. Figure e shows the percentage of identical CD4+, CD8+, and γδ+ TCR sequences shared between the blood and CNS on each day post-immunization. Figure f shows the frequency of the major populations (groups 1–4) of thymus-derived γδ+ T17 (tTγδ17) cells in the blood and CNS on different days post-immunization. Figure g shows the corresponding paired TCRγ and TCRδ sequences that define each major population (groups 1–4) of tTγδ17 cells. [Figure 8]Figure 1 shows the simultaneous activation of all T cells after EAE immunization. The clonally expanded CD8 TCRs are not specific for myelin peptides or proteins. (a)–(f) C57BL / 6J mice were immunized for EAE induction, and cells from the blood, draining LN, and spleen were isolated and analyzed for the total frequency of activated (CD44 high) (a, c, e) and naive (CD62L high) (b, d, f) CD4+ and CD8+ cells. Data are mean ± sem and represent two independent experiments. *P = 0.046, **P = 0.0023, ***P = 0.0002, ***P < 0.0001, one-way analysis of variance (ANOVA) followed by Dunnett's post hoc multiple comparison test. (g) Nine clonally expanded CD8 TCRs (EAE1-CD8 to EAE9-CD8) were retrovirally transduced and expressed in 58αβ- / - cells. Untransduced and transduced cell lines were stained with fluorochrome-conjugated anti-TCRβ and anti-CD3 to determine TCR surface expression. For h, untransduced and transduced EAE-CD8 TCR cell lines were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 12–16 h and surface stained with the activation marker CD69. For i, untransduced 58αβ- / - or OT-1 TCR-transduced cell lines were stimulated with BMDCs pulsed with SIINFEKL peptide or whole OVA protein for 12–16 h, washed, and stained for CD69. For j, unstimulated 58αβ- / - or EAE-CD8 TCR-transduced cell lines were stimulated with a pool of peptides (PP1–PP7) consisting of MOG, MBP, PLP, MAG, and SIINFEKL peptides and examined for CD69 expression (CD69 for EAE1-CD8 TCR is indicated in the figure). Peptides were of variable length (8-12 nucleotides). Each peptide pool contained 50 peptides. Data are representative of three independent experiments. [Figure 9]Generation and functional validation of the H2-Db yeast peptide-MHC library. (a) Schematic diagram of a mouse class I MHC H2-Db displayed on yeast as β2m, α1, α2, and α3, with peptides covalently attached to the MHC N-terminus. (b) Design of the peptide library displayed by the H2-Db. The design is based on the structure of 6218 TCR (SSLENFRAYV, DbPA224) (PDB accession 3PQY) bound to the H2-Db-restricted acid polymerase peptide 224-233. (c) Mutations (α2-W131 to α2-G131) required for proper folding of the H2-Db displayed on yeast are shown. The mutations were derived from error-prone mutagenesis. (d) Designs for two different lengths of H2-Db libraries are shown. For the nine amino acid (9MER) library, residues P1 through P9 were randomized, with limited diversity at MHC anchor positions P5 (Asn, N) and P9 (Met, Ile, and Leu, M / I / L). For the ten amino acid (10MER) library, residues P1 through P10 were randomized, with limited diversity at MHC anchor positions P5 (Asn, N) and P10 (Met, Ile, and Leu, M / I / L). TCR contact residues are colored pink, and MHC anchor residues are colored red or blue. e-g. Selection of the PA224-H2-Db error-prone library with soluble TCR6218. Increased cMyc expression among the induced yeast peptide-H2-Db error-prone library at different rounds of selection (RD1–RD4) (e, g) and soluble TCR6218 tetramer staining in the error-prone H2-Db library after RD4 (g) are shown. Each TCR was screened once on the yeast library. [Figure 10]Figure 1 shows in vitro and in vivo characterization of surrogate peptide-specific CD8+ T cells after EAE. For a, Jurkat αβ- / - T cells expressing 6218, EAE6, and EAE7-CD8 TCRs were stained with the corresponding yeast library-enriched pMHC tetramers (SSLENFRAYV, ASRSNRYFWL, SMRPNHFFFL, YQPGNWEYI, and HDRVNWEYI, respectively). For b, spleen and LN cells were isolated from non-immunized mice (n = 4) or mice immunized with MOG (n = 5), MOG + SP (n = 5), or SP (n = 5), and cells were enriched for SP-specific CD8+ T cells bearing pMHC tetramers. Representative flow cytometry gating strategies are shown for different cell surface markers and tetramer-specific cells. For c, representative flow cytometry data are shown for activation status (defined as CD44+CD62L−) on SP-specific CD8+ T cells (ASR, HDR, SMRP, and YQP-tet+) derived from wild-type and different immune groups (MOG, MOG+SP, and SP). For d, the activation / effector phenotype of SP-specific CD8 T cells (ASR, HDR, SMRP, and YQP-tet) from wild-type (n = 5) and different immunization groups (MOG (n = 3), MOG + SP (n = 4), and SP (n = 3)) is quantified (n = 5 mice per group). *P = 0.0169, **P = 0.0020, ****P < 0.0001, one-way analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparison test. Data are mean ± s.e.m. C57BL / 6J mice were immunized for EAE with an emulsion containing MOG35-55, CFA + PTX, with (n = 10) or without (n = 10) influenza peptide (SSLENFRAYV). Clinical scores after immunization were recorded. Data are mean ± s.e.m. and represent two independent experiments. [Figure 11]CD8+ T cell-specific SP immunization suppresses MOG35-55-specific CD4+ T cells and induces CD8+ T cells with a regulatory phenotype. For a–c, C57BL / 6J mice were immunized with an emulsion containing MOG35-55, CFA, and PTX (n=5), or MOG35-55, CFA, PTX, and SP (n=5). Spleens and LN cells were isolated, stained, and enriched for MOG35-55I-AbpMHC-specific CD4+ T cells and an irrelevant tetramer from unimmunized mice (a) and mice 10 days after immunization (b, c). Representative FACS plots for different groups are shown. For d-g, spleen and LN cells were isolated from non-immunized mice (n = 5) (d) and mice 10 days after immunization with MOG (n = 5) (g), MOG + SP (n = 5) (f), or SP alone (n = 4) (e). They were then stained and enriched for SP-specific CD8+ T cells using pMHC tetramers. Representative FACS dot plots of CD8+ T cells with the control phenotype (CD44+CD122+Ly49+) from each group are shown. For h, tetramer-positive (i.e., ASR, HDR, SMRP, and YQP-tet+) CD8+ T cells were subgated for CD122, CD44, and Ly49. The frequency of CD122+CD44+Ly49+ cells among SP-specific cells is shown between different immunization groups. ***P = 0.0002, one-way analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparison test. Data are means±sem and represent two independent experiments. [Figure 12]The CD8+ T cells elicited after MOG+SP immunization were specific, and their suppression was mediated by perforin. Adoptive transfer of CD122+CD44+Ly49+ abrogated EAE, and SP caused a more severe inflammatory uveitis than IRBP peptide alone. (a-g) C57BL / 6J mice were immunized with an emulsion containing MOG35-55, CFA, and PTX; MOG35-55, CFA, PTX, and SP (b); or MOG35-55, CFA, PTX, and influenza peptide (e). Spleen and LN cells were isolated from mice 10 days after immunization, and cells were enriched for CD4+ or CD8+ T cells or APCs by FACS. CD4+ T cells from MOG-immunized mice were labeled with CTV and cocultured with APCs from MOG-immunized mice in the absence (a) or presence of CD8+ T cells from wild-type mice (c), or CD8+ T cells from mice immunized with MOG+SP (b), CFA+PTX (d), MOG+influenza peptide (e), or perforin knockout (PENKO) mice immunized with MOG+SP (f). For g, CTV-labeled CD4+ T cells from mice immunized with MOG35-55, CFA+PTX were cocultured with CD8+ T cells from mice immunized with MOG35-55, CFA, PTX+SP in the presence of anti-Qa-1b antibody (10 μg ml-1). For h, CTV-labeled CD4+ T cells from mice immunized with OVA329-337, CFA, and PTX were cocultured with CD8+ T cells from mice immunized with MOG35-55, CFA, and PTX and SP. After 7 days of coculture, cells were washed, stained for surface markers, and analyzed for CD4+ T cell proliferation (CTV dilution). Representative data are from two independent experiments. For i, C57BL / 6J mice were immunized with MOG35-55, CFA, and PTX and SP (n=10). 10 days after immunization, spleen and LN cells were isolated, stained, and enriched for CD8+ T cells, followed by FACS for Ly49+ and Ly49- cells.Sorted Ly49+ and Ly49- cells were adoptively transferred (8 million cells per mouse) into C57BL / 6J mice (n = 5 mice per group) at the time of immunization. Clinical scores after adoptive transfer and immunization are shown. ****P < 0.0001, regression analysis by two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc multiple comparison test. Data are mean ± SEM and are representative of two independent experiments. For j-b, in eyes of wild-type, untreated mice, the retina showed a normal layered pattern and no leukocytes were present in the vitreous. For k, after subcutaneous injection of IRBP peptide antigen, there was a mild inflammatory reaction in 40% of eyes, with activated leukocyte infiltration in the vitreous (red arrows) and mild destruction of photoreceptors in the outer nuclear layer of the retina (black arrows). For l, after subcutaneous injection of both IRBP and SP, there was a severe inflammatory reaction in 80% of eyes, with activated leukocyte infiltration of the vitreous (red arrow) and severe destruction of retinal photoreceptors (black arrow). INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RGC, retinal ganglion cell layer; RPE, retinal plexiform layer. Five C57BL / 6J mice were examined for each condition. EAU was induced, and the mice were euthanized 21 days after immunization. Mouse eyes were enucleated and fixed, and pupil-optic nerve sections were examined histologically. For m-o, C57BL / 6J mice were immunized with IRBP, CFA, and PTX, with or without SP. Spleen and LN cells were isolated from the mice 10 days after immunization, and the cells were enriched for CD4+ or CD8+ T cells or APCs by FACS. CTV-labeled CD4+ T cells from IRBP-immunized mice were cocultured with APCs from IRBP-immunized mice and purified CD8+Ly49+ T cells (n), CD8+Ly49- T cells (o), or no CD8+ T cells (m) from mice immunized with IRBP and SP. After 7 days of coculture, cells were washed, stained with surface markers, and analyzed for CD4+ T cell proliferation. [Figure 13]Figure 1 shows transcriptional profiling of Ly49+ vs. Ly49- cells. C57BL / 6J mice were immunized with SP, CFA, and PTX (n=3). Spleen and LN cells were isolated from D10 mice, enriched for CD8+ T cells by FACS, and sorted for Ly49- and Ly49+ cells, followed by bulk RNA-seq analysis. (a) shows a heatmap of differentially expressed genes (log2-transformed fold change >2 and corrected P<0.005) in Ly49+ / Ly49- RNA-seq samples. Columns indicate samples. Rows and columns are ordered based on hierarchical clustering. Normalized gene expression values ​​are centered for each gene by subtracting the mean value of all samples from each sample value (2-3 mice per group). (b) Gene oncology enrichment analysis of differentially expressed genes (log2-transformed fold change >2 and two-sided Benjamini-Hochberg corrected P < 0.005 from DESeq2) between Ly49+ and Ly49- RNA-seq samples. The Y-axis represents the top 30 enriched gene ontologies (genes from gene ontologies highlighted in green are in Supplementary Table 6). The X-axis value is the fraction of differentially expressed genes within that ontology. The dot color represents the significance of gene ontology enrichment (one-sided Fisher's exact test), and the dot size represents the number of differentially expressed genes. The plot was generated with the R package "clusterProfiler." (c) Volcano plot (3 mice per group) depicting the difference in gene expression between Ly49+ and Ly49- samples. Each dot represents a gene. The list of genes differentially expressed in CD4+ T regulatory cells 25 is colored red if the gene is expressed in both MOG and SP RNA-seq samples, and green if not. The horizontal dotted line is generated at -log10 (0.05), and the two vertical dotted lines represent the log2 (2) fold change. Genes with negative fold changes are highly expressed in Ly49+ cells. [Figure 14]Figure 1 shows the primary clonal expansion of CD8+ T cells in patients with recent-onset MS. For a-c, BMCs from healthy controls (HC) (n = 4) and patients with MS (n = 18) were stained and analyzed by flow cytometry to determine T cell frequencies. The frequencies of CD4+ (a), CD8+ (b), and γδ+ (c) T cells are shown. Data are mean ± sem. For d and e, brain-homing and activated (CD49d+CD29+HLA-DR+CD38+) CD8+ T cells were single-cell sorted from PBMCs from healthy controls (d) and patients with newly diagnosed MS (e). Cells were subjected to single-cell paired TCR sequencing. Pie charts showing the clonal expansion of CD8+ T cells in healthy controls (n = 10) and patients with MS (n = 18) are shown. The number of cells in which the β chain was successfully identified is indicated above the pie chart. For each TCR clone expressed by more than one cell (clonally expanded), the absolute number of cells expressing that clone (≧2, ≧5, ≧10, ≧20, and ≧50) is indicated by different color segments. [Figure 15] Figure 1 depicts the TCR repertoire of brain-homing and activated CD4+ T cells in patients with recent MS. For a and b, brain-homing and activated (CD49d+CD29+HLA-DR+CD38+) CD4+ T cells were single-cell sorted from PBMCs of healthy controls (a) and patients with recent MS (b). Cells were subjected to single-cell paired TCR sequencing. Pie charts depicting clonal expansion of CD4+ T cells in healthy controls (n = 10) and patients with MS (n = 18) are shown. The number of cells in which the β chain was successfully identified is indicated above the pie chart. For each TCR clone expressed by two or more cells (clonally expanded), the absolute number of cells expressing that clone (≥ 2, ≥ 5, ≥ 10, ≥ 20, and ≥ 50) is indicated by different color segments. [Figure 16]Figure 1 depicts the TCR repertoire of brain-homing and activated γδ T cells in a patient with recent MS. For a and b, brain-homing and activated (CD49d+CD29+HLA-DR+CD38+) γδ T cells were single-cell sorted from PBMCs of a healthy control (a) and a patient with recent MS (b). Cells were subjected to single-cell paired TCR sequencing. Pie charts depicting clonal expansion of γδ T cells in healthy controls and patients with MS are shown. The number of cells in which the δ chain was successfully identified is indicated above the pie chart. For each TCR clone expressed by two or more cells (clonally expanded), the absolute number of cells expressing that clone (≥2, ≥5, ≥10, ≥20, and ≥50) is indicated by different color segments. From single-cell sorted γδ T cells, RAR-related orphan receptor (ROR) transcripts were amplified using gene-specific primers and simultaneously sequenced for the γ and δ chains. For c, the number of γδ T cells that were positive for RORC transcripts is shown in healthy controls (n=10) and patients with MS (n=18). *P=0.0301, paired t-test. Data are mean±sem. [Figure 17]Frequency of KIR+ CD8+ T cells in human autoimmune diseases. (a) Representative contour plots (left) and summary histograms (right) show the frequency of KIR+ CD8+ T cells (CD3+ CD56-) in the peripheral blood of healthy controls (HC, N = 16) and patients with systemic lupus erythematosus (SLE, N = 22), multiple sclerosis (MS, N = 10), or celiac disease (CeD, N = 14), analyzed by flow cytometry. KIR+ cells were detected with PE-conjugated antibodies against KIR2DL1 (clone no. 143211), KIR2DL2 / L3 (Dx27), KIR2DL5 (UP-R1), KIR3DL1 (Dx9), and KIR3DL2 (clone no. 539304). *P < 0.05, one-way analysis of variance (ANOVA) corrected for multiple comparisons. (b) Correlation between the frequency of KIR+ CD8+ T cells and autoimmune CD4+ T cells (CD45RA-, CD62L-, PD-1+, CXCR3+, CD39+, CD38+, CD127-, CD25low, CD161+, and ICOS+ CD4+ T cells) in the blood of SLE patients (N=11). R2=0.8479, P=0.0002***. (c) Expression of KIR transcripts (KIR3DL1, KIR2DL3, and KIR2DL2) in CD8+ T cells from healthy kidneys versus SLE nephritic kidneys. (d) Expression of KIR transcripts (KIR3DL1, KIR2DL3, and KIR2DL2) in synovial CD8+ T cells and FOXP3 expression in synovial CD4+ T cells from rheumatoid arthritis (RA) and osteoarthritis (OA). [Figure 18]Figure 1 shows depletion of gliadin-specific CD4+ T cells by KIR+ CD8+ T cells. (a) Experimental schematic. (b) Representative contour plot showing tetramer binding of 715 CD4+ T cells after enrichment by MACS column, and a summary of the number of gliadin-specific CD4+ T cells (binding to HLA-DQ2.5 tetramers complexed with gliadin peptides) per million CD4+ T cells on day 6 (N = 5). *P < 0.05, **P < 0.01, Friedman test corrected for multiple comparisons. (c) Representative contour plot and scatter plot summary showing Annexin V binding of gliadin-specific (tetramer-positive) CD4+ T cells from cultures harvested on day 3. **P < 0.01, one-way analysis of variance (ANOVA) corrected for multiple comparisons. [Figure 19]Increased KIR+ CD8+ T cells in COVID-19 patients. a) Shows the frequency of autoimmune CD4+ T cells (CD45RA-, CD62L-, PD-1+, CXCR3+, CD39+, CD38+, CD127-, CD25low, CD161+, and ICOS+ CD4+ T cells) in healthy adults (N = 18) versus COVID-19 patients (N = 54) (left, *P < 0.05, unpaired t-test), and in healthy adults versus COVID-19 patients with mild (N = 23), moderate (N = 17), or severe (N = 13) disease (right, *P < 0.05, **P < 0.01, ****P < 0.0001, one-way analysis of variance (ANOVA) corrected for multiple comparisons). (b) Representative contour and scatter plot summary showing the percentage of KIR cells among CD8+ T cells derived from blood from healthy controls and COVID-19 patients with various disease severities. Left: ***P<0.001, unpaired t-test. Right: *P<0.05, ***P<0.001, ****P<0.0001, one-way analysis of variance (ANOVA) corrected for multiple comparisons. (c) Correlation between the frequencies of KIR+ CD8+ T cells and autoimmune CD4+ T cells in the blood of COVID-19 patients (N=54). r=0.2811, P=0.0377*. (d) Frequencies of autoimmune CD4+ T cells, KIR+ CD8+ T cells, and CD4+ Tregs (CD25-high, CD127-low) in the blood of COVID-19 patients with and without vasculitis. *P<0.05, ****P<0.0001, unpaired t-test. e) Expression of KIR transcripts (KIR3DL1, KIR3DL2, KIR2DL3, and KIR2DL1) in CD8+ T cells from bronchoalveolar lavage fluid of healthy controls and COVID-19 patients with moderate or severe disease. [Figure 20]Single-cell RNA-seq analysis of KIR+ CD8+ T cells in blood. (a-b) Single-cell RNA-seq analysis by 10x Genomics of total CD8+ T cells derived from the blood of healthy subjects (N = 10), MS patients (N = 6), and COVID-19 patients (N = 25). (a) UMAP plot of eight subpopulations identified by unsupervised clustering based on the expression of marker genes in each cluster. (b) UMAP plot showing the distribution of KIR+ CD8+ T cells (expressing KIR3DL1, KIR3DL2, KIR2DL1, or KIR2DL3) and KIR- CD8+ T cells derived from healthy controls (HCs), MS patients, and COVID-19 patients. (c-e) KIR+CD8+ T cells in the blood of healthy controls (N = 10) and patients with MS (N = 2), SLE (N = 6), and CeD (N = 5) were sorted for single-cell RNA-seq using the Smart-seq2 protocol and analyzed using the R package "Seurat." (c) UMAP plots showing the separation of KIR+CD8+ T cells into six clusters (top) and the distribution of expanded (two or more cells expressing the same TCR) and non-expanded (cells expressing a unique TCR) cells (bottom). (d) UMAP plots of KIR+CD8+ T cells from MS, SLE, CeD, and HC are shown, with expanded and non-expanded cells annotated with different colors (expanded: red, non-expanded: blue, other diseases: gray). (e) Heatmap showing the expression of the top 10 differentially expressed genes in each cluster. The categories of each gene group are annotated on the left. f) Demonstration of the role of KIR+CD8+ T cells in autoimmune disorders and infections, where KIR+CD8+ T cells suppress pathogenic CD4+ T cells arising from autoreactivity in autoimmune diseases or cross-reactivity through cytotoxic activity against self-antigens in infectious diseases. DETAILED DESCRIPTION OF THE INVENTION

[0022] Before the present methods are described, it is to be understood that this invention is not limited to particular methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present invention is limited only by the appended claims.

[0023] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within this stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any specifically excluded limit in the stated range. As used in this specification and the appended claims, the articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to those described herein can also be used to carry out or test the present invention, preferred methods and materials are described below.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material in connection with which the publication is cited.

[0025] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0026] General methods in molecular and cellular biochemistry are described in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0027] The present invention has been described in terms of particular embodiments discovered or contemplated by the inventors to comprise preferred modes for carrying out the invention. Those skilled in the art will understand, in light of this disclosure, that numerous modifications and variations can be made in the particular embodiments exemplified without departing from the intended scope of the invention. All such modifications are intended to be within the scope of the appended claims.

[0028] Compositions and methods are provided for the characterization, use, and manipulation of antigen-specific, class I MHC-restricted, CD8+ regulatory T cells.

[0029] The method of the present invention can be used for preventive or therapeutic purposes. As used herein, the term "treating" refers to both the prevention of recurrence and the treatment of an existing condition. For example, the prevention of autoimmune disease can be achieved by administering a drug before the occurrence of a recurrence. As used herein, "treatment" encompasses any treatment of disease in mammals, particularly humans, and includes (a) preventing the occurrence of a disease or condition in a subject who may be predisposed to the disease or condition but has not yet been diagnosed with it, (b) inhibiting disease symptoms, i.e., preventing their development, or (c) alleviating disease symptoms, i.e., causing regression of the disease or condition. Treatment of ongoing disease, in which treatment stabilizes or improves the patient's clinical condition, is particularly targeted.

[0030] "Inhibiting" the onset of a disorder means either reducing the likelihood of the onset of the disorder or completely preventing the onset of the disorder. Reducing the severity of relapse means that the clinical signs associated with relapse are less severe in the presence of treatment than in untreated disease. As used herein, onset can also refer to relapse in patients with ongoing relapsing-remitting disease. The method of the present invention can be specifically applied to patients diagnosed with inflammatory diseases, including, for example, autoimmune diseases. Treatment can be aimed at treating or reducing the severity of relapse, which is an exacerbation of an existing condition.

[0031] As used herein, "diagnosis" generally includes determining a subject's susceptibility to a disease or disorder, determining whether a subject is currently affected by a disease or disorder, prognosis of a subject affected by a disease or disorder (e.g., identifying the disease state, stage of the disease, or responsiveness of the disease to treatment), and the use of therapeutic assays (e.g., monitoring a subject's condition to provide information regarding the efficacy or effectiveness of treatment).

[0032] The term "biological sample" encompasses a variety of sample types obtained from an organism and can be used in diagnostic or monitoring assays. The term includes blood, cerebrospinal fluid, and other liquid samples of biological origin, as well as solid tissue samples, such as biopsy samples or tissue cultures or cells derived therefrom and their progeny. The term also includes samples that have been manipulated in any way after procurement, such as by treatment with reagents, solubilization, or enrichment for particular components. The term encompasses clinical samples, including cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0033] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, e.g., humans, non-human primates, mice, rats, guinea pigs, rabbits, etc.

[0034] The term "agent," as used herein, includes any substance, molecule, element, compound, entity, or combination thereof. It includes, but is not limited to, for example, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, etc. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms "agent," "substance," and "compound" can be used interchangeably.

[0035] "Favorable conditions" shall have a meaning that depends on the context in which the term is used. That is, when used in connection with an antibody, the term shall refer to a condition that allows the antibody to bind to its corresponding antigen. When used in connection with contacting an agent with a cell, the term shall refer to a condition that allows the agent, which is capable of doing so, to enter the cell and perform its intended function. In one embodiment, the term "favorable conditions" as used herein refers to a physiological condition.

[0036] A "subject" or "patient" in the context of the present teachings is generally a mammal. Mammals other than humans can be advantageously used as subjects that represent animal models of inflammation. The subject can be male or female.

[0037] "Analyzing" includes determining a set of values ​​associated with a sample by measuring a marker in a sample (e.g., the presence or absence of a marker or constitutive expression level) and comparing said measurements to measurements in a sample or set of samples from the same subject or other control subjects. In particular, the cell surface markers of the present teachings can be analyzed by any of a variety of conventional methods known in the art. "Analyzing" can include, for example, performing a statistical analysis to determine whether a subject is a responder or non-responder to a therapy (e.g., administration of a regulatory peptide treatment described herein).

[0038] "Pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" refer to excipients, diluents, carriers, and adjuvants that are generally safe, non-toxic, and not biologically or otherwise undesirable and that are useful in the preparation of pharmaceutical compositions, and include excipients, diluents, carriers, and adjuvants that are acceptable for veterinary and human pharmaceutical use. As used herein and in the claims, "pharmaceutically acceptable excipients, diluents, carriers, and adjuvants" includes both one and more than one such excipient, diluent, carrier, and adjuvant.

[0039] As used herein, "pharmaceutical composition" is meant to encompass compositions suitable for administration to a subject, e.g., a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants that can induce an undesirable response in a subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). Pharmaceutical compositions can be designed to be administered to a subject or patient in need thereof via a number of different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intrathecal, intramuscular, subcutaneous, etc.

[0040] "Unit dosage" refers to a physically discrete unit suited as a unitary dosage for a particular individual to be treated. Each unit can contain a predetermined quantity of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for unit dosage forms can be dictated by (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds.

[0041] "Pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and useful in preparing the desired pharmaceutical composition, and includes excipients acceptable for veterinary and human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous.

[0042] "Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the compound are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, e.g., sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts include those formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and methanesulfonic acid and benzenesulfonic acid). Also included are acid addition salts formed with any alkane and arenesulfonic acid. Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, e.g., C 1~6 Examples include alkyl esters. When two acidic groups are present, the pharmaceutically acceptable salt or ester can be a mono-acid-mono-salt or ester, or a di-salt or ester; similarly, when more than two acidic groups are present, some or all of such groups can be salified or esterified. The compounds named in this invention can exist in unsalted or unesterified form, or salified and / or esterified form, and the naming of such compounds is intended to include both the original (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain compounds named in this invention may exist in two or more stereoisomeric forms, and the naming of such compounds is intended to include all single stereoisomers and all mixtures of such stereoisomers (whether racemic or not).

[0043] The terms "pharmaceutically acceptable" and "physiologically acceptable," and grammatical variations thereof, are used interchangeably to refer to compositions, carriers, diluents, and reagents, and indicate that these materials can be administered to a human without producing undesirable physiological effects that would prohibit administration of the composition.

[0044] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0045] As used herein, the term "in combination" refers to the use of two or more prophylactic and / or therapeutic agents. The use of the term "in combination" does not limit the order in which prophylactic and / or therapeutic agents are administered to a subject with a disorder. A first prophylactic or therapeutic agent can be administered to a subject with a disorder before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent.

[0046] disease state In some embodiments, the methods of the invention involve treating, isolating cell populations derived therefrom, or diagnosing individuals "at risk" of developing an inflammatory disease or in the "early stages" of an inflammatory disease. "At risk" of developing an inflammatory disease includes (1) individuals who are at high risk of developing an inflammatory disease, and (2) individuals who exhibit a "full clinical" disease state but do not meet the diagnostic criteria for an inflammatory disease (and therefore are not formally considered to have an inflammatory disease).

[0047] An individual who is "at high risk" for developing an inflammatory disease (also referred to as "at risk" for developing) is one who, compared to the general population, has a higher likelihood of developing an inflammatory disease or a disease associated with inflammation. Such individuals may have the following characteristics: a family history of an inflammatory disease; the presence of a particular genetic variant (gene) or combination of genetic variants that predisposes the individual to such an inflammatory disease; the presence of physical findings, laboratory test results, imaging findings, or marker test results (also referred to as "biomarker" test results) associated with the development of an inflammatory disease, or the presence of marker test results associated with the development of a metabolic disease; the presence of clinical signs associated with an inflammatory disease; the presence of certain symptoms associated with an inflammatory disease (although the individual is often asymptomatic), the presence of markers of inflammation (also referred to as "biomarkers"); and the presence of a specific condition associated with an inflammatory disease or inflammation. or other findings indicating an increased lifetime likelihood of developing a disease associated with inflammatory disease. Most individuals at increased risk of developing an inflammatory disease or disease associated with inflammation are asymptomatic and do not suffer from any symptoms associated with the disease for which they are at increased risk.

[0048] Individuals at high risk for developing an inflammatory disease or an inflammation-related disease include, but are not limited to, individuals who exhibit a "full clinical disease state." A pre-disease state can be diagnosed based on presenting symptoms, physical findings, laboratory findings, imaging findings, and other findings that result in an individual meeting diagnostic criteria for an inflammatory disease and therefore being formally diagnosed. An individual with "pre-clinical disease" exhibits findings suggesting that the individual is in the process of developing an inflammatory disease, but does not exhibit findings, including symptoms, clinical findings, laboratory findings, and / or imaging findings, necessary to meet the diagnostic criteria for a formal diagnosis of an inflammatory disease. In some embodiments, individuals exhibiting a pre-clinical disease state possess a gene variant or combination of gene variants that increases their risk of developing the disease compared to individuals who do not possess that gene variant or combination of gene variants. In some embodiments, these individuals have laboratory findings, physical findings, symptoms, or imaging findings that increase their risk of developing an inflammatory disease. In some embodiments, individuals with a pre-clinical disease state are asymptomatic. In some embodiments, individuals with a preclinical disease state exhibit increased or decreased levels of expression of particular genes, particular proteins, inflammatory markers, metabolic markers, and other markers.

[0049] In certain embodiments, the present invention is directed to the treatment of individuals with established inflammatory or inflammation-related diseases. Inflammatory diseases can be diagnosed based on an individual exhibiting symptoms, signs, clinical features, laboratory test results, imaging test results, biomarker results, and other findings that allow a physician to formally diagnose the individual as having an inflammatory disease, and the findings can be linked to the CD4+ receptors that cause the disease. + Specific expansion of T cells, expansion of γδT cells, and associated regulatory CD8 + This may include the expansion of T cells.

[0050] In some embodiments, an established inflammatory disease is an inflammatory disease in which an individual has been formally diagnosed by a doctor for more than six months. In established inflammatory diseases, the signs or symptoms of the disease may be more severe, for example, compared with the symptoms of an individual diagnosed with early inflammatory diseases. In established inflammatory diseases, the disease process may cause tissue or organ damage. As described herein, in certain embodiments, determining inflammation in an individual with established disease may include analyzing the individual for the presence of at least one marker indicating the presence of inflammation.

[0051] Inflammatory diseases are considered to be diseases that show clinical signs (abnormal clinical markers) such as visible inflammation, including pain, swelling, heat, and redness, and in the context of the present invention, include antigen-specific pathogenic CD4+ T cells as the causative agent. Inflammatory diseases include, but are not limited to, autoimmune diseases, and may further include diseases with specific T cell-mediated components. Autoimmune diseases include, for example, MS, SLE, RA, IDDM, NMO, etc.

[0052] Inflammatory demyelinating diseases of the central nervous system are of particular interest and include, but are not limited to, multiple sclerosis (MS), neuromyelitis optica (NO), and experimental acquired encephalitis (EAE). Demyelinating diseases can be initiated by peptides of myelin-associated proteins, such as MOG, MBP, and MAG. Demyelinating inflammatory diseases of the peripheral nervous system include acute inflammatory demyelinating polyradiculoneuropathy, acute motor axonal neuropathy, acute motor and sensory axonal neuropathy, Miller-Fisher syndrome, and Guillain-Barré syndrome (GBS) with acute panautonomic neuropathy subtype, chronic inflammatory demyelinating polyneuropathy (CIDP) with the classic CIDP subtype, CIDP with diabetes, and CIDP / monoclonal immunoglobulin of undetermined significance. These include MGUS, sensory-type CIDP, multifocal motor neuropathy (MMN), multifocal acquired demyelinating sensory and motor neuropathy or Lewis-Sumner syndrome, multifocal acquired sensory and motor neuropathy, and acquired distal demyelinating sensory neuropathy. Although not traditionally classified as an inflammatory disease, ALS has been found to have increased numbers of CD49e macrophages and may be treated by the methods described herein.

[0053] Multiple sclerosis is characterized by various symptoms and signs of CNS dysfunction, accompanied by remissions and recurrent exacerbations. Classifications for analysis using the methods of the present invention include relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS). The most common symptoms are paresthesia in one or more limbs, trunk, or one side of the face; weakness or clumsiness in the legs or hands; or visual disturbances, such as partial blindness and pain in one eye (retrobulbar optic neuritis), decreased visual acuity, or scotoma. Other common early symptoms include ophthalmoplegia resulting in double vision (diplopia), temporary weakness in one or more limbs, slight limb stiffness or unusual fatigue, mild gait disturbance, difficulty with bladder control, dizziness, and mild emotional disturbance, all of which indicate scattered central nervous system involvement and often occur months or years before the disease is recognized. Excessive fever can exacerbate symptoms and signs.

[0054] Neuromyelitis optica (NMO), or Devic's disease, is an autoimmune inflammatory disorder of the optic nerve and spinal cord. Although inflammation can affect the brain, the disorder differs from multiple sclerosis in that it has a different response pattern to treatment, possibly a different pattern of autoantigens, and the involvement of different lymphocyte subsets.

[0055] The primary symptom of Devic's disease is loss of vision and spinal cord function. In other etiologies of optic neuritis, visual impairment usually manifests as decreased visual acuity, but visual field defects or loss of color vision may occur alone or precede formal loss of vision. Spinal cord dysfunction can lead to muscle weakness, decreased sensation, or loss of bladder and bowel control. Spinal cord damage can range from inflammatory demyelination to necrotic damage of white and gray matter. The inflammatory lesions in Devic's disease have been classified as type II lesions (complement-mediated demyelination), but they differ from MS pattern II lesions in their prominent perivascular distribution. Thus, the pattern of inflammation is often quite different from that seen in MS.

[0056] Rheumatoid arthritis (RA) is a chronic syndrome generally characterized by symmetric inflammation of peripheral joints, which can result in progressive destruction of articular and periarticular structures with or without common symptoms (Firestein (2003) Nature 423(6937):356-61; McInnes and Schett. (2011) N Engl J Med.365(23):2205-19). The cause is unknown. A genetic predisposition has been identified and, in some populations, has been localized to a pentapeptide within the HLA-DRβ1 locus of class II histocompatibility complexes. Environmental factors may also play a role.

[0057] Notable immunological abnormalities that may be important in pathogenesis include antibodies and immune complexes found in synovial fluid cells and vasculitis. Plasma cells produce antibodies that contribute to these complexes. Lymphocytes infiltrating the synovial tissue are primarily helper T cells, capable of producing proinflammatory cytokines. Macrophages and their cytokines (e.g., tumor necrosis factor, granulocyte-macrophage colony-stimulating factor) are also abundant in diseased synovium. Increased adhesion molecules contribute to the migration and retention of inflammatory cells in the synovial tissue. An increase in macrophage-derived lining cells is prominent, along with several lymphocytic and vascular changes in early disease.

[0058] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by butterfly rash, oral ulcers, photosensitivity, seizures, low white blood cell counts, low platelet counts, seizures, a positive antinuclear antibody (ANA) test, and positive results for other autoantibodies. SLE is an autoimmune disease often characterized by polyclonal B cell activation, which results in a variety of antiprotein and nonprotein autoantibodies that lead to immune complexes and inflammation that contribute to tissue damage, but also pathological CD4 + T cells may also be involved. SLE has a variable course characterized by exacerbations and remissions, making it difficult to study. For example, some patients present primarily with skin rash and joint pain, may experience spontaneous remissions, and may require few medications. At the other end of the spectrum are patients with severe and progressive renal involvement (glomerulonephritis and encephalitis) requiring treatment with high doses of steroids and cytotoxic drugs such as cyclophosphamide.

[0059] Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, involve an autoimmune attack of the intestine, causing chronic diarrhea, frequent bleeding, and other symptoms of colon dysfunction.

[0060] Systemic sclerosis (SSc, or scleroderma) is an autoimmune disease characterized by fibrosis of the skin and viscera and widespread vascular involvement. Patients with SSc are classified according to the degree of skin sclerosis. Patients with limited SSc have thickened skin on the face, neck, and distal extremities, whereas patients with diffuse SSc also involve the trunk, abdomen, and proximal extremities. Compared with patients with localized disease, patients with diffuse disease tend to develop visceral involvement earlier in the course of the disease (Laing et al. (1997) Arthritis Rheum. 40:734-42). The majority of patients with diffuse SSc who develop severe visceral involvement do so within the first 3 years after diagnosis, coinciding with progressive fibrosis of the skin (Steen and Medsger (2000) Arthritis Rheum. 43:2437-44). Common manifestations of diffuse SSc, which cause substantial morbidity and mortality, include interstitial lung disease (ILD), Raynaud's phenomenon and digital ulcers, pulmonary arterial hypertension (PAH) (Trad et al. (2006) Arthritis. Rheum. 54:184-91), musculoskeletal symptoms, and cardiac and renal involvement (Ostojic and Damjanov (2006) Clin. Rheumatol. 25:453-7). Current therapies focus on treating specific symptoms, but disease-modifying drugs that target the underlying pathogenetic mechanisms are lacking.

[0061] Autoimmune hepatitis is a disease in which the body's immune system attacks liver cells. This immune response causes liver inflammation, also known as hepatitis. Genetic factors may predispose some people to autoimmune diseases. Four subtypes of autoimmune hepatitis are recognized, but the clinical utility of distinguishing between subtypes is limited: (1) positive ANA and SMA, elevated immunoglobulin G (classical form, responds well to low-dose steroids); (2) positive LKM-1 (typically female children and teenagers, disease can be severe), LKM-2, or LKM-3; (3) positive antibodies to soluble liver antigens (this group behaves like group 1) (anti-SLA, anti-LP); and (4) no detectable autoantibodies (approximately 20%) (of debatable relevance / importance) (Krawitt et al., 2014). et al. Autoimmune hepatitis. New England Journal of Medicine, 1996 334(14):897-903).

[0062] Many degenerative diseases have an underlying inflammatory component; examples of such degenerative diseases include osteoarthritis (OA), Alzheimer's disease (AD), and macular degeneration.

[0063] Osteoarthritis (OA) affects approximately 27 million people in the United States and is treated by primary care physicians. OA accounts for 25% of consultations and half of all NSAID prescriptions. OA is a chronic arthropathy characterized by the destruction and potential loss of articular cartilage, along with other joint changes, including bone remodeling, which may include bone hypertrophy (osteophyte formation), subchondral sclerosis, and the formation of subchondral cysts. OA is considered a breakdown of the synovial joint. OA results in the degeneration of the joint, including the articular cartilage and subchondral bone, resulting in mechanical abnormalities and impaired joint function. Symptoms may include joint pain, tenderness, stiffness, sometimes effusion, and impaired joint function. A variety of causes can initiate the process leading to cartilage loss.

[0064] Alzheimer's disease (AD) is the most common neurodegenerative disease in the population. AD affects approximately 10% of people aged 65 years or older and nearly 50% of people aged 85 years or older. It is estimated that approximately 22 million people will suffer from AD by 2025. AD is characterized by slowly progressive dementia. A definitive diagnosis of AD is made when the triad of dementia, neurofibrillary tangles, and senile plaques is discovered postmortem. Senile plaques are consistently found in the brains of patients with Alzheimer's disease. The main component of senile plaques is amyloid beta protein (Aβ). Aβ is a 42-amino acid peptide derived from amyloid precursor protein (APP), a transmembrane glycoprotein with various physiological roles, including cell proliferation, adhesion, cell signaling, and neurite outgrowth. APP is normally cleaved within the Aβ domain to generate secretory fragments. However, an alternative process leads to APP cleavage, generating soluble Aβ, which can accumulate in senile plaques. Currently available drugs are central cholinesterase inhibitors that aim to increase the concentration of postsynaptic acetylcholine in the brain. These drugs offer minimal clinical benefit in only a few cognitive parameters.

[0065] Although wet macular degeneration, associated with retinal neovascularization and vascular leakage, is more common in the dry form, also known as age-related macular degeneration (AMD). AMD is a chronic disease associated with central vision loss, blurred vision, and eventual blindness. While the causes and risk factors for AMD are multifactorial, complement activation and activation of innate immunity, involving cytokine production by macrophages and microglia, have been implicated in the pathogenesis of AMD. Anti-inflammatory therapies, including corticosteroids, nonsteroidal anti-inflammatory drugs, methotrexate, rapamycin, and biologic agents, including TNF inhibitors and complement inhibitors, have been suggested to slow the progression of AMD (Wang et al., 2011. Eye (2011) 25, 127-139). However, these treatments are not curative, and because AMD is a chronic, nonfatal disease, their use is limited by the risk of toxicity.

[0066] IDDM is a cell-mediated autoimmune disease that results in the destruction of insulin-secreting beta cells and overt hyperglycemia. T lymphocytes invade the islets of Langerhans and specifically destroy insulin-producing beta cells. B-cell depletion leads to an inability to regulate blood glucose levels. Overt diabetes occurs when blood glucose levels rise above a certain level, usually approximately 250 mg / dL. In humans, a long presymptomatic period precedes the onset of diabetes. During this period, there is a gradual loss of pancreatic beta cell function. Disease progression can be monitored in susceptible individuals diagnosed by family history and genetic analysis. The most significant genetic effect is seen in genes in the major histocompatibility complex (IDDM1), but other loci, including the insulin gene region (IDDM2), also show association with the disease.

[0067] Regarding type II diabetes and metabolic syndrome, type II diabetes is characterized by insulin resistance and hyperglycemia, which may subsequently lead to retinopathy, nephropathy, neuropathy, or other pathological conditions. In addition, diabetes is a known risk factor for atherosclerotic cardiovascular disease. Metabolic syndrome is characterized by hypertension, obesity, hyperlipidemia, and insulin resistance (manifested as overt diabetes or fasting hyperglycemia or impaired glucose tolerance). IFG refers to a group of factors that increase the risk of developing heart disease, diabetes, or other health problems. There is a well-characterized progression from a normal metabolic state to impaired fasting glucose (IFG: fasting glucose level >100 mg / dL) or impaired glucose tolerance (IGT: 2-hour glucose level of 140-199 mg / dL after a 75-gram oral glucose challenge). Both IFG and IGT are considered prediabetic states, and more than 50% of subjects with IFG progress to overt type 2 diabetes within an average of three years. Insulin resistance is caused, at least in part, by chronic low-grade inflammation. Macrophages accumulate in obese adipose tissue, where they produce TNF and other inflammatory cytokines in response to stimulation by saturated fatty acids and circulating lipopolysaccharide (LPS). Furthermore, TNF inhibition can reverse insulin resistance.

[0068] Atherosclerosis and atherosclerotic cardiovascular disease are diseases of the arterial wall. They are characterized by the accumulation of fatty substances in the arterial wall, leading to the development of fatty plaques that can rupture, causing vascular occlusion and ischemia. When such vascular occlusion and ischemia occur in the coronary arteries, myocardial infarction can result. Atherosclerotic lesions contain a highly inflammatory environment characterized by the accumulation of inflammatory cells, including macrophages, and to a lesser extent T and B cells, as well as high levels of inflammatory cytokines, chemokines, and MMPs (Libby et al., Nature 2011.473(7347):3170-25). Atherosclerosis can also be associated with low-grade systemic inflammation, as evidenced by elevated levels of high-sensitivity CRP (hsCRP) in the blood; this abnormality can be partially counteracted by treatment with the drug rosuvastatin (Libby et al., Nature 2011.473(7347):3170-25).

[0069] In addition to autoimmune diseases, inflammatory diseases and diseases associated with inflammation include, but are not limited to, acne vulgaris, acne conglobata, acne fulminans, asthma, celiac disease, chronic prostatitis, ulcerative colitis, microscopic colitis, collagenous colitis, Crohn's disease, atopic dermatitis, diverticulitis, glomerulonephritis, interstitial cystitis, viral hepatitis including, but not limited to, hepatitis B and hepatitis C, interstitial cystitis, irritable bowel syndrome, reperfusion injury, sarcoidosis, amyloidosis, and transplant rejection including, but not limited to, heart, lung, kidney, pancreas, bone marrow, stem cell, skin, cornea, and islet cell transplants. Additional inflammatory diseases and diseases associated with inflammation include infectious diseases associated with inflammation, including, but not limited to, human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), syphilis, rickettsial diseases, Lyme disease, bacterial cellulitis, chronic fungal infections, ehrlichiosis, HHV-6, herpes simplex virus 1 and 2, strongyloidiasis, Epstein-Barr virus, cytomegalovirus, mycoplasma infections, Creutzfeldt-Jakob disease, onchocerciasis, nocardia, Whipple's disease, mycobacterial diseases, ringworm infections, and chronic infections with alphaviruses, including, but not limited to, chikungunya, Ross River virus, or other alphaviruses.Additional inflammatory and inflammation-related diseases include antiphospholipid syndrome, Hashimoto's thyroiditis, De Quervain's thyroiditis, Graves' thyroiditis, adrenalitis, type 1 diabetes, hypophysitis, pemphigus vulgaris, bullous pemphigoid, Eaton-Lambert syndrome, myasthenia gravis, Addison's disease, ankylosing spondylitis, alopecia areata, autoimmune hemolytic anemia, immune thrombocytopenic purpura, autoimmune hepatitis, Behçet's disease, cardiomyopathy, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune inner ear disease, cicatricial pemphigoid, Dego's disease, and dermatological disorders. Dermatitis / juvenile dermatitis, polymucositis, inclusion body myositis, Guillain-Barré syndrome, Meniere's disease, mixed connective tissue disease, pernicious anemia perivasculitis, polychondritis, polyglandular autoimmune syndrome, polymyalgia rheumatica, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, reactive arthritis, rheumatic fever, scleroderma, Sjogren's syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, periarteritis nodosa, uveitis, vitiligo, autoimmune Wilson's disease, and ulcers due to autoreactivity to clotting factors. These include, but are not limited to, blood disorders, chronic urticaria, vasculitis including but not limited to granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, Henoch-Schonlein purpura, hypersensitivity vasculitis, hypocomplementemic urticarial vasculitis, and polyarteritis nodosa.

[0070] Inflammatory conditions can also arise in the context of infections, including, but not limited to, bacterial, viral, protozoal, and fungal infections, where cross-reactivity between pathogen antigens and self-antigens leads to inflammation and unwanted CD4+ T cell activation against self-antigens. Viral pathogens of interest include, but are not limited to, coronavirus infections, such as SARS-CoV-1, SARS-CoV-2, and MERS-CoV, as well as other viruses that trigger enhanced inflammatory and autoreactive T cell responses. Other microorganisms of interest include, but are not limited to, Yersinia species, such as Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica; Franciscella species; Pasturella species; Vibrio species, such as V. cholerae and V. parahemolyticus; Legionella species, such as L. pneumophila; Listeria species, such as L. monocytogenes; and Mycoplasma. Mycobacterium species, such as M. tuberculosis and M. leprae; Rickettsia species, such as R. rickettsii and R. typhi; Chlamydia species, such as C. trachomatis, C. pneumoniae and C. psittaci; Helicobacter species, such as H. pylori. In addition, intracellular protozoan pathogens, such as Plasmodium species, Trypanosoma species, Giardia species, Toxoplasma species, and Leishmania species, may also be mentioned.

[0071] Identification and isolation of regulatory T cells Antigen-specific MHC class I-restricted T cells are shown herein to have a distinct timing for expansion following exposure to antigen and to have a distinct phenotype, which can be conveniently described as a cell surface phenotype that allows for ease of identification and isolation.

[0072] As described in the Examples, upon contact with an initiating antigen (e.g., immunization), total CD4 in the blood increases from about 8 to 12 days after immunization. + There is an increase in the frequency of T cells, which decreases over the next 7 to 10 days. + and γδ + There is a concomitant increase in the frequency of T cells, which is due to the CD4 + This pattern of synchronous behavior in these T cell populations was also observed in other tissues, such as the CNS. Tissues that are sources of T cells, such as the spleen and lymph nodes, showed a significant decrease in total CD4 by day 7. + , CD8 + , and γδ + The T cells may have different synchronous behavioral patterns, with a gradual decline in frequency followed by an increase in frequency. All three types of T cells showed increased clonal expansion beginning approximately 7 days after immunization. Clonally expanded CD4+ cells were activated, e.g., CD44 高 CD6L 低 and are specific for the initiating antigen. Clonally expanded CD8+ cells do not respond to the initiating antigen.

[0073] An isolated population of regulatory (CD8+KIR+) T cells can be used as a therapeutic, can be genetically engineered to express an exogenous TCR, can be screened to determine the antigen specificity of the TCR, etc. Cells can be isolated from a cell-containing biological sample derived from an individual of interest at any time, but are conveniently obtained at the time of maximal expansion in the blood. For example, after immune stimulation, including immunization, the peak of regulatory cells in the blood can be about 7, 8, 9, 10, 11, 12, or 13 days after immunization. The number of regulatory T cells in the blood is typically low, e.g., the total P Less than about 2%, less than about 1.5%, less than about 1%, less than about 0.5% of the BMC.

[0074] Markers of regulatory T cells include CD8 and typically include one or more KIR proteins, including, but not limited to, one or more of KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, and KIR3DL2, and may specifically include one or more of KIR2DL2, KIR2DL3, and KIR3DL1. Cells may be further characterized by expression of markers, including, but not limited to, CD38, HLA-DR, CD29, CD44, CD122, and the like. Cells may be activated, e.g., CD44 高 CD6L 低 The cells can also be characterized for expression of a TCR associated with regulatory T cells, for example, by binding to a multimeric peptide / MHC polypeptide.

[0075] Markers can be detected and / or used for cell selection or isolation by binding to an affinity reagent, e.g., a specific binding member (i.e., a second specific binding member) that specifically binds to the marker on the cell via chemical or physical means. Specific binding pairs of interest include carbohydrates and lectins, complementary nucleotide sequences, peptide ligands and receptors, effector and receptor molecules, hormones and hormone-binding proteins, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Specific binding pairs may also include analogs, derivatives, and fragments of the original specific binding member. For example, receptor and ligand pairs may include peptide fragments, chemically synthesized peptidomimetics, labeled proteins, derivatized proteins, and the like.

[0076] Particularly useful reagents are antibodies specific to markers present on desired cells (for positive selection) and undesired cells (for negative selection). Whole antibodies, or fragments such as Fab, F(ab)2, light chain or heavy chain fragments, may be used. Such selected antibodies may be polyclonal or monoclonal and are generally commercially available or, alternatively, easily produced by techniques known to those skilled in the art. The antibodies selected for use have low levels of nonspecific staining and typically have an affinity for the antigen of at least about 100 μM.

[0077] In one embodiment of the present invention, the antibody for selection is coupled to a plate, bead, magnetic reagent, etc., or labeled with a label that enables selection, such as a fluorescent label or mass tag. The exact method of coupling is not critical to the practice of the present invention, and many alternatives are known in the art. Direct coupling involves attaching the antibody to a plate, particle, magnetic reagent, etc. Indirect coupling can be achieved by several methods. The antibody can be coupled to one member of a high-affinity binding system, e.g., biotin, and the other member, e.g., a particle attached to avidin. Alternatively, a second-stage antibody that recognizes a species-specific epitope of the antibody, e.g., anti-mouse Ig, anti-rat Ig, etc., can be used.

[0078] Functionally relevant regulatory T cells can be purified from tissue samples or cultures, and purified cell populations are useful for gene expression analysis, drug screening assays, therapeutic purposes, in vitro culture and co-culture, etc.

[0079] The resulting cell composition may be highly purified, with the desired cells being at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or more of the desired cell type.

[0080] Isolation of the cell population of interest for either positive or negative selection utilizes affinity separation to provide a substantially pure population. Techniques for affinity separation include magnetic separation using antibody-coated magnetic beads, affinity chromatography, and chromatography using antibodies bound to monoclonal antibodies. or cytotoxic agents, such as complement and cytotoxins, used in conjunction with monoclonal antibodies, as well as "panning" using antibodies bound to a solid matrix, such as a plate, or other convenient technique. Any technique may be used that is not unduly detrimental to cell viability.

[0081] Positive immunoselection utilizes reagents that selectively bind, for example, CD8, inhibitory KIR proteins, etc., on the cell surface. Negative immunoselection is optionally performed to deplete cells of other lineages, e.g., CD4, B cell markers, monocyte markers, etc. Size, e.g., forward scatter, can be used to gate out non-lymphocyte blood cells. In some embodiments, two, three, four, five, or more negative immunoselection reagents are used, e.g., in a cocktail or separate negative selections. In some embodiments, a lineage cocktail is used that includes reagents for each negative selection, such as myeloid cells, B cells, CD4+ T cells, etc. If negative separation is used, it is often performed before positive selection to deplete the cell population of undesired cells. Positive selection is then performed.

[0082] A specific binding member, usually an antibody, is added to the cell suspension and incubated for a period of time sufficient to bind to the available antigen. Incubation is usually at least about 2 minutes and can be less than about 30 minutes. It is desirable to have a sufficient concentration of antibody in the reaction mixture so that the efficiency of separation is not limited by a lack of reagent. The appropriate concentration can be determined by titration.

[0083] The medium in which the cells are isolated can be any medium that maintains cell viability. Various media are commercially available and can be used depending on the properties of the cells, such as Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's Phosphate Buffered Saline (dPBS), RPMI, Iscove's Medium, PBS with 5 mM EDTA, etc. Cells can be placed in culture, formulated for therapy, and frozen.

[0084] The resulting compositions have a variety of uses in clinical therapy, research, development, and commercial purposes. For therapeutic purposes, for example, they can be administered in therapeutically effective amounts to selectively suppress unwanted pathogenic T cell responses, optionally after expansion in culture.

[0085] Expansion or activation in culture may utilize cytokines and / or antigen-presenting cells (APCs). Contacting may be performed in any suitable medium. If present, APCs may then be loaded with a suitable peptide antigen or protein, which is presented on the cell surface. The T cell to APC ratio, if present, may be any value from about 1:20 to about 20:1 and is not critical as long as the number of APCs is not limiting. A period of up to 8 days, 10 days, 12 days, or 14 days may be sufficient (see, e.g., Dudley et al., JCO 2005;23(10):2346-2357). Regulatory T cells primed in this manner may be used for any desired purpose, including experimental purposes for determining antigen specificity, cytokine profiling, etc., and for in vivo delivery.

[0086] Cytokines useful for in vitro expansion include, but are not limited to, one or more cytokines that enhance the proliferation of CD8+ T cells, including, but not limited to, type I IFN (IFNα and IFNβ), IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, IL-25, IL-27, IL-33, and the like. Cells may be cultured in conventional nutrient media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing cells. Any of these media may be supplemented with hormones, if desired. The medium may be supplemented with monocytes and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may be included at appropriate concentrations known to those of skill in the art. Culture conditions, such as temperature, pH, and the like, will be those previously used for the host cell selected for expression and will be apparent to those of skill in the art.

[0087] Ex vivo T cell activation can be achieved by procedures established in the art, including cell-based T cell activation, antibody-based activation, or activation using various bead-based activation reagents. Cell-based T cell activation can be achieved by exposing T cells to antigen-presenting cells such as dendritic cells or artificial antigen-presenting cells such as irradiated K562 cells. Antibody-based activation of T cell surface CD3 molecules with soluble anti-CD3 monoclonal antibodies also supports T cell activation in the presence of IL-2.

[0088] T cells can be cultured in contact with a surface that provides an agent that stimulates CD3 TCR complex-associated signals (e.g., an anti-CD3 antibody) and an agent that stimulates costimulatory molecules on the surface of T cells (e.g., an anti-CD28 antibody). Bead-based T cell activation can be achieved using commercially available T cell activation reagents, including, but not limited to, Invitrogen® CTS Dynabeads® CD3 / 28 (Life Technologies, Inc. Carlsbad, CA) or Miltenyi MACS® GMP ExpAct Treg beads or Miltenyi MACS GMP TransAct™ CD3 / 28 beads (Miltenyi Biotec, Inc.). Suitable conditions for T cell culture are well known in the art. Lin, et al. (2009) Cytotherapy 11(7):912-922; Smith, et al. (2015) Clinical & Translational Immunology 4:e31, published online January 16, 2015. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0089] The regulatory T cells thus isolated and optionally expanded are optionally genetically modified, for example, to express a TCR other than the native TCR. Regulatory T cells expanded during an immune response to a vaccine can be modified to express a TCR specific for suppressing, for example, an autoimmune disease, a transplantation antigen, etc. Transduction of T cells with an expression vector can be achieved using techniques including, but not limited to, co-incubation of host T cells with a viral vector, electroporation, and / or chemically enhanced delivery.

[0090] Alternatively, engineered TCRs can be inserted, for example, by CRISPR / Cas9, meganucleases, engineered I-CreI homing endonucleases, etc. See, e.g., Eyquem et al. (2017) Nature 543:113-117, and Georgiadis et al. (2018) Mol. Ther. 26:1215-1227.

[0091] To determine the suitability of a cell composition for therapeutic administration, the cells can first be tested in a suitable animal model. At one level, the cells are evaluated for their ability to survive and maintain their phenotype in vivo. The cell composition is administered to an immunodeficient animal (e.g., nude mice, or animals rendered immunodeficient chemically or by irradiation). Tissue is harvested after a period of regrowth and evaluated for the presence or absence of the administered cells or their progeny.

[0092] For therapeutic purposes, the cells can be autologous or allogeneic, in which case the MHC restriction of the regulatory T cells matches the MHC type of the recipient.

[0093] An effective dose of regulatory cells is infused into the recipient, allowing them to contact CD4+ T cells in their natural environment, such as lymph nodes. The dosage and frequency may vary depending on the agent, the mode of administration, etc. Those skilled in the art will understand that such guidelines will be adjusted according to individual circumstances. The dosage may also vary depending on local administration, such as intranasal, inhalation, etc., or systemic administration, such as intramuscular (im), intraperitoneal (ip), intravenous (iv), etc. The enhanced immune response may be manifested by an increase in the cytolytic response of regulatory T cells against target cells present in the recipient, such as a reduction in the symptoms of an autoimmune disease.

[0094] Regulatory T cells can be provided in pharmaceutical compositions suitable for therapeutic use, for example, human treatment. Therapeutic preparations containing such cells can be frozen or in the form of an aqueous solution, and can be prepared for administration with physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). The cells are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the drug delivery site, the method of administration, the administration schedule, and other factors recognized by physicians.

[0095] The cells can be administered by any suitable means, usually parenterally, including intramuscular, intravenous (bolus or slow infusion), intraarterial, intraperitoneal, intrathecal, or subcutaneous administration.

[0096] Regulatory T cells can be infused into a subject in any physiologically acceptable medium, usually intravascularly, but they can also be introduced into any other convenient site where the cells can find a suitable site for growth. Generally, at least about 10 4 cells / kg, at least approximately 10 5 cells / kg, at least approximately 10 6 cells / kg, at least approximately 10 7 Cells / kg, or more, may be administered, usually limited by the number of T cells obtained during harvest.

[0097] The course of treatment can be a single dose or multiple doses over a period of time. In some embodiments, the cells are administered in a single dose. In some embodiments, the cells are administered in two or more divided doses, administered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120, or 180 days. The amount of cells administered in such a divided dosing protocol can be the same in each administration, or can be provided at different levels. A multi-day administration protocol over a period of time can be provided by a skilled artisan (e.g., a physician) who monitors the administration of cells, taking into account the subject's response to treatment, including the side effects of treatment and their control, as discussed above.

[0098] Screening for antigen specificity The selected regulatory T cells can be used as a source of sequences encoding TCRs that provide antigen-specific suppression of unwanted CD4+ T cell-mediated responses. The TCR-encoding sequences can be isolated by any convenient method, for example, as detailed in the Examples.

[0099] The TCR of interest can be expressed in soluble form and multimerized for use as a selective binding agent. The soluble protein can be a single chain or, more commonly, a heterodimer. In some embodiments, the soluble TCR is modified by adding a biotin acceptor peptide sequence to the C-terminus of one polypeptide. After biotinylation with the acceptor peptide, the TCR can be multimerized by binding to a biotin-binding partner, such as avidin, streptavidin, traptavidin, neutravidin, etc. The biotin-binding partner can include a detectable label, such as a fluorophore, mass label, etc., or can be bound to particles, such as paramagnetic particles. Selection of the ligand bound to the TCR can be performed by flow cytometry, magnetic selection, etc., as known in the art.

[0100] TCR multimers are utilized in binding assays to libraries of diverse peptide antigens. The peptide ligands are typically about 8 to about 20 amino acids in length, typically about 8 to about 18 amino acids, about 8 to about 16 amino acids, about 8 to about 14 amino acids, about 8 to about 12 amino acids, about 10 to about 14 amino acids, or about 10 to about 12 amino acids. It is understood that a completely random library represents an extraordinary number of possible combinations. In some methods, diversity is limited to residues that anchor the peptide to the MHC binding domain, referred to herein as MHC anchor residues. The location of the anchor residues within the peptide is determined by the specific MHC binding domain. Diversity can also be limited at other positions, as known from binding studies, e.g., TCR anchors. At least 10 6 Pieces, at least 10 7 pieces, more generally at least 10 8 different peptide ligands are present in the library.

[0101] The MHC proteins used in the library can be from any mammalian or avian species. Of particular interest are human HLA proteins. HLA proteins include the class II subunits HLA-DPα, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα, and HLA-DRβ, as well as the class I proteins HLA-A, HLA-B, HLA-C, and β2-microglobulin.

[0102] The MHC binding domain is typically a soluble form of a normally membrane-bound protein. The soluble form is derived from the native form by deletion of the transmembrane domain. Advantageously, the protein is truncated to remove both the cytoplasmic and transmembrane domains. In some such embodiments, the binding domain is subjected to mutagenesis and selected for amino acid changes that enhance the solubility of the single-chain polypeptide without altering the peptide-binding contacts. For class I proteins, the binding domain may include the α1, α2, and α3 domains of class I alleles, including, but not limited to, HLA-A, HLA-B, HLA-C, H-2K, H-2D, and H-2L, in combination with β2-microglobulin. Up to about 10, usually up to about 5, amino acids of the transmembrane domain may be included, but preferably none of the amino acids of the transmembrane domain are included. The deletions are such that they do not interfere with the domain's ability to bind to peptide ligands.

[0103] The library of diverse sequences is generated and inserted into a vector suitable for the host cell of interest, which may be, but is not limited to, suitable for expression in yeast cells, and the yeast cells can be induced to express the polypeptide library. Upon introduction into the host cell, expression of the library is induced, and the cells are maintained for a period of time sufficient to provide cell surface display of the library polypeptides.

[0104] Selection of peptides that bind to the regulatory TCR is performed by combining the multimerized TCR with a population of host cells expressing the library. Rounds of selection are performed until the selected population has a signal above background, usually at least three times. ,More often, at least four selection rounds are performed.

[0105] After the final round of selection, polynucleotides are isolated from the selected host cells and the sequences of the selected peptide ligands are determined, typically by high-throughput sequencing.

[0106] The sequencing platform that can be used in the present disclosure includes but is not limited to pyrosequencing, sequencing by synthesis, single molecule sequencing, second generation sequencing, nanopore sequencing, sequencing by ligation or sequencing by hybridization.Preferred sequencing platform is commercially available from Illumina (RNA-Seq) and Helicos (digital gene expression or "DGE")."Next generation" sequencing methods include, but are not limited to, the following: 1) methods and apparatus described in 454 / Roche Lifesciences, Margulies et al., Nature (2005) 437:376-380 (2005), and U.S. Patent Nos. 7,244,559, 7,335,762, 7,211,390, 7,244,567, 7,264,929, and 7,323,305; and 2) Helicos BioSciences. Corporation (Cambridge, MA), U.S. Patent Application No. 11 / 167046, and U.S. Patent Nos. 7,501,245, 7,491,498, 7,276,720, and U.S. Patent Application Publication Nos. 2009 / 0061439, 2008 / 0087826, 2006 / 0286566, 2006 / 0024711, 2006 / 0024678, 2008 / 0213770, and 2008 / 0103058; 3) Applied Biosystems (e.g., SOLiD Sequencing); 4) Dover Systems (e.g., Polonator G.007 sequencing), 5) Illumina, such as those described in U.S. Patent Nos. 5,750,341, 6,306,597, and 5,969,119, and 6) Pacific These methods and devices include, but are not limited to, those commercially available from Biosciences, U.S. Patent Nos. 7,462,452, 7,476,504, 7,405,281, 7,170,050, 7,462,468, 7,476,503, 7,315,019, 7,302,146, 7,313,308, and U.S. Patent Application Publication Nos. 2009 / 0029385, 2009 / 0068655, 2009 / 0024331, and 2008 / 0206764. All references are incorporated herein by reference. Such methods and devices are provided herein as examples and are not intended to be limiting.

[0107] As shown in the examples, the peptide antigens thus identified may be natural peptides in an individual or may be surrogate peptides that specifically activate regulatory T cells of interest. Peptides, termed regulatory peptides, are useful as screening tools and are particularly useful as therapeutic agents that activate regulatory T cells. Regulatory peptides can be used to activate T cells in vitro or in vivo.

[0108] Inflammatory diseases, including autoimmune diseases, discussed herein can be treated by administering a therapeutically effective amount of a regulatory peptide, or an active fragment or derivative thereof, to a subject. The peptide can be administered as a single agent, as a cocktail of both peptides, or in combination with a second therapeutic agent.

[0109] Regulatory peptides typically contain at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids or more, and may further include fusion polypeptides known in the art in addition to the sequences provided. Suitable regulatory peptides also include derivatives, variants, and biologically active fragments of naturally occurring regulatory peptides. Regulatory peptide sequences can be designed sequences derived from mutagenesis in diverse peptide libraries. TCR specificity can be conformational, and thus peptides that activate regulatory T cells of interest can have sequences that are essentially unrelated to naturally occurring peptides.

[0110] Regulatory peptides can be modified for various purposes, for example, into a wide variety of other oligopeptides or proteins. For example, post-translational modifications, such as prenylation, acetylation, amidation, carboxylation, glycosylation, pegylation, etc. Such modifications can also include glycosylation modifications, such as those made during polypeptide synthesis and processing or during further processing steps, for example, by modifying the glycosylation pattern of the polypeptide by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes. In some embodiments, variants of the invention include variants having phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine, or phosphothreonine.

[0111] The ability of a regulatory peptide to regulate lymphocyte activity can be determined, for example, by the ability of the peptide to induce a cytotoxic effect on activated pathogenic lymphocytes, as disclosed in the Examples provided herein.

[0112] In some embodiments, the regulatory peptide is provided as a fusion protein, e.g., fused in-frame with a second polypeptide. In some embodiments, the second polypeptide can increase the size of the fusion protein, e.g., to prevent rapid clearance of the fusion protein from circulation. In some other embodiments, the second polypeptide is part or all of an Fc region. In some other embodiments, the second polypeptide is any suitable polypeptide substantially similar to Fc, e.g., providing increased size and / or additional binding or interaction with Ig molecules. These fusion proteins can facilitate purification and exhibit increased half-life in vivo. Fusion proteins with disulfide-bonded dimeric structures (due to IgG) can also be more efficient at binding and neutralizing other molecules than the monomeric secreted protein or protein fragment alone.

[0113] In some other embodiments, the regulatory peptide variants of the present invention include variants that have been further modified to improve resistance to proteolysis, optimize solubility, or further qualify as therapeutic agents. For example, the variants of the present invention further include analogs containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids can replace some or all of the amino acid residues.

[0114] Polypeptides can be prepared by cell-free translation systems or synthetic in vitro synthesis using conventional methods known in the art. Various commercially available synthesis devices are available, such as automated synthesizers from Applied Biosystems, Inc., Foster City, Calif., Beckman. Synthesizers can be used to substitute naturally occurring amino acids with unnatural amino acids. The specific sequence and preparation method are determined by convenience, economy, required purity, etc.

[0115] The polypeptides may also be isolated and purified according to conventional methods of recombinant synthesis. A lysate of the expression host may be prepared, and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, the composition used will be free of contaminants related to the preparation method of the product and its purity. For purposes of this specification, the desired product will comprise at least 20% by weight, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight. Typically, percentages are based on total protein.

[0116] The regulatory peptides can be provided in pharmaceutical compositions suitable for therapeutic use, e.g., human treatment. In some embodiments, the pharmaceutical compositions comprise one or more therapeutic entities of the present invention, or pharmaceutically acceptable salts, esters, or solvates thereof. In some other embodiments, the pharmaceutical compositions of the present invention comprise one or more therapeutic entities of the present invention in combination with another therapeutic agent.

[0117] Therapeutic entities are often administered as pharmaceutical compositions containing an active therapeutic agent and other pharmaceutically acceptable excipients. The preferred form depends on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition can also contain a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also contain other carriers or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0118] In yet some other embodiments, the pharmaceutical compositions may also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers (e.g., latex-functionalized Sepharose™, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0119] Also provided are methods of combination therapy, which may provide additive or synergistic benefits. The regulatory peptide combination can be achieved with a second drug selected from one or more of the general classes of drugs commonly used in the non-antigen-specific treatment of autoimmune diseases, including corticosteroids and disease-modifying drugs, or antigen-specific drugs. Corticosteroids, such as prednisone, methylpredisone, prednisolone, and solumedrol, have both anti-inflammatory and immunoregulatory activities. They can be administered systemically or by local injection. Corticosteroids are useful in early disease as temporary adjunctive therapy while waiting for disease-modifying drugs to exert their effects. Corticosteroids are also useful as chronic adjunctive therapy in patients with severe disease.

[0120] Disease-modifying drugs are also useful in combination therapy. These drugs include methotrexate, leflunomide, etanercept, infliximab, adalimumab, anakinra, rituximab, CTLA4-Ig (abatacept), antimalarials, gold salts, sulfasalazine, d-penicillamine, cyclosporine A, cyclophosphamide, azathioprine, and others. Treatment for MS may include interferon-beta, copaxone, and anti-VLA4, which reduce relapse rates. MS is also treated with immunosuppressants, including methylprednisolone, other steroids, methotrexate, cladribine, and cyclophosphamide.

[0121] Combination therapy may be administered sequentially in a stepwise manner, provided in a co-administration formulation, or co-administration may be simultaneous. "Co-administration" of a known therapeutic agent with a pharmaceutical composition of the present invention refers to administration of the drug and regulatory peptide at a time such that both the known drug and the composition of the present invention have a therapeutic effect. Such co-administration may involve simultaneous (i.e., concurrent), prior, or subsequent administration of the drug relative to administration of a compound of the present invention. Those skilled in the art will be able to determine the appropriate timing, sequence, and dosage of administration for particular drugs and compositions of the present invention. There is no difficulty in doing so.

[0122] The regulatory peptides can function as active ingredients in pharmaceutical compositions formulated for the treatment of the various disorders described above. The active ingredient is present in a therapeutically effective amount, i.e., in an amount sufficient when administered to treat a disease or condition mediated by inflammatory lymphocytes, particularly by reducing their activity. The composition can also include various other agents to improve delivery and efficacy, for example, to improve delivery and stability of the active ingredient.

[0123] For example, depending on the desired formulation, the composition may also contain a pharmaceutically acceptable, non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, buffered water, physiological phosphate-free saline, Ringer's solution, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also contain other carriers, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The composition may also contain additional substances that approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and surfactants. The composition may also contain any of a variety of stabilizers, such as antioxidants.

[0124] Peptides can be complexed with a variety of well-known compounds that enhance the in vivo stability of the polypeptide or otherwise enhance its pharmacological properties (e.g., increase the polypeptide's half-life, reduce its toxicity, enhance solubility or uptake). Examples of such modifying or complexing agents include sulfate, gluconate, citrate, and phosphate. The polypeptides of the composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0125] Further guidance regarding suitable formulations for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0126] The pharmaceutical compositions may be administered for prophylactic and / or therapeutic treatment. Toxicity and therapeutic efficacy of the active ingredients can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, for example, by LD 50 (a dose lethal to 50% of the population) and ED 50 This involves determining the dose that is therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50 Compounds that exhibit large therapeutic indices are preferred.

[0127] The data obtained from cell culture and / or animal studies can be used in formulating a range of dosages for humans. The dosage of the active ingredient is typically selected so that it is within the ED range with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0128] The pharmaceutical compositions described herein can be administered in a variety of different ways, including by administering the composition with a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, or transdermal means.

[0129] Formulations suitable for parenteral administration, such as by intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous or non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0130] The components used to formulate pharmaceutical compositions are preferably highly pure and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are preferably sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is preferably substantially free of any potentially toxic substances, such as any endotoxins, that may be present during the synthesis or purification process. Compositions for parenteral administration are also preferably sterile, substantially isotonic, and made under GMP conditions.

[0131] The regulatory peptide composition may be administered in a single dose or in multiple doses, typically administered daily, every other day, weekly, twice weekly, monthly, for a period sufficient to reduce the severity of the inflammatory disease, which may include multiple doses over a period of time, e.g., 1, 2, 3, 4, 6, 10, or more doses.

[0132] Determination of a therapeutically or prophylactically effective amount can be performed based on animal data using conventional calculation methods. In one embodiment, a therapeutically or prophylactically effective amount contains about 0.1 mg to about 1 g of protein. In another embodiment, an effective amount contains about 1 mg to about 100 mg of protein. In a further embodiment, an effective amount contains about 10 mg to about 50 mg of protein. Effective doses depend at least in part on the route of administration. Doses can be about 0.1 μg / kg, about 1 μg / kg, about 10 μg / kg to about 100 μg / kg of patient body weight.

[0133] In the method of use, an effective dose of the agent of the present invention is administered alone or in combination with an additional active agent for the treatment of the conditions listed above. The effective dose can be about 1 ng / kg, 10 ng / kg, 100 ng / kg, 1 μg / kg, 10 μg / kg, 25 μg / kg, 50 μg / kg, 100 μg / kg, 250 μg / kg, 500 μg / kg, 750 μg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 250 mg / kg, 500 mg / kg, 750 mg / kg, etc. Dosages can be administered multiple times as needed, e.g., every 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, daily, every 2 days, every 3 days, weekly, etc. Dosages may be administered orally.

[0134] The compositions may be administered in a single dose or in multiple doses, typically administered daily, every other day, weekly, twice weekly, monthly, for a period sufficient to reduce the severity of the inflammatory disease, which may include multiple doses over a period of time, e.g., 1, 2, 3, 4, 6, 10, or more doses.

[0135] Determination of therapeutically or prophylactically effective amounts of agents according to the present methods can be performed based on animal data using conventional calculation methods. The effective dose will depend, at least in part, on the route of administration.

[0136] The present invention has been described with reference to particular embodiments discovered or contemplated by the inventors to comprise preferred modes for carrying out the invention. Those skilled in the art will appreciate that this disclosure will enable them to In light of this, it should be understood that numerous modifications and variations can be made in the specific embodiments exemplified without departing from the intended scope of the invention. By considering biological functional equivalence, changes can be made in the protein structure without affecting the biological action in kind or amount. All such modifications are intended to be within the scope of the appended claims.

[0137] Example 1 Opposing T cell responses in experimental autoimmune encephalomyelitis In experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), induction generates a continuous wave of clonally expanded CD4+, CD8+, and γδ+ T cells in the blood and central nervous system. In MS patients, we also observed a major expansion of CD8+ T cells. In EAE, we found that most expanded CD4+ T cells are specific for the induction of the myelin peptide MOG35-55. In contrast, peptide ligands derived from a yeast peptide-MHC display library for some clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results demonstrate that induction of autoreactive CD4+ T cells leads to the counter-recruitment of regulatory CD8+ T cells.

[0138] Here, we explored whether a coordinated T cell response occurs in EAE and found that it occurs in both the blood and the central nervous system (CNS). Expanded CD4+ T cells were largely specific for the MOG35-55 peptide, as expected, whereas clonally expanded CD8+ T cells failed to respond to myelin peptides or proteins.

[0139] To identify target antigens, we screened six CD8+ TCRs on a class I MHC molecule H2-Db yeast p-MHC display library and obtained surrogate peptides for two of these TCRs. We found that these significantly reduced the severity of EAE rather than exacerbating it. Further analysis indicated that these T cells represent a unique subset of regulatory CD8+ T cells that suppress MOG35-55-specific CD4+ T cell proliferation. The induction of autoreactive CD4+ T cells in EAE triggers a countervailing wave of regulatory CD8+ T cells. In newly diagnosed MS patients, TCR analysis demonstrated a highly pronounced CD8+ T cell clonal expansion in activated brain-homing T cells and a similar bias toward an IL-17 phenotype in γδ+ T cells. CD4+ clonal expansion was generally modest. Most importantly, pathogenic CD4+ and γδ+ cell responses countered by regulatory CD8+ T cell responses appear likely to be a common phenomenon across autoimmune diseases.

[0140] result We describe the recruitment of three distinct T cell types after EAE induction. Here, we performed an extensive investigation of T cell dynamics after EAE induction in the blood, spleen, lymph nodes (LNs), and CNS-infiltrating lymphocytes (Figure 1a and Figure 6). We observed a gradual and significant increase in the frequency of total CD4+ T cells in the blood from day 0 to day 10 post-immunization (PI), peaking around day 10, declining to below baseline levels by day 15, and finally recovering by day 17. A similar decline and recovery in the frequency of total CD4+ T cells was observed again at days 19 and 21, respectively (Figure 1b). We also observed a similar significant increase in the frequency of total CD8+ and γδ+ T cells in the blood at day 10 PI, and the rate and magnitude of decline and increase of these cells precisely matched that of CD4+ T cells (Figure 1b). This synchronous behavioral pattern in these T cell populations was also observed in the CNS (Figure 1c). Splenic and LN T cells were collected from D0 to D7. CD4+, CD8+, and γδ+ T cell frequencies showed a distinct pattern of synchronous behavior, with a gradual decline, followed by an increase in frequency by D17, and another decline between D17 and D30 (Fig. 1d and 1e). In parallel, effector cells (CD44 高 CD6L 低 ) (Figures 8a, 8c, and 8e) and naive cells (CD44 低 CD62L 高 ) There were corresponding changes in the frequency of PI (Figures 8b, 8d, and 8f).

[0141] CD4+, CD8+, and γδ+ T cells underwent clonally expanded expansion after EAE induction in both the blood and CNS. To determine whether these waves of T cells constitute a focal immune response, we investigated the effector (CD44 高 CD6L 低 Single-cell paired TCR sequencing (Figure 2a) of CD4+, CD8+, and γδ+ T cells was performed at various time points (Figure 2b and Table 1). All three T cell types showed increasing clonal expansion beginning at D7 as the disease progressed (Figure 2c and Figures 9a-c). As expected, identical TCRs were rare or absent between mice, but we found identical TCR sequences were shared between the blood and CNS at each day within each mouse, with maximal sequence sharing at D15 (Figure 9d). Among γδ+ T cells, we found that nearly all clonally expanded, and some non-clonal, γδ+ T cells in the blood and CNS were enriched for a TCR known to be expressed by natural γδ+ T cells (nTγδ17)13 (Figure 9e), with levels peaking at D15 and representing 96% of total γδ+ T cells in the CNS (Figures 9e and 9f).

[0142] Clonal expansion of CD8+ T cells does not respond to myelin antigens. To determine whether the expanded CD4+ T cell clones respond to the MOG35-55 peptide, we performed a SKWαβ - / -We expressed four of these CD4+ TCRs (Table 2) on IgG1 cells and stained them with MOG35-55I-Ab peptide-MHC tetramers. All four of these CD4+ TCRs bound to the tetramer (Figure 3a-d), reinforcing the preference for MOG-specific CD4+ T cells in this disease and providing a way to enrich for clonally expanded T cells important in the immune response, as activated / effector cells (CD44 高 CD6L 低 We validated our strategy of sequencing the .

[0143] To investigate the antigen specificity of CD8+ T cells in EAE, we clonally expanded 58 αβ- / - cells expressing nine of the common CD8+ TCRs (Figure 10a and Table 3). These CD8+ TCRs (EAE1-CD8 to EAE9-CD8) were cocultured with bone marrow-derived dendritic cells (BMDCs) pulsed with a myelin protein-derived peptide pool (Table 4), and their activation was measured. Surprisingly, none of the 350 myelin peptides stimulated any of the CD8+ TCR cell lines (Figure 3e). Cells expressing an ovalbumin-specific TCR (OT-1) cocultured with BMDCs loaded with the ovalbumin-derived SIINFEKL peptide resulted in robust activation (Figure 3e and Figure 10c), as well as anti-CD3 and anti-CD28 stimulation (Figure 10b). Myelin proteins also failed to stimulate any of the CD8+ TCR cell lines, whereas OT-1 T cells were robustly stimulated with ovalbumin protein (Fig. 3e and Extended Data Figs. 4c and 4d), suggesting that only a minority of the activated and clonally expanded CD8+ T cells induced during EAE are specific for myelin antigens.

[0144] H2-D b Regarding the generation of yeast peptide-MHC libraries, Garcia and colleagues recently developed a yeast peptide-MHC library system for the identification of αβ TCR ligands. To discover peptide antigens for these EAE-CD8 TCRs, we used the SIINFEKL or SSLENFRAYV peptides to target H2-K as previously described.b and H2-D b Constructs were designed (Figures 11a and 11b). Although the initial constructs were correctly delivered to the yeast surface, they did not bind to the cognate TCR, indicating incorrect folding. To rescue proper folding, we used H2-K Both the full-length constructs of H2-K and H2-Db were subjected to error-prone mutagenesis. b Although we were unsuccessful in the single H2-D b Having found that the mutation restored TCR recognition (Fig. 11c and 11e-g), we generated two different peptide libraries with this mutation, mutating 9 and 10 aa inserts with limited diversity in the two key MHC-binding anchor residues (Fig. 11a-d). The estimated diversity of both peptide libraries was 5 × 10 8 It was.

[0145] Class I peptide immunization protects mice from EAE. b Six of the clonally expanded EAE-CD8 TCRs (Table 3) were used to screen the yeast-pMHC library. After four rounds of selection, the influenza-specific control (6218), EAE6, and EAE7 TCRs showed robust tetramer staining (Figures 4a-c and Table 5). We obtained a perfect match between the influenza TCR and its known peptides (Figure 4d), but failed to find a match in the mouse genome for the two EAE TCR peptides. Nevertheless, they were not associated with H2-D b can still serve as an important surrogate when complexed with

[0146] To characterize these CD8 surrogate peptides (SPs), we used Jurkat αβ expressing 6218, EAE6, and EAE7-CD8 TCRs. - / -Cells were stained with the corresponding yeast library enriched pMHC tetramers (SSLENFRAYV for 6218, ARSNRYFWL and SMRPNHFFFL for EAE6-CD8, and YQPGNWEYI for EAE7-CD8) and showed robust staining (Figure 12a). Furthermore, upon analyzing the enriched peptide sequences of EAE7-CD8 TCR, we noticed that the 36th enriched peptide (HDRVNWEYI) was highly similar to the top enriched YQPGNWEYI peptide. EAE7-CD8 TCR cell lines were stained with YQPGNWEYI and HDRVNWEYI H2-D, which showed robust tetramer staining. b Staining was performed with pMHC tetramer (Fig. 12a).

[0147] To determine the immune responses elicited by these SPs, we immunized mice with either adjuvant (CFA+PTX), MOG35-55, all four peptides together [YQPGNWEYI (YQP), HDRVNWEYI (HDR), ASRSNRYFWL (ASR), and SMRPNHFFFL (SMRP) SP immunization], or MOG35-55 with all four peptides together (MOG+SP immunization). We analyzed CD8+ T cells from spleens and LNs 10 days PI using SP-H2-D. b We also enriched MOG-specific CD4+ T cells from mice immunized with MOG35-55 using tetramers (Fig. 12b). 35-55 WT, MOG, and WT-specific CD4+ T cells were induced (Fig. 5a and 5c). 35-55We detected very few SP-specific CD8+ T cells in mice immunized with either IFN-γ or adjuvant. However, MOG+SP immunization increased the frequency of CD8+ T cells specific for ASR, HDR, and SMRP (Figures 5b and 5d). Compared to WT mice, SP immunization induced a higher frequency of HDR- and SMRP-specific CD8+ T cells, with no change in the number of ASR- and YQP-specific CD8+ T cells (Figures 5b and 5d). A higher percentage of SP-specific CD8+ T cells exhibited an activated and effector phenotype after immunization (Figures 12b-d). Thus, these SPs identified a pre-existing pool of specific CD8+ T cells in mice that could be activated, some of which would expand upon immunization.

[0148] To test the effect of these peptides on EAE, we induced EAE with or without SP. MOG immunization induced severe disease in 100% of mice, whereas addition of SP with MOG resulted in much less severe or no disease, with only a 30% incidence of very mild disease and most mice showing no symptoms at all. (Fig. 5e and Table 6). 35-55 Immunization of another group of mice with + influenza peptides did not result in a significant difference in the severity of EAE (Fig. 12e). We tested the preventive or therapeutic effects of these peptides on EAE by immunizing mice with SP one week before or after MOG immunization (MOG and SP challenge, respectively), and with both of these challenges, mice had less severe disease (Figs. 5f and 5g and Table 7). Overall, MOG + SP immunization significantly improves EAE.

[0149] Immunization with class I peptides inhibits MOG 35-55 This reduction in severity by the addition of SP is a key factor in the development of MOG. 35-55 To investigate whether MOG directly affected specific CD4+ T cells, we investigated whether MOG directly affected specific CD4+ T cells during MOG or MOG+SP immunization. 35-55 Using I-Ab tetramer, MOG35-55 The frequency of specific CD4+ T cells in the spleen and LN was analyzed. As expected, MOG immunization increased the frequency of MOG-specific CD4+ T cells. 35-55 This resulted in an increase in the number of I-Ab tetramer+ CD4+ T cells (Figures 6a and 6b and Figure 13a). Interestingly, the frequency of these T cells was significantly reduced in MOG+SP-immunized mice (Figures 6a and 13b and 13c).

[0150] To examine this in vitro, we investigated the effects of MOG on the immune system in the presence or absence of CD8+ T cells derived from MOG+SP or SP-immunized mice. 35-55 The magnitude of CD4+ T cell proliferation was quantified using CellTrace™ Violet dye (CTV) to label CD4+ T cells from MOG-immunized mice co-cultured and stimulated with MOG in the absence of CD8+ T cells. 35-55 MOG35-55-specific CD4 T cells proliferated robustly against MOG35-55 (Fig. 6c). However, addition of CD8+ T cells from either MOG+SP- or SP-immunized mice resulted in a significant decrease in the proliferative capacity of CD4+ T cells (Fig. 6d), indicating that both conditions elicited CD8+ T cells that actively suppressed the proliferation of MOG35-55-specific CD4+ T cells. However, CD8+ T cells from either immunized WT, MOG+influenza peptide, or adjuvant-immunized mice proliferated robustly against MOG35-55. 35-55 MOG+SP-induced CD8+ T cells did not suppress the proliferation of stimulated CD4+ T cells (Figure 14a-d). Furthermore, MOG+SP-induced CD8+ T cells did not suppress the proliferation capacity of ovalbumin-specific CD4+ T cells, suggesting that the suppression was antigen-specific (Figure 14h).

[0151] Class I peptide immunization induces a unique subset of regulatory CD8+ T cells. Interestingly, we found that MOG+SP and SP immunization induced significantly higher frequencies of CD8+ T cells expressing CD44, CD122, and Ly49, which are considered markers of Qa-1b-restricted regulatory CD8+ T cells (Fig. 6e). Furthermore, among individual SP-specific CD8+ T cells, there was a significant increase in the frequency of CD8+ T cells with these markers (Fig. 13d-h). We found that the EAE CD8-TCR described here is not Qa-1b-restricted, as we failed to find any effect of anti-Qa-1b antibodies on CD8 suppression of MOG-specific CD4+ T cells (Fig. 14g).

[0152] CD8+ T cells with this phenotype express MOG in vitro 35-55 To determine whether specific CD4+ T cells can be actively suppressed, CD4+ T cells from immunized MOG were co-cultured with total CD8+ T cells or purified CD8+CD44+CD122+Ly49+ (Ly49+) or CD8+CD44+CD122+Ly49- (Ly49-) T cells from MOG+SP-immunized mice. We found that total CD8+ T cells as well as Ly49+CD8+ T cells from MOG+SP-immunized mice actively suppressed specific CD4+ T cells. 35-55 Furthermore, we adoptively transferred Ly49+ and Ly49- cells into mice before EAE induction and found that Ly49+ cells from MOG+SP-immunized mice significantly reduced EAE, and Ly49- cells significantly reduced EAE. We found that the transfer of cells had no effect (Fig. 14i), indicating that Ly49+ cells can not only suppress CD4+ T cells in vitro but also suppress EAE in vivo.

[0153] We also tested the effect of SP on another autoimmune disease model, experimental autoimmune uveitis (EAU). We induced EAU in C57BL6 / J mice using standard methods and compared EAU pathology with that in mice immunized with human interphotoreceptor binding protein (IRBP) + SP. IRBP-immunized mice developed a mild inflammatory response in 40% of the mice (Figures 15a and 15b). Interestingly, IRBP + SP immunization resulted in a more severe inflammatory response in a much larger proportion of mice (80%) (Figure 15c). We also performed in vitro suppression assays and found that CD4 + T cells robustly proliferated in the absence of CD8 + T cells from IRBP + SP-immunized mice (Figure 15d), and neither Ly49 + nor Ly49 - CD8 + T cells from IRBP + SP mice were able to suppress the proliferation of IRBP-specific CD4 + T cells (Figures 15e and 15f). Thus, EAE SP appears to be specific for the disease.

[0154] Qa-1b-restricted regulatory cells have been shown to mediate their effects through perforin, which is important for cytotoxicity. To test this possible mechanism in our system, we used MOG 35-55 We cocultured CD4+ T cells from immunized mice with CD8+ T cells from perforin knockout mice immunized with MOG+SP and found that this completely abolished their suppressive ability (Fig. 14f). Furthermore, we performed RNA-seq analysis of SP-specific CD8+ T cells from MOG- and MOG+SP-immunized mice, as well as CD8+ (Ly49+ vs. Ly49-) T cells from MOG+SP-immunized mice. Gene ontology enrichment analysis of differentially expressed genes between Ly49+ and Ly49- indicated genes involved in various T cell functions (Figs. 16a and 16b and Table 8).

[0155] Interestingly, SP-specific CD8+ T cells showed a significant upregulation of Ly49 genes, which are mostly inhibitory, as well as genes related to CD8+ T cell effector and memory functions, in addition to NK cell receptor genes (Fig. 6j). In addition, Ly49+ and SP-specific CD8+ T cells also express many genes associated with regulatory CD4+ T cells (Fig. 16c). Overall, our results suggest that SP immunization inhibits pathogenic MOG through cytotoxicity. 35-55 These findings strongly suggest that CD8+ T cells with a regulatory phenotype are induced that suppress specific CD4+ T cells and ultimately confer resistance to EAE.

[0156] To determine whether there are any similarities between MS and multiple sclerosis, we first directly determined the frequencies of CD4+, CD8+, and γδ+ T cells in recently diagnosed MS patients and found no difference in the frequency of total T cells in peripheral blood compared with healthy controls (HCs) (Figures 17a-c). However, similar to EAE and celiac disease, we performed single-cell TCR sequencing of activated brain-homing (CD38+HLA-DR+CD49d+CD29+) CD4+, CD8+, and γδ+ T cells from PBMCs of newly diagnosed MS patients and HCs (Table 9). We observed a large oligoclonal expansion of CD8+ T cells in MS patients compared with HCs (Figure 18a). We also observed only a small oligoclonal expansion of CD4+ T cells in MS patients (Figure 19a). Furthermore, we found that γδ + T cells also underwent clonally expanded expansion in MS patients and HCs (FIG. 20a).

[0157] The oligoclonal expansion of γδ+ T cells in MS has been noted previously. Unlike mouse nTγδ17 cells, which are largely "preprogrammed" in the thymus, the differentiation of human γδ17 T cells and their TCRs is poorly understood. In vitro activation of Vγ9δ2 T cells with TCR-specific agonists in the presence of cytokines has been shown to induce ROR-related orphan receptor gamma (RORγ) expression and IL-17 production. Indeed, we detected a significant increase in the frequency of RORC transcript-positive γδ T cells in MS patients (Figure 20b). Thus, in a chronic autoinflammatory environment such as MS, some of these expanded γδ T cells may produce IL-17 and contribute to the pathogenesis of diseases such as EAE.

[0158] In summary, we demonstrated that the simultaneous recruitment of oligoclonal CD4+, CD8+, and γδ+ T cells parallels not only EAE but also newly diagnosed MS patients. Furthermore, when we systematically characterized each of these cell types, we found that two of the three were likely pathogenic. Specifically, the γδ+ T cells that dominate the response are well-known producers of IL-17, a pro-inflammatory cytokine known to be important in EAE pathology, whereas the CD4+ T cell response is primarily MOG-specific and a major driver of EAE pathology.

[0159] In contrast to these two cell types, clonally expanded CD8+ T cells exhibit regulatory functions. These T cells have a distinct phenotype and express the classical class I MHC molecule H2-D. b We extend our observations centered on Qa-1b-restricted regulatory CD8+ T cells to include peptides presented by

[0160] More importantly, this study demonstrates the value of studying T cell specificity and activity "from scratch": identifying the most active T cells in a given response by single-cell paired TCR sequencing, using both activation markers and clonal expansion as key indicators, followed by ligand identification using either yeast display libraries or reporter cells transfected with candidate antigens and relevant TCR pairs. This contrasts with traditional methods, which typically involve knowing (or guessing) the relevant antigen.

[0161] In summary, the work presented here demonstrates the existence of a subset of CD8+ T cells capable of suppressing pathogenic CD4+ T cells in mice and humans, resulting in their synergistic recruitment during disease induction. Determining the ligands of these regulatory CD8+ T cells in autoimmune diseases has significant therapeutic value.

[0162] method Experimental animals. Female C57BL / 6J mice (designated either B6 or WT) and female perforin knockout mice (stock number: 002407) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The experimental procedures used in this study were approved by the Animal Care and Use Committee of Stanford University.

[0163] Human samples. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy blood donations from the Stanford Blood Center. Healthy human subjects were men and women aged 22–47 years. PBMCs from multiple sclerosis patients were obtained from the Multiple Sclerosis Center at the University of California, San Francisco (UCSF). The UCSF Committee on Human Research approved the protocol, and informed consent was obtained from all participants. Detailed information on the patient population included in this study is shown in Table 9.

[0164] Production of soluble TCRs. Soluble TCRs were produced as previously described. TCR-variable mouse constant human (VmCh) chimeras containing engineered C-domain disulfides were cloned into the pAcGP67a insect expression vector (BD Biosciences, 554756) encoding either a C-terminal acidic GCN4-zipper-biotin acceptor peptide (BAP)-6xHis tag (for the α chain) or a C-terminal basic GCN4 zipper-6xHis tag (for the β chain). Each chain also encoded a 3C protease site between the C-terminus of the TCR ectodomain and the GCN4 zipper, allowing for zipper cleavage. Baculovirus for each TCR construct was generated in SF9 cells via cotransfection of BD Baculogold linearized baculovirus DNA (BD Biosciences, 554739) with Cellfectin II (Life Technologies, 10362-100). TCR α and β chain viruses were administered in various ratios in small volumes (2 mL) of High Co-infections were performed in Five cells to find a ratio that ensured a 1:1 α:β stoichiometry.

[0165] To prepare soluble TCRs, 1 L of High Five cells was infected with the appropriate ratio of TCRα and TCRβ viruses for 48 hours at 28°C. The collected culture medium was conditioned with 100 mM Tris-HCl (pH 8.0), 1 mM NiCl2, and 5 mM CaCl2, and the resulting precipitate was removed by centrifugation. The medium was then incubated with Ni-NTA resin (QIAGEN 30250) at room temperature for 3 hours and eluted in 1x HBS + 200 mM imidazole (pH 7.2). The TCRs were then site-specifically biotinylated by adding recombinant BirA ligase, 100 μM biotin, 50 mM bicine pH 8.3, 10 mM ATP, and 10 mM magnesium acetate, followed by overnight incubation at 4°C. The reaction was then purified by size exclusion chromatography using an AKTA Purifier (GE Healthcare) on a Superdex 200 column (GE Healthcare). Peak fractions were pooled and then tested for biotinylation using an SDS-PAGE gel shift assay. Proteins were typically 100% biotinylated.

[0166] Mouse yeast display H2-D b Peptide library production, tag enrichment, staining, and selection. The single-chain trimer (SCT) H2-Db yeast construct was synthesized as an N-terminal fusion to the yeast surface protein Aga2p. Full-length SCT H2-D b The constructs were cloned into the vector pYAL. These constructs consist of the Aga2p leader sequence followed by a 9-10 mer peptide sequence, a Gly-Ser(GGGGS)3 linker, a mouse β2-microglobulin (β2M) sequence, a second glycine linker (GGGGS)4, and a mouse H2-D bThe SCTH2-Db MHC construct contained the heavy chain sequence, either a Myc or HA epitope tag, a third glycine linker (GGGGS)3, and the Aga2 protein. The SCTH2-Db MHC construct was then electroporated into EBY-100 yeast as described above and induced for expression in SGCAA, pH 4.5 medium at 20°C for 24-72 hours until maximal epitope tag staining was observed (typically 40%-70% of the total population). Full-length H2-Db MHC constructs were then electroporated into EBY-100 yeast as described above and induced for expression in SGCAA, pH 4.5 medium at 20°C for 24-72 hours until maximal epitope tag staining was observed (typically 40%-70% of the total population). b Yeast constructs were mutagenized as previously described. Briefly, constructs were mutagenized via error-prone PCR (Genemorph II kit, Agilent 200550). The error-prone constructs were ligated into the pYAL vector, with a final error rate of approximately 4-5 nucleotide substitutions per kbp, as judged by sequencing clones. Yeast libraries were generated by electroporation of competent EBY-100 cells via homologous recombination of the linear pYAL-cMyc / HA vector. The final library contained approximately 5x10 8 The yeast transformants included:

[0167] As previously mentioned, the pMHC constructs were prepared by using mutagenic primers that allow all 20 amino acids to be sequenced via the NNK codon instead of running along the peptide. The peptide library was generated in the same manner as the error-prone library, except that it was dammed. The library allowed only limited diversity at known MHC anchor residues to maximize the number of correctly folded and displayed pMHC clones in the library. H2-D bFor the P5 and P9 anchors, the P5 and P9 anchors were restricted to Asn (N) and Met / Ile / Leu (M / I / L), respectively, using the AAC and MTS codons. The resulting PCR products were used as templates for a second PCR reaction in which 50 nucleotides of sequence homologous to the vector were added to both ends of the PCR product. 50 μg of this second PCR product and approximately 10 μg of linearized vector were then purified and used to electroporate yeast to generate each library. Prior to selection on the H2-Db 9MER and 10MER pMHC libraries, each was enriched for its respective epitope tag to maximize the percentage of yeast in the initial pool with correctly folded and displayed pMHC molecules displayed on their surface. To achieve this, each library was divided into 1 × 10 7 The cells were separately induced in 500 mL of SGCAA for 24–72 h at 20 °C at a starting density of cells / mL. When maximal epitope tag staining was observed, approximately 1.4 × 10 9 Induced yeast cells were washed once in PBS + 0.5% BSA and 1 mM EDTA (PBE buffer) and resuspended in 5 mL of PBE with 200 μL of Miltenyi streptavidin microbeads (Miltenyi, 130-048-101). The cell and bead mixture was incubated for 1 hour at 4 °C with rotation, washed again in PBE, resuspended in 5 mL of PBE, and passed through a cell strainer onto a pre-wetted MACS LD column (Miltenyi 130-042-901). After the column was completely emptied, it was washed twice with 2 mL of PBE and the flow-through was collected.

[0168] Isolate the cells from the flow-through by centrifugation and incubate with 80 µL of anti-cMyc AlexaFluor647 antibody or anti-HA AlexFluor647 antibody (Cell The cells were resuspended in 5 mL of PBE with ATP (signaling, 2233, and 3444) and incubated at 4 °C for 1 h with rotation. The cells were washed and resuspended in 5 mL of PBE. 220 μL of Miltenyi anti-AlexaFluor 647 microbeads (Miltenyi, 130-091-395) was added, and the mixture was incubated at 4 °C for 30 min with rotation, protected from light. The cells were then washed, resuspended in 6 mL of PBE, and split equally between two pre-wetted MACS LS columns (Miltenyi, 130-042-401). After the columns were completely emptied, each column was washed twice with 3 mL of PBE, and the flow-through was retained. The cells were eluted from the columns using 5 mL of PBE per column. A small fraction of the eluate (5–20 μL) was retained, and AlexaFluor 647 staining was compared with that of the flow-through for quantification of tag enrichment. The remaining eluted cells were pooled, collected by centrifugation, and resuspended in a total of 40 mL of SDCAA medium. The cell density was measured spectrophotometrically at 600 nm. The cell density was then adjusted to below OD by adding SDCAA, and the yeast was cultured overnight at 30°C. The cells were passaged for another round of overnight growth in SDCAA.

[0169] For tag elution, the yeast cells were cultured in 500 mL of SGCAA at 20°C. To stain pMHC in the TCR tetramer, biotinylated TCR was incubated with streptavidin conjugated to AlexaFluor 647, AlexaFluor 488, or phycoerythrin at a 5:1 ratio on ice for 5 minutes to ensure complete tetramer formation. Yeast cells were then stained with 250 nM tetramer plus anti-Myc-AlexaFluor 488 or anti-HA-AlexaFluor 488 antibodies (Cell Signaling, 2279 or 2350, respectively) on ice for 3 hours, washed twice with ice-cold PBE buffer, and then analyzed via flow cytometry (Accuri C6 flow cytometer). All yeast selections and sequencing of the yeast libraries were performed as previously described.

[0170] H2-D bList of primers used for the library. H2-D b Error prone line For generating the random H2-Db library, the forward primer was 5'-TGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTAGCGT TTTAGCAAGCAGCCTGGAGAACTTCAGAGCCTACGTGG-3' and the reverse primer was 5'-GAACAAAAGCTTATCTCCGAAGAAGACTTG-3'. For the random H2-Db library, the forward primer for the 9MER HA library (initial randomization PCR) was 5'-TCAATATTTTCTGTT ATTGCTAGCGTTTTAGCANNKNNKNNKNNKAACNNKNNKNNKMTSGGTGGAGGAG GTTCTG-3' and the reverse primer for the 9MER HA library (initial randomization PCR) was 5'-TCCACCACCACCAGC GTAGTCTGGAACGTCGTATGGGTAGGATCCCTCCCA-3'. To add overlap for homologous recombination with the linearized pYAL vector, forward primer: 5'-ATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTG TTATTGCTAGCGTTTTAGCA-3', reverse primer: 5'-TCCACCACCACCAGCGTAGTCTGGAACGTCGTATG GGTAGGATCC CTCCCA-3'.

[0171] Class I and Class II peptide monomer production, tetramerization, and tetramer enrichment. For peptide I-Ab monomer production, peptide-I-Ab monomers were produced as previously described. Briefly, IA bThe extracellular portion of the α-chain was linked to an acidic zipper at the C-terminus, followed by AviTag (GLNDIFEAQKIEWHE) and a 6x histidine tag. The peptides myelin oligodendrocyte glycoprotein (MOG) 38-48 (GWYRSPFSRVV) or ovalbumin (OVA) 327-337 (VHAAHAEINEA) were tethered to the N-terminus of the I-Ab β-chain, followed by a basic zipper and a 6x histidine tag. A disulfide trap was introduced through oxidation of the cysteine ​​at position p+2 and cysteine ​​at position 72 of the I-Ab α-chain, mutated from valine, to ensure proper peptide binding.

[0172] The α-chain and peptide-β-chain were separately cloned into the pAcGP67A vector by Gibson Assembly (New England Biosciences, E2611S). Baculovirus for each construct was generated in SF9 cells via co-transfection of BD BaculoGold linearized baculovirus DNA (BD Biosciences, 554739) with Cellfectin II (Life Technologies, 10362-100). The α- and β-chain viruses were co-infected in small volumes (2 mL) of High Five cells at various ratios to find a ratio that ensured a 1:1 α:β stoichiometry.

[0173] To prepare soluble monomers, 1 L of High Five cells was infected with the appropriate ratio of α and β viruses for 48 hours at 28°C. The collected culture medium was conditioned with 100 mM Tris-HCl (pH 8.0), 1 mM NiCl2, and 5 mM CaCl2, and the resulting precipitate was removed by centrifugation. The medium was then incubated with Ni-NTA resin (QIAGEN, 30250) at room temperature for 3 hours and eluted in 1x HBS + 200 mM imidazole (pH 7.2). The TCR was then site-specifically biotinylated by adding recombinant BirA ligase, 100 μM biotin, 50 mM bicine pH 8.3, 10 mM ATP, and 10 mM magnesium acetate, and incubating overnight at 4°C. The reaction was then purified by size exclusion chromatography using an AKTA Purifier (GE Healthcare) on a Superdex 200 column (GE Healthcare). Peak fractions were pooled and then tested for biotinylation using an SDS-PAGE gel shift assay. Proteins were typically 100% biotinylated.

[0174] Peptide-H2-D b Monomer production. Peptide-H2-D b Monomers were refolded with the appropriate peptide and human β2-microglobulin as previously described. b and human β2-microglobulin were separately expressed in the form of inclusion bodies in BL21DE3 (ThermoFisher, C600003). b In the construct, H2-D bThe α chain was linked to an AviTag and a 6X histidine tag. Refolding was performed using rapid dilution. After biotinylation with BirA, the proteins were purified by size-exclusion chromatography (Superdex 20010 / 300GL) and stored at -80°C. For YQPGNWEYI (YQP), HDRVNWEYI (HDR), ASRSNRYFWL (ASR), and SMRPNHFFFL (SMRP), we individually refolded and purified the monomers. For peptide-H2-Db monomers of the 6218 influenza peptides (QGLSNMRVRL, VGLENMRVRL, VSLRNMRSYL, and SSLENFRAYV), the H2-Db was refolded with a photocleavable peptide (FAPGNY-Anp-AL), and the target peptide was exchanged for the H2-Db upon UV cleavage of FAPGNY-Anp-AL.

[0175] Peptide-MHC tetramer formation. All tetramers were freshly prepared as described above. Briefly, for tetramerization, the amounts of fluorophore-conjugated streptavidin and pMHC monomer were mixed at a 4:1 molar ratio. One-fifth the amount of fluorophore-conjugated streptavidin was added to the monomer solution every 10 min at room temperature.

[0176] Enrichment of tetramer-positive T cells in mice and cell lines. Single-cell suspensions of spleen and LN cells were prepared from non-immunized or immunized mice and resuspended in 200 μL of FACS buffer (Ca2+ / Mg2+-free sterile PBS, 0.5% BSA, 0.5 mM EDTA) containing Fc block (1:100) and 10 μM biotin. I-Ab-MOG 38-48 Tetramer (15nM), I-Ab-OVA 327-337 Tetramer (15nM), H2-D b -ASR tetramer (25nM), H2-D b -SMRP tetramer (10nM), H2-D b -YQP tetramer (25 nM) and H2-D bA tetramer concentration of 1-HDR tetramer (25 nM) was used for staining the cells. Cells were stained with the tetramer at room temperature for 1 hour and washed with FACS buffer. 38-48 and I-Ab-OVA 327-337 For tetramers, enrichment of tetramer-positive cells was performed using the EasySep™ PE Positive Selection Kit (STEMCELL Technologies, 18557). When cells were stained with both I-Ab tetramers and H2-Db tetramers, enrichment of tetramer-positive cells was performed using anti-PE microbeads (Miltenyi Biotec, 130-048-801) and anti-His microbeads (Miltenyi Biotec, 130-094-258) according to the manufacturer's instructions.

[0177] After tetramer enrichment, cells were surface stained with an antibody cocktail for 20 minutes at 4°C. Stained cells were washed using FACS buffer and analyzed on an LSR II (Becton Dickinson) or single-cell sorted / bulk-sorted on a FACS Aria Fusion SORP (Becton Dickinson). Lentivirally TCR-transduced Jurkat TCRαβ - / - Cell lines were stained with tetramer at a concentration of 20 nM in FACS buffer containing 10 μM biotin for 1 hour at room temperature, followed by surface staining with the appropriate antibody for 20 minutes at 4°C. After surface staining, cells were washed with FACS buffer and then transferred to an LSR. The analysis was performed using a Becton Dickinson HPLC-MS / MS HPLC system.

[0178] Single-cell mouse and human TCR sequencing and data analysis. All human TCR primers used were previously published. All mouse TCR primer sequences are provided in Table 1. TCR sequencing was performed according to previously established protocols.

[0179] Induction and evaluation of EAE. EAE was actively induced in C57BL / 6J mice according to a previously established protocol. Briefly, for EAE induction, 200 μg of MOG from the yeast libraries ASRSNRYFWL, SMRPNHFFFL YQPGNWEYI, and HDRVNWEYI was used. 35-55 Mice were subcutaneously injected with an equal volume of complete Freund's adjuvant (CFA; Sigma-Aldrich, F5881) supplemented with 200 μg of Mycobacterium tuberculosis H37Ra (Difco Laboratories, 231141) or CD8-specific SP into the right and left hind flanks. On the day of immunization and two days after immunization, each mouse received 200 ng of PTX (List Biological Laboratories, 180) via intraperitoneal injection. Starting on day 5 post-injection, mice were scored daily for clinical signs of EAE as follows: 0: no clinical manifestations of disease; 1: tail flaccidity without hind limb weakness; 2: hind limb weakness; 3: complete hind limb paralysis and tail flaccidity; 4: tail flaccidity and hind limb paralysis with urinary or fecal incontinence; and 5: moribund. Clinical quantitative characteristic variables were assessed as previously described.

[0180] Isolation of CNS-infiltrating mononuclear cells. CNS-infiltrating cells were isolated according to a previously established protocol. Briefly, on different days after immunization, animals were perfused with saline, and the brain and spinal cord were removed. A single-cell suspension was obtained and passed through a 70 μm strainer. Mononuclear cells were obtained by centrifugation through a Percoll gradient (37% / 70%) and collected from the interface. The cells were washed, labeled with fluorescent dye-conjugated antibodies, and analyzed by flow cytometry.

[0181] TCR, H2-D by lentiviral transduction b , and Qa-1b expression. TCRα, β, H2-D bThe α and β2M constructs were cloned into lentiviral constructs. For TCR expression, the alpha and beta TCR lentiviral constructs were transfected separately into 293X cells. Viruses were harvested 72 hours post-transfection and transfected into Jurkat αβ cells. - / - or SKWαβ - / - Cells were transduced. SKW or Jurkat cells were enriched for the highest expression of TCRαβ by using Miltenyi anti-APC selection (Miltenyi 130-090-855). A similar strategy was used for transduction and expression of H2-D cells, except that T2 cells were used for transduction and expression. b and β2M expression.

[0182] T cell stimulation assay. T cell stimulation assays were performed as described above. All T cell peptide stimulation experiments were performed in 96-well round-bottom plates in a total volume of 200 μl. T2, K562 cells, or BMDCs were pulsed with 10–100 μg of peptide for 45 minutes, washed once, and plated (10,000 cells / well). TCR-expressing cell lines (100,000 cells / well) were cocultured with APCs for 18 hours. At the end of stimulation, cells were harvested, washed, stained for TCRβ, human CD3, and CD69, and analyzed for activation on an LSR II (Becton Dickinson).

[0183] In vitro proliferation / suppression assay. Spleen and LN cells were collected from WT or immunized mice. Single-cell suspensions were prepared, and RBCs were lysed using Ack lysis buffer (ThermoFisher Scientific A1049201). Total CD4+ (Miltenyi 130-049-201) and CD8+ (Miltenyi 130-049-401) T cells were positively purified using Miltenyi kits according to the established manufacturer's protocol, followed by FACS sorting. Similarly, antigen-presenting cells were isolated using the Miltenyi Pan dendritic cell (DC) isolation kit (Miltenyi 130-100-875). After CD4+ T cell enrichment, cells were counted and stained with CellTrace™ Violet dye (ThermoFisher Scientific A1049201) according to the manufacturer's instructions. The antibody was labeled with 1000kJ / mL (scientific C34557).

[0184] In vitro proliferation / suppression assays were performed according to previously published protocols. Briefly, labeled CD4+ T cells were incubated with pan DCs (0.75 × 106 cells / well) with or without CD8+ T cells (1:1 ratio, 0.25 × 106 cells / well). 6 In some suppression experiments, cells were pre-incubated with 10 μg / ml of anti-Qa-1b neutralizing antibody (6A8.6F10.1A16, BD Biosciences). Cells were cultured in a total volume of 200 μl in 96-well round-bottom plates. CD4+ T cells were co-cultured with MOG 35-55 On day 7, cells were washed, stained with surface antibodies, and analyzed on an LSR II (Becton Dickinson).

[0185] Adoptive transfer. EAE was actively induced in C57BL / 6J mice with 200 μg of MOG derived from the yeast libraries ASRSNRYFWL, SMRPNHFFFL YQPGNWEYI, and HDRVNWEYI. 35-55 +An emulsion containing CD8-specific PPT and 200 μg of Mycobacterium tuberculosis Mice were immunized by contact with an equal volume of complete Freund's adjuvant (CFA; Sigma-Aldrich, F5881) supplemented with H37Ra (Difco Laboratories, 231141). On the day of immunization and two days after immunization, each mouse received 200 ng of PTX (List Biological Laboratories, 180) via intraperitoneal injection. Ten days after immunization, spleens and lymph nodes were harvested, and CD8+ cells were obtained using a CD8 enrichment kit and sorted for CD44+CD122+Ly49+ (Ly49+) and CD44+CD122+Ly49- (Ly49-) cells.

[0186] FACS-purified Ly49+ and Ly49- cells (8 million cells / mouse) were adoptively transferred at the time of active MOG immunization, and mice were scored daily for clinical signs of EAE starting on day 5 or later, as previously described.

[0187] Induction and evaluation of EAU. Experimental autoimmune uveitis (EAU) was induced in mice as previously described. Briefly, for EAE induction, an emulsion containing 300 μg of human interphotoreceptor binding protein (IRBP) peptide 1-20 in CFA (1:1 v / v) and 0.2 μg of PTX on day 0 and again on day 2 was subcutaneously injected into the posterior right and left flanks of mice. Mice were euthanized 21 days after immunization. The eyeballs were removed from the mice's eyes, fixed, and pupil-optic nerve sections were examined histologically as previously described.

[0188] Whole-transcriptome sequencing and data analysis. Whole-transcriptome sequencing was performed as described previously, and T cells were bulk-sorted directly in Trizol (Qaigen). RNA was extracted using the RNeasy Plus Micro Kit (Qiagen). After analysis on the 2100 Bioanalyzer, the resulting libraries were sequenced on a HiSeq4000 platform (Illumina). For each sample in the whole-transcriptome sequencing library, 75-base pair paired-end reads were obtained from the sequencer. Each sample condition was completed in triplicate, except for the WT sample, which generated a single sample. Read quality was determined using FastQC 0.11.4. Using TopHat v2.0.13, we aligned the reads to the mouse reference genome (NCBI / assembly GRCm38). On average, 90% of the reads aligned to the reference genome. One MOG+SP sample was removed from downstream analysis as an identified outlier. Differential gene expression analysis and read count normalization used as input for the heatmap were determined via DESeq2. TPM values ​​were calculated with RSEM v1.3.0. Heatmaps were generated using the R package "pheatmap." Gene ontology analysis plots were generated with the R package "enrichplot." Data availability: RNA-seq data and yeast p-MHC selection data are available under the accession number G Deposited in the Gene Expression Omnibus (GEO) data repository under SE 130975. Source data for each figure are provided.

[0189] [Table 1-1]

[0190] [Table 1-2]

[0191] [Table 1-3]

[0192]

Table 1-4

[0193]

Table 2

[0194]

Table 3-1

[0195]

Table 3-2

[0196]

Table 4-1

[0197]

Table 4-2

[0198]

Table 5-1

[0199]

Table 5-2

[0200]

Table 6

[0201] Summary of Illumina totals per round of 6218, EAE6, and EAE7-CD8 TCR selection. Unique peptide sequences corresponded to reads that were in-frame without a stop codon. Fraction of unique peptides refers to the total sequencing reads per RD divided by the unique peptide sequences for that RD. Corrected fold enrichment refers to the fold enrichment of peptides per RD selection normalized to the total number of reads from the naive RD.

[0202] [Table 7]

[0203] [Table 8]

[0204] [Table 9-1]

[0205] [Table 9-2]

[0206] [Table 10]

[0207] [Table 11]

[0208] Participants in the trial were from the University of California, San Francisco. This forms an ongoing prospective observational study at the CA Multiple Sclerosis Center, San Francisco, California. Patients were recruited if suspected of having MS or presented within 24 hours and 90 days of an initial event indicative of MS. Patients with clinically isolated syndrome (CIS) were included if they met the Magnetic Resonance Imaging in Multiple Sclerosis (MAGNIMS) criteria (Polman et al., 2011). 1). Eligibility criteria included no prior treatment with MS disease-modifying therapy or broad-spectrum immunosuppressants, nor treatment with corticosteroids within the past 30 days. Peripheral blood lymphocytes were prepared by Ficoll gradient and frozen in liquid nitrogen within 2 hours of phlebotomy. Age- and sex-matched healthy control PBMCs were obtained from the Stanford Blood Center (Stanford University, Stanford, CA). Ancestry: 1 - European American, 2 - African American, 3 - Hispanic, 4 - Asian. HLA-DRB1*15:01: 1 - carrier and 2 - noncarrier. PP, primary progressive MS, RR, relapsing-remitting MS, CIS, clinically isolated syndrome. HC, healthy control.

[0209] Related Publications International Multiple Sclerosis Genetics Consortium et al.Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.Nature 476,214-219(2011) Fallang, L.-E. et al. Nat. Immunol. 10, 1096-1101 (2009) Sollid,et al.Immunogenetics 64,455-460(2012) Zamvil, S. et al. Nature 317, 355-358 (1985) Blankenhorn,E.P.et al.Ann.Neurol.70,887-896(2011) Skulina,C.et al.PNAS 101,2428-2433(2004) Babbe,H.et al.J.Exp.Med.192,393-404(2000) Blink,S.E.&Miller,S.D.Curr.Mol.Med.9,15-22(2009) Han,A.et al.Proc.Natl.Acad.Sci.U.S.A.110,13073-13078(2013) Birnbaum,M.E.et al.Cell 157,1073-1087(2014) Gee,M.H.et al.Cell 172,549-556.e16(2018) Han,et al.Nat.Biotechnol.32,684-692(2014) Wei,Y.-L.et al.Front Immunol 6,118(2015) Langrish,C.L.et al.J.Exp.Med.201,233-240(2005) Kroenke et al.J.Exp.Med.205,1535-1541(2008) Ben Nun et al.European Journal of Immunology 11,195-199(1981) Jager et al.The Journal of Immunology 183,7169-7177(2009) Denton,A.E.et al.The Journal of Immunology 187,5733-5744(2011) Day,E.B.et al.PNAS 108,9536-9541(2011). Moon,J.J.et al.Immunity 27,203-213(2007) Kim,H.-J.&Cantor,H.Semin.Immunol.23,446-452(2011) Lu,et al.Proc.Natl.Acad.Sci.USA105,19420-19425(2008) Kim,H.-J.et al.Proc.Natl.Acad.Sci.USA108,2010-2015(2011) Agarwal,RK&Caspi,RRMethods Mol.Med.102,395-419(2004) Zemmour,D.et al.Nat.Immmunol.19,291-301(2018) Hvas,et al.J.Neuroimmunol.46,225-234(1993) Wucherpfennig,KWet al.PNAS 89,4588-4592(1992) Gandhi,R.,Laroni,A.&Weiner,HL J.Neuroimmunol.221,7-14(2010) Caccamo,N.et al.Blood 118,129-138(2011) Moens,E.et al.J.Leukoc.Biol.89,743-752(2011) Sutton,CEet al.Immunity 31,331-341(2009) Price et al.PLoS ONE 7,e39750(2012) Harrington,LEet al.Nat.Immunol.6,1123-1132(2005) Elias,et al.Int Immunol 11,957-966(1999) Kumar et al.J.Exp.Med.184,1609-1617(1996) Hu,D.et al.Nat.Immunol.5,516-523(2004) Panoutsakopoulou,V.et al.J.Clin.Invest.113,1218-1224(2004) Davis&Brodin Rebooting Human Immunology.Annual Review of Immunology 36,843-864(2018) O’Shea,et al.Current Biology 3,658-667(1993) Birnbaum,M.E.et al.Cell 157,1073-1087(2014) Adams,J.J.et al.Immunity 35,681-693(2011) Nelson,R.W.et al.Immunity 42,95-107(2015) Stadinski,B.D.et al.PNAS 107,10978-10983(2010) Altman,J.D.et al.Science 274,94-96(1996) Grotenbreg,G.M.et al.PNAS 105,3831-3836(2008) Krementsov,D.N.et al.Ann.Neurol.75,50-6 6(2014) Tennakoon,D.K.et al.J.Immunol.176,7119-7129(2006) Bian,Y.et al.PLOS Pathogens 13,e1006384(2017) Mahajan et al.J Vis Exp e3184-e3184(2011).doi:10.3791 / 3184 Mamedov,M.R.et al.Immunity 48,350-363.e7(2018) Love,M.I.,Huber,W.&Anders,S.Genome Biol.15,31(2014) Li,B.&Dewey,C.N.BMC Bioinformatics 12,323(2011) Kolde,RR package version1.0.8.(2015) Yu,G.clusterProfiler:An universal enrichment tool for functional and comparative study.doi:10.1101 / 256784

[0210] Example 2 CD8+KIR+ cells in human diseases A small subset of CD8+ T cells expressing Ly49 protein in mice can suppress autoimmunity in models of demyelinating diseases. Here, we demonstrate that CD8+ T cells expressing killer cell immunoglobulin-like receptors (KIRs), the functional counterparts of the Ly49 family, are expressed in the blood and inflamed tissues of patients with a wide variety of autoimmune diseases. + It has been shown that the frequency of T cells is significantly increased in celiac disease (CeD). - Not KIR + CD8 + T cells were isolated from patient leukocytes in vitro and expressed pathogenic gliadin-specific CD4 + This, together with gene expression data, demonstrated that these cells express mouse Ly49 + CD8 + Furthermore, in COVID-19 patients who may suffer from significant clinical problems suggestive of autoimmunity, the inventors found that KIR + CD8 + We found that elevated levels of T cells correlated with disease severity and the development of vasculitis, a common complication of COVID-19, but that CD4+ regulatory T cells did not show this trend. Additionally, we found that CD4 regulatory T cells, which have a phenotype characteristic of gliadin-specific cells, were expressed in CeD. + We also found an increase in regulatory CD8 T cells, which are also elevated in some autoimmune diseases. +This defines and characterizes T cell subsets that we speculate are common features of infectious disease responses and also exist in autoimmunity, functioning to control autoreactive or otherwise pathogenic T cells. These data also suggest that many of the complications of COVID-19 are the result of various types of autoimmunity.

[0211] Although most CD8+ T cells are directed toward controlling pathogen-infected or cancer cells, there has been long-standing evidence in mice that some cells can also suppress autoimmune responses. This potential regulatory function of CD8+ T cells was first implicated in CD8+ T cell depletion in experimental autoimmune encephalomyelitis (EAE), a mouse model of human multiple sclerosis (MS). The Ly49 family of inhibitory C-type lectin-like receptors ubiquitously present on natural killer (NK) cells was identified as a unique surface marker for this regulatory CD8+ T cell subset, and the transcription factor Helios was identified as an essential control element for their differentiation and function in mice. Recently, we demonstrated that clonally expanded CD8+ T cells during EAE recognize peptides bound to H2-Db and that these peptides stimulate Ly49+CD8+ regulatory T cells to suppress disease. This extends the original observation to include classical class I MHC interactions beyond Qa-1, indicating a general mechanism of peripheral tolerance. Herein, we identify CD8+ T cells expressing killer cell immunoglobulin-like receptors (KIRs), the functional counterparts of the mouse Ly49 family in humans, as a novel CD8+ T cell subset that targets pathogenic CD4+ T cells in celiac disease (CeD) and other autoimmune disorders and infectious diseases.

[0212] Increased KIR+ CD8+ T cells in human autoimmune diseases. Both mouse Ly49 and human KIR receptors bind to class I MHC molecules, typically contain inhibitory tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tails, and are ubiquitously expressed on NK cells as well as a small subset (1-5%) of CD8+ T cells. We analyzed CD8+ T cells expressing inhibitory KIRs in the peripheral blood of patients with autoimmune diseases and age / gender-matched healthy controls (HCs). In particular, we found that KIR3DL1 and KIR2DL3 are two major KIR subtypes expressed by a small subset of human CD8+ T cells. We found a significant 10-fold increase in KIR+ CD8+ T cells in subsets of patients with MS and systemic lupus erythematosus (SLE), compared with healthy controls, and to a lesser extent in CeD (Figure 17a). Furthermore, the frequency of KIR+CD8+ T cells in the blood of SLE patients positively correlated with the frequency of potentially autoreactive CD4+ T cells with a phenotype previously found to be unique to gliadin-specific CD4+ T cells in CeD (CD45RA-CD62L-PD-1+CXCR3+CD39+CD38+CD127-CD25lowCD161+ICOS+) (Figure 17b), indicating the expansion of KIR+CD8+ T cells synchronous with the intensity of the CD4+ T cell-driven autoimmune response. Next, we investigated whether KIR+CD8+ T cells are also present in inflamed tissues of autoimmune diseases. We utilized publicly available single-cell RNA-seq data from SLE kidneys and rheumatoid arthritis (RA) synovium previously generated by the Accelerating Medicines Partnership RA / SLE program. First, we identified CD8+ T cells expressing KIR transcripts (KIR3DL1, KIR2DL3, and KIR2DL2) in the kidney and synovium. Notably, we observed that the number of KIR+ CD8+ T cells was significantly increased in the kidneys of patients with SLE compared with healthy kidneys (Fig. 17c).Furthermore, we detected a higher frequency of KIR+CD8+ T cells in the synovial tissue of RA patients compared with osteoarthritis (OA) patients, and the proportion of synovial FOXP3+CD4+ Treg cells was similar between RA and OA (Fig. 17d). Although both RA and OA cause arthritis, RA is a classic autoimmune disease, whereas OA is not, suggesting that KIR+CD8+ T cells may be more important than CD4+ Tregs in suppressing autoimmune inflammation.

[0213] KIR+CD8+ T cells are the functional and phenotypic equivalent of mouse Ly49+CD8+ T cells. Next, we investigated whether KIR+CD8+ T cells are the functional counterpart of mouse Ly49+ regulatory CD8+ T cells. Previously, we found that Ly49+CD8+ T cells suppress myelin oligodendrocyte glycoprotein (MOG)-specific CD4+ T cells in a perforin-dependent manner, indicating cytotoxicity as the mechanism of suppression. Deamidated gliadin, derived from dietary gluten, is a CD4+ T cell antigen that causes autoimmune enteropathy in human CeD. Therefore, we explored whether KIR+CD8+ T cells could suppress gliadin-specific CD4+ T cells from CeD patients. CD8+ T cells were purified from peripheral blood mononuclear cells (PBMCs) of HLA-DQ2.5+CeD patients. These KIR+CD8+ and KIR-CD8+ T cells were sorted and activated overnight with anti-CD3 / CD28 microbeads, then cultured at a 1:30 ratio with the CD8-depleted fraction of PBMCs in the presence of 250 μg / mL deamidated gluten. Cultures were harvested on day 6 and enriched for gliadin-specific CD4+ T cells. The activity of gliadin-specific CD4+ T cells was quantified using PE-labeled HLA-DQ2.5 tetramers complexed with different gliadin peptides (Fig. 18a). In the absence of KIR+CD8+ T cells, deamidated gluten significantly stimulated the expansion of gliadin-specific CD4+ T cells. Importantly, stimulated KIR+CD8+ T cells significantly reduced the number of gliadin-specific CD4+ T cells without affecting the number of total CD4+ T cells, whereas KIR-CD8+ T cells did not (Fig. 18b). We also measured Annexin V binding on day 3 (Fig. 18a) and found increased staining of gliadin-specific CD4+ T cells in the presence of KIR+CD8+ T cells (Fig. 18c), indicating that these T cells suppress pathogenic CD4+ T cells by direct killing. This effect of KIR+CD8+ T cells targets only a small fraction of CD4+ T cells, as it had no discernible effect on CD4+ T cell proliferation in response to anti-CD3 stimulation.

[0214] To further investigate whether KIR+CD8+ T cells are the phenotypic equivalent of mouse Ly49+ T cells in humans, we performed RNA sequencing (RNA-seq) analysis of KIR+ versus KIR-CD8+ T cells from patients with MS and compared them with mouse Ly49+CD8+ T cells in EAE (a mouse model of human MS). There were 778 differentially expressed genes (adjusted P<0.05, fold change>2) between KIR+ and KIR-CD8+ T cells, of which 300 were upregulated and 478 were downregulated in KIR+CD8+ T cells. Notably, KIR+ CD8+ T cells showed significant upregulation of inhibitory KIR receptor genes as well as cytotoxic molecules (e.g., GZMH, GZMB, PRF1, and GNLY), NK-related genes (e.g., NKG7, NCR1, and the KLR family), and cell trafficking molecules (e.g., CX3CR1, which mediates leukocyte migration to inflamed tissues and is involved in tissue damage-mediated brain inflammation, and the brain-homing receptor ITGB1). Additionally, KIR+ CD8+ T cells had higher transcript levels of Helios (encoded by Ikzf2), a transcription factor associated with the regulatory function of both CD4+ and CD8+ T cells. Meanwhile, KIR+ CD8+ T cells downregulated naive / memory T cell-associated molecules such as CCR7, SELL, TCF7, and IL7R, indicating that they may have entered a program for effector T cell differentiation. Interestingly, KIR+ CD8+ T cells have low expression of the costimulatory receptor CD28, one of the key characteristics of regulatory CD8+ T cell populations in mice and humans. Gene ontology enrichment analysis of these differentially expressed genes showed enrichment for T cell activation, proliferation, migration, and differentiation. Furthermore, gene set enrichment analysis (GSEA) revealed that approximately half of the top 200 genes upregulated in Ly49+ CD8+ T cells (including the cytotoxic molecule GZMB, KLRC family genes, CX3CR1, ITGB1, and IKZF2) were also elevated in KIR+ CD8+ T cells.Previously, we found that Ly49+CD8+ T cells express 16 of 60 genes conserved in CD4+ regulatory T cells (Tregs), and these same Treg signature genes23 were also enriched in KIR+CD8+ T cells in GSEA analysis. Overall, our RNA-seq analysis indicates that KIR+CD8+ T cells from MS patients share many similarities with Ly49+CD8+ T cells from EAE mice.

[0215] Furthermore, we also performed RNA-seq on KIR+ and KIR-CD8+ T cells from healthy subjects and patients with other autoimmune diseases, specifically CeD and SLE, to determine whether there are common characteristics shared by KIR+CD8+ T cells across different conditions. We identified a set of 963 genes that were differentially expressed (adjusted P<0.05, fold change>2) between KIR+ and KIR-CD8+ T cells from all subjects, including HC, MS, CeD, and SLE patients. Many of these overlapped with differentially expressed genes previously defined in MS. However, larger fold changes in these genes were associated with a higher frequency of KIR+CD8+ T cells. This was observed in patients with pulmonary fibrosis. Consistent with the transcriptional profile, KIR+ CD8+ T cells exhibited high protein expression levels of granzyme B, perforin, CX3CR1, KLRG1, CD244, TIGIT, T-bet, and Helios proteins, and low levels of CCR7, CD27, and CD28, as measured by flow cytometry. In addition, we compared KIR+ and KIR- CD8+ T cells in kidney or synovium for expression of the same genes enriched in circulating KIR+ CD8+ T cells. Similar to these cells, both kidney and synovium KIR+ CD8+ T cells upregulated KLRG1, CD244, TIGIT, CX3CR1, PRF1, GZMB, and IKZF2, while downregulated CD28 and CCR7. Overall, our results demonstrate that KIR+CD8+ T cells are the functional and phenotypic equivalent of murine Ly49+CD8+ T cells in humans, with conserved characteristics in both healthy subjects and patients with autoimmune conditions.

[0216] Increased KIR+CD8+ T cells correlate with immune dysregulation in COVID-19 patients. Previously, it was thought that most self-specific T cells were eliminated in the thymus, but recent studies have shown that this is not the case; many such cells survive and reside in the periphery in both humans and mice. We speculate that this is because the constant threat of infectious diseases throughout human history led to the assumption that even autoreactive T cells might be required to respond to specific pathogens, necessitating a full T cell repertoire. Consistent with this are classic experiments showing that infections or treatments that mimic them (e.g., complete Freund's adjuvant) can activate self-specific T cells. There is also anecdotal evidence that many patients report infections shortly before the onset of illness. Therefore, we were interested in analyzing patients with infectious diseases to determine whether KIR+CD8+ T cells were induced as part of the response.

[0217] In particular, new reports indicate that infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may lead to excessive production of proinflammatory cytokines and the emergence of autoimmune-related complications, especially in patients with severe disease. Therefore, we analyzed the frequencies of both KIR+CD8+ T cells and autoimmune CD4+ T cells with phenotypes characteristic of CeD-derived gliadin-specific cells in the peripheral blood of coronavirus disease 2019 (COVID-19) patients compared with age- and sex-matched healthy subjects collected before the pandemic. Similar to previous findings in influenza-infected patients, the frequency of autoimmune CD4+ T cells was increased in COVID-19 patients, particularly in those with moderate or severe disease (Figure 19a). The proportion of KIR+CD8+ T cells was also elevated in COVID-19 patients and correlated with disease severity (Figure 19b) as well as the frequency of autoimmune CD4+ T cells (Figure 19c). Furthermore, both autoimmune CD4+ T cells and KIR+ CD8+ T cells were increased in COVID-19 patients with vasculitis or embolism, and to a lesser extent in patients with acute respiratory distress syndrome (ARDS) (Figure 19d). These are common complications of the disease and are likely caused by excessive inflammation. These two T cell subsets are associated with autoimmune-related immunopathology during SARS-CoV-2 infection. However, CD25 T cells were significantly elevated in COVID-19 patients compared with healthy donors or COVID-19 patients with different disease severity or complications. 高 CD127 低 CD4 + No significant differences in the levels of Tregs were observed by the inventors, which is likely due to the KIR + CD8 + We suggest that T cells are a unique regulatory mechanism that is upregulated during SARS-CoV-2 infection to specifically control cross-reactivity against self-antigens.

[0218] We also utilized publicly available single-cell RNA-seq data of bronchoalveolar immune cells from COVID-19 patients and healthy controls to investigate whether KIR+CD8+ T cells are also present in the bronchoalveolar lavage fluid (BALF) of COVID-19 patients. CD8+ T cells expressing KIR transcripts (KIR3DL1, KIR3DL2, KIR2DL3, or KIR2DL1) were detected in BALF from COVID-19 patients with moderate or severe disease, but not in BALF from healthy controls (Figure 19e), indicating that KIR+ CD8+ T cells are also induced at the site of infection.

[0219] Commonalities and Heterogeneity of KIR+ CD8+ T Cells. To better understand the functional properties of this type of cell under different conditions, we used the Seurat package to integrate single-cell RNA-seq data (generated on the 10xGenomics platform) of peripheral blood CD8+ T cells from healthy subjects, MS patients, and COVID-19 patients. Total CD8+ T cells were projected onto a 2D UMAP and, after unsupervised clustering, eight subpopulations were identified based on gene expression. KIR+ CD8+ T cells from different conditions (healthy, MS, and COVID-19) formed distinct clusters with high expression of effector genes (GZMB and PRF1) and KIR transcripts (Figures 20a and 20b), revealing the commonalities of KIR+ CD8+ T cells across physiological and disease states, as well as their uniqueness compared to other CD8+ T cells.

[0220] To better understand the similarities and heterogeneity of KIR+ CD8+ T cells under different conditions and explore the mechanisms of their suppressive activity against pathogenic CD4+ T cells, we performed single-cell RNA-seq using the Smart-seq2 protocol on 4,512 KIR+ CD8+ T cells sorted from the blood of age- and sex-matched healthy subjects (N = 10) and patients with MS (N = 2), SLE (N = 6), or CeD (N = 5). In parallel, we also analyzed their TCR α and β sequences. Unsupervised clustering of these KIR+ CD8+ T cells by Seurat identified six clusters: clusters 1–3 primarily contained expanded KIR+ CD8+ T cells (two or more cells expressing the same TCR), while clusters 5 and 6 consisted of unexpanded cells expressing unique TCRs (Figure 20c). Expanded KIR+ cells in clusters 1–3 had higher transcripts for genes related to cytotoxic molecules (e.g., GZMH, GZMB, and PRF1) and effector T cells (e.g., FCGR3A, FGFBP2, and CX3CR1). Cluster 2, more restricted to expanded KIR+ cells from MS patients, showed higher levels of type I IFN-responsive genes (including IFIT1, IFIT2, IFIT3, MX1, RSAD2, and ISG15). Cluster 3, specific to expanded KIR+ cells from a subset of HC and SLE patients, showed higher expression of genes involved in glycolysis (e.g., GAPDH, GPI, ENO1, and PGK1) (Figure 20d–e). Cells in cluster 4 were in a transitional state with loss of memory-related features. Clusters 5 and 6 (restricted to unexpanded KIR+CD8+ T cells) exhibited memory and naive indices, respectively (Fig. 20e), and accounted for a small proportion of total KIR+CD8+ T cells. T cell clones expressing the same TCR can be found in different clusters, indicating possible lineage relationships.Additionally, clonally expanded KIR+ CD8+ T cells in COVID-19 patients (identified from previous 10x Genomics scRNA-seq) showed higher expression of cytotoxic genes while downregulating naive or memory-related genes compared to non-expanded KIR+ CD8+ T cells. Thus, in parallel with clonal expansion, KIR+ CD8+ T cells lose their naive or memory attributes, enter an effector T cell differentiation program, and then suppress autoreactive CD4+ T cells through cytotoxicity. While there are common features shared by KIR+ CD8+ T cells from healthy subjects and different diseases, there is also heterogeneity associated with different diseases or treatments (i.e., upregulated type I IFN signaling and glycolysis in clusters 2 and 3).

[0221] Consideration Here, we characterize KIR+CD8+ T as a novel regulatory CD8+ T cell subset in humans, which may contribute to pathogenic CD4+ T cells arising from autoreactivity in autoimmune disorders or cross-reactivity to self-antigens in infectious diseases via cytotoxicity. + Similar to the perforin- or Fas / FasL-dependent suppression of autoreactive CD4 T cells by mouse Ly49 CD8 T cells, human KIR CD8 T cells targeted pathogenic CD4 T cells via their cytolytic activity. This is because expanded KIR CD8 T cells significantly upregulated cytotoxic molecules, leading to increased apoptosis of gliadin-specific CD4 T cells observed in the presence of KIR CD8 T cells. We frequently observed an increased frequency of KIR CD8 T cells in the blood and inflamed tissues of patients with autoimmune diseases, which in SLE positively correlates with potentially autoreactive CD4 T cells with a rare phenotype specific to gliadin-specific cells derived from CeD. This expansion of KIR CD8 T cells, which correlates with the incidence of these autoimmune responses, may function as a negative feedback mechanism, ameliorating pathogenesis by killing autoreactive T cells.

[0222] Increased KIR+CD8+ T cells, along with CD4+ T cells with the same phenotype as gliadin-specific cells in the CeD, which may represent autoreactive clones that cross-react with pathogens, have been found in COVID-19 patients and have been associated with autoimmune-related complications. Therefore, the primary role of KIR+CD8+ T cells may be to control autoreactive T cells that arise during the course of infectious disease due to their cross-reactivity to antigens expressed by specific pathogens. This allows the organism to maintain as complete a peripheral T cell repertoire as possible to protect itself from potential infection by pathogens, while still precisely controlling T cell clones that are cross-reactive to self-antigens.

[0223] Interestingly, clonally expanded KIR+CD8+ T cells are also found in the peripheral blood of healthy subjects, sharing a gene expression signature with those of patients with autoimmune diseases or COVID-19. This indicates that at least some of this type of T cell is continuously active, although not at the extremely high levels seen in COVID-19 patients or some autoimmune subjects. Activation of KIR+CD8+ T cells may be a specific control mechanism for maintaining peripheral tolerance, even in healthy individuals. The fact that CD4+ regulatory T cells are not elevated in COVID-19 patients indicates that their role in peripheral tolerance is quite different and perhaps more general, whereas KIR+CD8+ T cells may be specifically directed toward maintaining tolerance during infection.

[0224] In summary, we identify KIR+CD8+ T cells as a novel human CD8+ T cell subset, similar to Ly49+CD8+ T cells in mice, capable of killing pathogenic T cells while excluding other cells. They are active across a wide range of autoimmune diseases (e.g., MS, SLE, and CeD), at least in some infectious diseases (e.g., COVID-19), and to a lesser extent in healthy adults. This type of peripheral tolerance is likely complementary to, and distinct from, CD4+ regulatory T cells, which represent a distinct lineage of T cells and appear generally inactive in COVID-19 patients. Thus, the KIR+CD8+ T cells and characteristics described herein are useful for understanding key dynamics in both immune dysregulation phenomena and therapeutic applications. We also note an increase in a rare phenotype of CD4+ T cells in COVID-19 patients, characteristic of gliadin-specific cells that are pathogenic in celiac disease and elevated in other autoimmune diseases. Together, these data indicate that many of the sequelae in COVID-19 patients may be autoimmune in nature.

[0225] Human Samples: The cohort of patients with autoimmune disorders in this study met the classification criteria for systemic lupus erythematosus (SLE), celiac disease (CeD), or multiple sclerosis (MS), respectively. Blood collection from patients with SLE, CeD, or MS was covered under IRB-14734 (Stanford University Immunological and Rheumatic Disease Database: Disease Activity and Biomarker Study), IRB-20362 (Studying the Molecular Factors Involved in Celiac Disease Pathogenesis), and IRB-8629 (Understanding Mechanisms of Allergy and Immunology Study). Blood from healthy subjects was requested from the Stanford Blood Center or collected from healthy volunteers under IRB-40146. The above protocol was approved by the Stanford University Office of Research Compliance. PBMCs from MS patients were also obtained from the Multiple Sclerosis Center at the University of California, San Francisco (UCSF) in collaboration with the UCSF Committee on Human Research. All participants were randomly assigned to a veterinary practice (VVL) and a veterinarian (VVL) at UCSF. All participants provided informed consent.

[0226] PBMCs were isolated from blood by density gradient centrifugation (Ficoll-Packe, GE Healthcare). Regarding COVID-19 patients and sample collection, enrollment included adults with RT-PCR-positive COVID-19. Informed consent was obtained from each patient or, if the patient was unable to provide consent, from the patient's legally authorized representative. Participants were excluded if they were taking experimental medications (i.e., drugs not approved by regulatory authorities for use in COVID-19). COVID-19 disease severity was defined as described in the literature. Blood collection from COVID-19 patients was covered by IRB-14734 and NCT 04373148. Handling of COVID-19 PBMCs for flow cytometry analysis was covered by APB-3343-MD 0620. The above IRB and APB protocols were approved by Stanford University's Office of Research Compliance. Clinical metadata was obtained from the Stanford Clinical Data Electronic Medical Record System according to the permission of consenting participants, and disease definitions and diagnoses were used according to Harrison's Principles of Internal Medicine, 20e.

[0227] Flow cytometry analysis. CD8a (RPA-T8), CD56 (5.1H11), KIR3DL1 (Dx9), KIR2DL2 / L3 (Dx27), KIR2DL5 (UP-R1), TIGIT (A15153G), KLRG1 (SA231A2), CD244 (C1.7), CX3CR1 (2A9-1), CD28 (CD28.2), CD27 (O323), CCR7 (G043H7), T-bet (4B10), Helios (22F6), Granzyme B (QA16A02), Perforin (B-D48), PD-1 (EH12.2H7), CD25 ( Fluorescent dye-conjugated anti-human antibodies for M-A251, CD39 (A1), CD161 (HP-3G10), CD38 (HIT2), ICOS (C398.4A), CXCR3 (G025H7), CD45RA (HI100), CD4 (RPA-T4), and CD62L (DREG-56) (Biolegend); CD3 (UCHT-1), TCRβ (IP26), and CD127 (HIL-7R-M21) (BD); KIR2DL1 (clone #143211) and KIR3DL2 (clone #539304) (R&D) were used for staining. Frozen cell samples were thawed and washed with 10% FBS in RPMI with benzonase (Sigma-Aldrich, 1:10,000). After centrifugation at 50g, cells were treated with 1:20 diluted FcR block (BD) in FACS buffer (0.5% BSA, 2mM EDTA in PBS) for 10 minutes, followed by staining with antibodies against surface molecules (30 minutes, 4°C). For intracellular staining, cells were fixed and permeabilized with an intracellular fixation and permeabilization buffer set (eBioscience), followed by staining with antibodies against intracellular antigens (30 minutes, 4°C). Cells were acquired on an LSR II flow cytometer (BD), and data were analyzed using FlowJo X. Dead cells were identified using a viability stain (LIVE / DEAD™ Fixable Near-IR Dead Cell Imaging Kit). Stain, ThermoFisher).

[0228] Functional assay. Chymotrypsin singlet digest was deamidated with recombinant human transglutaminase 2 as described previously. On day 0, PBMCs were isolated from the blood of HLA-DQ2.5+CeD patients. CD8+ T cells were purified from PBMCs using CD8 microbeads (Miltenyi) according to the manufacturer's instructions, stained with flow antibodies, and viable CD3+CD56-CD8+KIR+ or KIR- T cells were sorted using a FACSAria Fusion flow cytometer (BD). Sorted KIR+ or KIR-CD8+ T cells were stimulated for 18 hours with anti-CD3 / CD28 beads (Gibco) supplemented with 50 U / mL IL-2 at a 1:1 ratio (1 μL of beads per 4 × 104 cells) in a 96-well plate. CD8-PBMCs were stimulated with 250 μg / mL deamidated gluten or left unstimulated at 3 x 105 to 1 x 106 / 100 μL per well supplemented with 50 U / mL IL-2. X-VIVO15 with gentamicin L-Gln (Lonza) supplemented with 10% human AB serum (Sigma-Aldrich) was used as the culture medium. After 18 hours, anti-CD3 / CD28 beads were removed from CD8+ T cells using a magnet, and KIR+ or KIR-CD8+ T cells were added to the CD8-PBMC culture at a 1:30 ratio. 50 U / mL IL-2 was added to the culture on day 3. Cells were harvested on day 6 and stained with 10 μg / mL HLA-DQ2.5 tetramer complexed with four disease-associated and immunodominant gliadin T cell epitopes (DQ2.5-glia-α1a, QLQPFPQPELPY; DQ2.5-glia-α2, PQPELPYPQPE; DQ2.5-glia-ω1, QQPFPQPEQPFP; DQ2.5-glia-ω2, FPQPEQPFPWQP) for 5 min at room temperature.

[0229] Magnetic bead enrichment of tetramer-positive CD4+ T cells was performed as previously described. Cells were washed with FACS buffer and then stained with antibodies against surface molecules for 30 minutes at 4°C. After two washes with FACS buffer, 10% of the cells were reserved for FACS analysis, while 90% were labeled with anti-PE microbeads and subjected to magnetic bead enrichment of PE-conjugated tetramer-positive cells using a single MACS column according to the manufacturer's protocol (Miltenyi). Cells were also collected on day 3 and Annexin V binding on gliadin-specific CD4+ T cells was measured (BD). All cells were acquired on an LSR II flow cytometer (BD), gated on viable CD3+CD4+CD8- TCRαβ+ cells, and analyzed using FlowJo X software. The frequency of tetramer-positive cells was calculated by dividing the number of tetramer+ CD4+ T cells after enrichment by the number of CD4+ T cells in the sample before enrichment multiplied by 9 (to account for the fact that 90% of the cells were used for enrichment).

[0230] Bulk RNA-seq gene expression quantification and data analysis. Bulk RNA sequencing was performed as previously described. Viable KIR+ or KIR-CD8+ T cells were bulk-sorted directly into 350 μL of TRIzol (Qiagen) using a FACSAria Fusion flow cytometer (BD). Total RNA was extracted from TRIzol samples using chloroform separation and isopropanol precipitation, followed by cleanup using the RNAeasy Plus Mini Kit (Qiagen). After analysis on the 2100 Bioanalyzer, sequencing libraries were purified using the RiboGone Mammalian rRNA Depletion Kit (Clontech) and SMARTer Str. The bulk RNA sequencing library was prepared using the Binding RNA-seq kit (Clontech). The resulting libraries were sequenced on the HiSeq 4000 platform (Illumina) at the Stanford Functional Genomics Facility. For each sample in the bulk RNA sequencing library, 75-base pair paired-end reads were obtained from the sequencer. We aligned the reads to the human reference genome (NCBI GRCh38) using STAR v2.7.0e50. Gene counts were quantified and normalized to Salmon51 (TPM). Differential gene expression analysis was determined via the DESeq function in the DESeq2R package.

[0231] Heatmaps were generated using seaborn.clustermap in Python. Gene ontology analysis plots were generated with the R package "clusterProfiler." To generate gene sets for gene set enrichment analysis (GSEA), we selected the top 200 genes upregulated in Ly49+CD8+ T cells compared with Ly49-CD8+ T cells in EAE mice, as well as previously reported CD4+ Treg signature genes identified in mice. These mouse genes were converted to their human homologs and organized into gene sets for subsequent GSEA analysis of human KIR+ versus KIR-CD8+ T cells.

[0232] Single-cell RNA-seq analysis of kidney and synovial tissue. Unique molecular identifier (UMI) count matrices for cells in kidney (accession code SDY997) or synovial tissue (accession code SDY998) generated by CEL-Seq2 were downloaded from the ImmPort repository, and downstream analysis was performed using the Seurat 3.0 package. Cells with fewer than 1,000 detected genes, more than 5,000 detected genes, or more than 25% mitochondrial genes were discarded. CD8+ T cells (expressing CD3D, CD3E, CD8A, and CD8B transcripts) and CD4+ T cells (expressing CD3D, CD3E, and CD4 transcripts) were selected for standard downstream procedures of log-normalization, variable gene selection, and data scaling.

[0233] Single-cell RNA-seq analysis of bronchoalveolar immune cells. Filtered expression matrices of single-cell RNA-seq of immune cells from bronchoalveolar lavage fluids of six severe and three moderate COVID-19 patients and three healthy controls, generated by 10xGenomics, were downloaded from Gene Expression Omnibus under accession number GSE145926. CD8+ T cells were identified for downstream analysis using the Seurat3.0 package.

[0234] Analysis of single-cell RNA-seq data generated by 10xGenomics. Single-cell RNA-seq data from blood-derived T cells from healthy subjects (N = 10), MS patients (N = 6), and COVID-19 patients (N = 25) using a microfluidic droplet platform (10xGenomics Chromium Single Cell 5' paired-end chemistry) were demultiplexed, aligned to the GRCh38 reference genome, and converted to a gene count matrix using CellRanger 3.1.0. Downstream analysis was performed using the Seurat 3.0 package. Cells with fewer than 800 detected genes, more than 3,000 detected genes, or more than 10% mitochondrial genes were discarded. CD8+ T cells (expressing CD8A and CD8B but not TRDC transcripts) from each individual were selected for further analysis. To ensure comparable counts across cells, gene counts were normalized to 10,000 reads per cell and then log-transformed. We identified highly variable genes for each individual and then used Seurat's integrated anchor detection algorithm to identify the integrated gene expression data from all individuals. We performed PCA dimensionality reduction on the integrated data, then clustered cells with the Louvain algorithm and used UMAP to refine the data. We used the Wilcoxon rank-sum test implemented in the "FindConservedMarkers" function of the Seurat package to identify marker genes of canonical cell types that were conserved across conditions.

[0235] Single-cell RNA-seq gene expression quantification and data analysis using Smart-seq2. Single-cell RNA-seq of blood KIR+ CD8+ T cells (viable CD3+ CD56- CD8+ TCRαβ+ KIR+ cells) was performed using the Smart-seq2 protocol with several modifications. Briefly, single cells were sorted into 96-well plates containing 5 μL lysis buffer in each well (0.8 U / μL RNase inhibitor (Clontech), approximately 5,000 molecules of ERCC (External RNA Control Consortium) spike-in RNA (Ambion), 0.08% BioUltra Triton X-100 (Sigma-Aldrich), 2 μM oligo-dT30VN (Integled DNA Technologies, 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′), and 2 mM Qiagen dNTP mix). Immediately after sorting, plates were sealed with aluminum seals (Axygen), centrifuged, flash-frozen on dry ice, and stored at -80°C. Prior to reverse transcription, plates were thawed on ice and thawed at 72°C for 3 minutes. Five microliters of a reaction mixture containing 10 mM DTT, 2 μM TSO (Exiqon, 5′-AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG-3′), 20 U / μL SMARTscriebe reverse transcriptase (Takara), 2 U / μL RNase inhibitor (Clontech), and 2× single-stranded buffer was added to each well. Reverse transcription was performed by incubating the wells at 42°C for 90 minutes, 50°C for 2 minutes, and 42°C for 2 minutes in a thermal cycler (Eppendorf). Reverse transcription was terminated by heating at 70°C for 15 minutes.Subsequently, 15 μL of PCR mixture containing 1.67× KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602) and 0.17 μM IS PCR primer (IDT, 5'-AAGCAGTGGTATCAACGCAGAGT-3') was added to each well, and second-strand synthesis was performed on a thermal cycler (Eppendorf) using the following program: 1) 98°C for 3 minutes, 2) 22 cycles of 98°C for 20 seconds, 67°C for 15 seconds, and 72°C for 6 minutes, and 3) 72°C for 5 minutes. One μL of the cDNA product was used for the TCR PCR reaction. The remaining 24 μL of the cDNA product was then transferred to a Biomek FX. P Purification was performed using AMPure XP beads (Beckman Coulter) on an automated workstation (Beckman Coulter). 15.6 μL of Ampure XP beads (0.65x) were added to each sample and mixed by pipetting up and down 30 times. The mixture was incubated at room temperature for 5 minutes to allow DNA to bind to the beads. The 96-well plate was then placed on a magnet for 5 minutes, and the liquid was removed while the sample was still on the magnet. The beads were washed twice with 180 μL of 80% (vol / vol) ethanol solution and air-dried on the magnet for 6 minutes. 25 μL of water was added to each well, mixed by pipetting up and down 10 times, and incubated at room temperature for 3 minutes. The plate was then placed on the magnet for 3 minutes, and the supernatant was transferred to a new 96-well plate. Finally, 2 μL of the supernatant was subjected to quality control using capillary electrophoresis on a fragment analyzer (Agilent Technologies) by the Stanford Protein and Nucleic Acid Facility.

[0236] The cDNA in the 96-well plate was transferred to a 384-well low volume serial dilution (LVSD) plate (TTP Labtech) and incubated with a Mosquito X1 liquid handler (TTP The DNA was diluted to 0.16 ng / μL using a 500 kJ / mL ELISA kit (Illumina, FC-131-1096). Illumina sequencing libraries were prepared using a Mosquito HTS liquid handler (TTP Labtech) as previously described. Briefly, the DNA was diluted to 0.16 ng / μL using a Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096). Tagging was performed with 4 μL of double-stranded cDNA. Each well was mixed with 0.8 μL of Nextera tagging DNA buffer (Illumina) and 0.4 μL of Amplicon Tagment mix (Illumina), then incubated at 55°C for 10 minutes. The reaction was stopped by adding 0.4 μL of neutralized tagging buffer (Illumina) and centrifuging at 3,000 g for 5 minutes at room temperature. Index PCR reactions were performed by adding 0.8 μL of premixed 5 μM i5 and i7 unique dual index primers (IDT, customized) and 1.2 μL of Nextera NPM mix (Illumina). PCR amplification was performed in a C1000 Touch™ thermal cycler with a 384-well reaction module (Bio-Rad) using the following program: 1) 72°C for 3 minutes, 2) 95°C for 30 seconds, 3) 12 cycles of 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 minute, and 4) 72°C for 5 minutes. After library preparation, wells from each library plate were pooled using a Mosquito HTS liquid handler (TTP Labtech). After pooling, the wells were purified twice using 0.65x and 1x AMPure XP beads (Beckman Coulter), respectively. Library quality was assessed using an Agilent 2100 bioanalyzer and normalized to 5 nM. Libraries were sequenced on a Hiseq4000 sequencing system (Illumina) at the Stanford Functional Genomics Facility, obtaining 150-bp paired-end reads.

[0237] The Stanford Functional Genomics Facility extracted and generated FASTQ files for each cell, distinguished by unique dual-index adapters. Reads were aligned to the GRCh38 genome using STAR v2.6.1d. Transcript abundance was quantified using HTSeq v0.5.4p5.

[0238] Standard procedures for filtering, log-normalization, variable gene selection, dimensionality reduction, and clustering were performed using the Seurat 3.0 package. Briefly, cells with fewer than 800 detected genes, more than 5,000 detected genes, or more than 15% mitochondrial genes were discarded. To make counts comparable across cells, the number of genes was normalized to 10,000 reads per cell and then log-transformed. After PCA dimensionality reduction, cells were clustered by running the Louvain algorithm and visualized using UMAP. Differential expression analysis was performed using the Wilcoxon rank-sum test implemented in the "FindAllMarkers" function of the Seurat package. Significantly differentially expressed genes were defined as those with a log fold change >0.5 and a Bonferroni-corrected p-value <0.05.

[0239] Single-cell TCR-seq. TCR-seq was performed using a single-cell paired TCR sequencing method previously developed by the inventors, with minor modifications. Briefly, for the first TCR reaction, 1 μL of the Smart-seq2 cDNA product was pre-amplified with HotStarTaq DNA polymerase (Qiagen) using multiplex PCR with multiple Vα and Vβ region primers and Cα and Cβ region primers. A 25-cycle first PCR reaction was performed according to the manufacturer's instructions, using the following cycling conditions: 95°C for 15 minutes; 94°C for 30 seconds, 62°C for 1 minute, 72°C for 1 minute (25 cycles); 72°C for 10 minutes; 4°C. A 1 μL aliquot of the first reaction was then used as template for a second 12 μL PCR using HotStarTaq DNA polymerase (Qiagen) with multiple internally nested TCR Vα, TCR Vβ, TCR Cα, and Cβ primers. The cycling conditions were 95°C for 15 minutes; 25 cycles of 94°C for 30 seconds, 64°C for 1 minute, and 72°C for 1 minute; 72°C for 7 minutes; and 4°C. One microliter of the second PCR product was loaded onto an Illumina MiSeq platform incorporating the barcode. The resulting PCR product was used as a template for a third 20 μL PCR reaction, allowing sequencing on the platform. For the third and final PCR reaction for TCR sequencing, amplification was performed with HotStarTaq DNA polymerase for 36 cycles using a 5' barcoded primer (0.05 μM) containing the common 23-base sequence, a third internal nested Cα and / or Cβ primer, and a 3' barcoded primer (0.05 μM) containing the sequence of the Illumina paired-end primer. The cycling conditions were 95°C for 15 min; 36 cycles of 94°C for 30 s, 66°C for 30 s, and 72°C for 1 min; 72°C for 10 min; and 4°C. The PCR products were combined in equal volume proportions and run on a 1.2% agarose gel. A band around 350–380 bp was excised and gel-purified using a Qiaqick gel extraction kit (Qiagen). This purified product was then sequenced on the Miseq platform (Illumina) and 2 × 250 bp reads were obtained.

[0240] In vitro cell proliferation assay: CD8+ T cells were purified from PBMCs of healthy donors using CD8 microbeads (Miltenyi) according to the manufacturer's instructions, stained with flow antibodies, and viable CD3+CD56-CD8+KIR+ or KIR- T cells were sorted using a FACSAria fusion flow cytometer (BD). Sorted KIR+ or KIR-CD8+ T cells were stimulated with anti-CD3 / CD28 beads (Gibco) supplemented with 50 U / mL IL-2 at a 1:1 ratio (1 μL of beads per 4 × 104 cells) in a 96-well plate for 18 hours. CD8- PBMCs were labeled with CellTrace Violet (CTV, ThermoFisher) according to the manufacturer's instructions. 1 μg / mL of anti-CD3 (UCHT-1) was coated overnight at 4°C in 50 μL of PBS per well of a 96-well plate. After removal of anti-CD3 / CD28 microbeads, KIR+ and KIR-CD8+ T cells were mixed with CTV-labeled CD8-PBMCs at a 1:30 ratio and cultured in 96-well plates pre-coated with 1 μg / mL anti-CD3. After 4 or 6 days, cells were harvested, and the dilution of CTV in CD4+ T cells was analyzed by flow cytometry.

[0241] Statistical Analysis. No specific statistical method was used to predetermine sample size. All results are presented as mean ± SEM. The significance of differences between groups was analyzed as indicated in the figure legends. Correlation coefficients with two-tailed Pearson P values ​​were determined in correlation analyses. A P value of <0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism software version.

[0242] Each publication cited herein is incorporated by reference in its entirety for all purposes.

[0243] It is understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0244] It should be noted that as used in this specification and the appended claims, the articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, a reference to "the culture" includes one or more cultures and equivalents thereof known to those skilled in the art, and so forth. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0245] cross reference This application claims the benefit of U.S. Patent Application No. 62 / 882,810, filed August 5, 2019, which is incorporated herein by reference in its entirety.

Claims

1. CD4 responding to the initiating antigen ＋ selectively suppress the activity of T cells that are not themselves activated by the initiating antigen, CD8 ＋ K.I.R. ＋ An isolated population of human regulatory T cells.

2. 2. The population of regulatory T cells of claim 1, wherein the cells express one or more of KIR2DL2, KIR2DL3, and KIR3DL1.

3. 3. The population of regulatory T cells of claim 1 or 2, formulated in a therapeutically effective unit dose.

4. The population of regulatory T cells according to any one of claims 1 to 3, wherein CD8+KIR+ human regulatory T cells are expanded in vivo in response to immunization with said initiating antigen.

5. 5. The population of regulatory T cells of claim 4, wherein the T cells are enriched from a sample obtained from an individual after immunization.

6. The population of regulatory T cells of claim 5, wherein the sample is peripheral blood, CSF, synovial fluid, or a biopsy sample.

7. 6. The population of regulatory T cells of claim 5, wherein the sample is obtained from 10 to 20 days after immunization.

8. 6. The population of regulatory T cells of claim 5, wherein the sample is obtained from 14 to 16 days after immunization.

9. The population of regulatory T cells of claim 3 , wherein the starting antigen is used in an immunization step.

10. The population of regulatory T cells according to any one of claims 1 to 9, wherein the initiating antigen is an autoantigen.

11. The population of regulatory T cells according to any one of claims 1 to 10, wherein the regulatory T cells are enriched from a sample obtained from an individual after the onset of an autoimmune disease.

12. 12. The population of regulatory T cells of claim 11, wherein the sample is peripheral blood, CSF, synovial fluid, or a biopsy sample.

13. The population of regulatory T cells of claim 11, wherein the sample is obtained from 10 to 20 days after the onset of immunity.

14. The population of regulatory T cells according to any one of claims 1 to 10, wherein the regulatory T cells are enriched from a sample obtained from an individual after infection.

15. The population of regulatory T cells of claim 14, wherein the infection is SARS-CoV-2 infection.

16. The population of regulatory T cells according to claim 8 , wherein an antigen other than a self-antigen is used in the immunization step.

17. 10. The method of claim 1, wherein the regulatory T cells are engineered to express a TCR other than the native TCR.

7. A population of regulatory T cells according to claim 6.

18. 18. The population of regulatory T cells of any one of claims 1 to 17, wherein the isolated population is enriched by affinity selection against CD8 and an inhibitory KIR protein.

19. 19. The population of regulatory T cells of claim 18, wherein the inhibitory KIR protein is one or more of KIR2DL2, KIR2DL3, and KIR3DL1.

20. The population of regulatory T cells according to any one of claims 4 to 19, wherein the cell population is expanded in vitro.

21. 2. The population of regulatory T cells of claim 1, wherein the isolated population is generated by in vitro contact with an immunogen.

22. 22. The population of regulatory T cells of claim 21, wherein the immunogen is a regulatory peptide.

23. 23. The population of regulatory T cells of any one of claims 1 to 22, wherein the cells are restricted to MHC class I antigens.

24. 24. The population of regulatory T cells of any one of claims 1 to 23, wherein the cells are restricted to an MHC class I antigen associated with an autoimmune disease.

25. 20. The population of regulatory T cells of any one of claims 1 to 19, wherein the cells are restricted to an MHC class I antigen other than an MHC associated with an autoimmune disease.

26. The population of regulatory T cells of claim 1 , wherein the T cells are expanded in vivo after immunization with a regulatory peptide.

27. CD4 responding to target antigen ＋ CD8 that selectively suppresses the activity of T cells that are not themselves activated by the target antigen ＋ KIR (*) ＋ 1. A method for expanding a population of human suppressor T cells in vivo in an individual, comprising: immunizing said individual with a regulatory peptide.

28. A method for obtaining a population of regulatory T cells, the method comprising enriching from an individual for a cell population according to any one of claims 4 to 27.

29. 27. A method for screening a population of regulatory T cells according to any one of claims 4 to 26 for antigen specificity, comprising: expressing the T cell receptor (TCR) of said regulatory T cells as a soluble multimer; contacting the soluble multimers in a binding assay against a library of diverse peptides in an MHC context and selecting MHC peptides that bind to the T cell receptor; performing multiple rounds of selection; identifying the antigen specificity of the regulatory T cells by sequencing the selected peptide antigen; A method comprising:

30. 27. A method of reducing CD4+ T cell responses to a target antigen in an individual, comprising administering an effective dose of a suppressor T cell population according to any one of claims 1 to 26.

31. 31. The method of claim 30, wherein the target antigen is an autoantigen.

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