PD-1+CD38HICD8+ T cells and uses thereof

Abstract

As used herein, PD-1 + CD38 hi CD8 + Methods are provided for targeting T cells to reduce apoptosis of leukocytes (e.g., lymphocytes). + CD38 hi CD8 + Methods for inducing apoptosis of leukocytes in a subject using adoptive cell therapy with T cells are provided. + CD38 hi CD8 + T cells are dysfunctional and have the ability to kill target cells that express the CD31 receptor, including CD4 and CD8 T cells and endothelial cells.

Description

[Technical Field]

[0001] Prior Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 380,989, filed October 26, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] PD-1 + CD38 hi CD8 + T cells are strongly associated with immunotherapy resistance in cancer. Depletion of these cells results in the proliferation of total and tumor antigen-specific CD8 T cells in the tumor microenvironment (TME). + Increasing the number of T cells, their activation, and effector function reverses resistance to immunotherapy, although the role of these cells in other pathologies remains unclear. Summary of the Invention [Means for solving the problem]

[0003] PD-1 + CD38 hi CD8 + T cells are dysfunctional and have the ability to kill target cells that express the CD31 receptor. These target cells include CD4 and CD8 T cells and endothelial cells. The killing mechanism is via CD38 binding to CD31 on the target cell. + PD1 + CD8 + This is due to the degranulation of high levels of granzyme B (GzmB) from T cells, which induces apoptosis in target cells. Therefore, these cells are called fratricidal CD8 T cells (T fratThese cells, which normally represent less than 12% of all CD8 T cells, are highly expressed in patients with advanced COVID-19 (e.g., up to 30-60%). They also kill autologous CD4 T cells and CD8 T cells in patients with advanced COVID-19 who are known to be severely lymphopenic. Furthermore, PD1 + CD38 hi CD8 + T cells are induced as a result of cAMP response element binding protein (CREB-1)-dependent bradykinin (BDK)-mediated immune signaling, leading to ARDS lesions in the lung. + CD38 hi CD8 + T cells are induced in autoimmune disease models and infusion of these cells reverses symptoms.

[0004] Provided herein are methods for blocking the binding of CD38 to CD31 on leukocytes and endothelial cells. + CD38 hi CD8 + Also provided herein are methods for preventing degranulation of Gzm B in T cells. Further provided herein are methods for preventing degranulation of PD-1 in adoptive cell therapy for the treatment of autoimmune diseases. + CD38 hi CD8 + Methods of using the T cells are provided.

[0005] For example, provided herein are methods for reducing apoptosis of leukocytes in a subject, comprising reducing binding of CD38-expressing cells to CD31-expressing leukocytes in the subject. In some methods, an agent that reduces binding of CD38-expressing cells to CD31-expressing leukocytes is administered to the subject. In some methods, for example, an agent that reduces binding of PD-1 in the subject is administered to the subject. + CD38 hi CD8 + PD-1 by depleting T cells + CD38 hi CD8 +The reduction in T cells may result in increased PD-1 expression on CD31-expressing leukocytes or endothelial cells in the subject. + CD38 hi CD8 + T cell binding was reduced.

[0006] In some methods, the CD31-expressing leukocytes are selected from the group consisting of T lymphocytes, dendritic cells, natural killer cells, and macrophages. In some methods, the CD31-expressing leukocytes are T lymphocytes. In some methods, the T lymphocytes are CD4+ T cells or CD8+ T cells.

[0007] In some methods, the CD38-expressing cells are CD8+ T cells. In some methods, the CD8+ T cells are PD-1+CD38hiCD8+ T cells. In some methods, the agent is an antibody that specifically binds to CD31. In some methods, the agent is an antibody that specifically binds to CD38. In some methods, the agent reduces the level of PD-1+CD38hiCD8+ T cells in the subject. In some methods, degranulation of GzmB in PD-1+CD38hiCD8+ T cells is reduced in the subject. In some methods, transfer of GzmB from PD-1+CD38hiCD8+ T cells to target cells (e.g., leukocytes, or endothelial cells) in the subject is reduced. In some methods, the subject has acute respiratory distress syndrome (ARDS) or an ARDS-associated infection (e.g., COVID-19).

[0008] Also provided are methods for treating an autoimmune disease in a subject, comprising administering to the subject a population of PD-1+CD38hiCD8+ T cells. Also provided are methods for treating an infectious disease in a subject, comprising administering to the subject a population of PD-1+CD38hiCD8+ T cells. Some methods further include generating the population of PD-1+CD38hiCD8+ T cells by contacting CD8+ T cells with a suboptimal antigen and / or a PD-1 inhibitor prior to administering the population of PD-1+CD38hiCD8+ T cells to the subject.

[0009] In some methods, CD8+ T cells are contacted with an agent that inhibits the interaction of PD-1 with its ligand(s) to generate a population of PD-1+CD38hiCD8+ T cells. In some methods, the population of PD-1+CD38hiCD8+ T cells is expanded prior to administration to the subject.

[0010] Any of the methods provided herein can further include administering a second therapeutic agent to the subject. In some methods, the second therapeutic agent is an immunomodulatory agent. In some methods, the immunomodulatory agent is an immunosuppressant or an immunostimulatory agent.

[0011] The details of one or more embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the description and the claims.

[0012] This application includes the following drawings. The drawings are intended to illustrate certain embodiments and / or features of the compositions and methods and to supplement any description(s) of the compositions and methods. The drawings do not limit the scope of the compositions and methods unless the written description expressly indicates otherwise. [Brief explanation of the drawings]

[0013] [Figure 1A] Differential regulation of PD1- and CD38-expressing CD8 T cell subtypes is shown. The percentages and mean fluorescence intensity (MFI) of IFN-γ+, CD40L+, and CD69+PD1+CD38hi effector T cells (Teff) and CD38 KD PD1+CD38hi CD8 T cells were determined by flow cytometry. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by one-way ANOVA (*P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001). Scrambled RNA (scRNA) was used as a control for CD38 siRNA. [Figure 1B]Differential regulation of CD8 T cell subtypes expressing PD1 and CD38 is shown. Recall responses in PD1+CD38hi cells re-challenged with Ova-V with and without CD38 KD (b) measured by the percentage of IFN-γ+, CD40L+, and CD69+ cells (c). For comparison, recall responses in Teff cells re-challenged with Ova are shown as the percentage of IFN-γ+ cells (c). [Figure 1C] Differential regulation of CD8 T cell subtypes expressing PD1 and CD38 is shown. Recall responses in PD1+CD38hi cells re-challenged with Ova-V with and without CD38 KD (b) measured by the percentage of IFN-γ+, CD40L+, and CD69+ cells (c). For comparison, recall responses in Teff cells re-challenged with Ova are shown as the percentage of IFN-γ+ cells (c). [Figure 1D] Differential regulation of PD1- and CD38-expressing CD8 T cell subtypes. Levels of pro- and anti-inflammatory cytokines and cytolytic molecules in in vitro generated PD1+CD38hi and TeffCD8 T cells were determined by flow cytometry. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). [Figure 1E] Differential regulation of PD1- and CD38-expressing CD8 T cell subtypes is shown. Levels of pro- and anti-inflammatory cytokines and cytolytic molecules were determined by flow cytometry in PD1+CD38hi and TeffCD8 T cells isolated in vivo from untreated TC-1 tumors in mice. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001). [Figure 1F]Differential regulation of PD1- and CD38-expressing CD8 T cell subtypes. RNA and ATAC-seq analysis of PD-1+CD38hi and Teff cells generated from OT1 CD8+ T cells. Principal component analysis (PCA) of RNA-seq analysis of PD-1+CD38hi and Teff CD8 T cells treated with IL-2. [Figure 1G] Differential regulation of PD1- and CD38-expressing CD8 T cell subtypes is shown. RNA and ATAC-seq analysis of PD-1+CD38hi and Teff cells generated from OT1 CD8+ T cells. Venn diagram showing pairwise comparison of up- and down-regulated gene analysis in IL-2-treated PD-1+CD38hi and TeffCD8 T cells (left), and GO pathway enrichment analysis (right) using differentially expressed up- and down-regulated genes in PD-1+CD38hi and TeffCD8 T cells (RNA-seq analysis). [Figure 1H] Differential regulation of PD1- and CD38-expressing CD8 T cell subtypes. Top 50 differentially expressed genes (DEGs) associated with the principal component 2 (PC2) axis in IL-2-treated PD-1+CD38hi and TeffCD8 T cells by RNA-seq analysis. [Figure 1I] Figure 1 shows differential regulation of PD1- and CD38-expressing CD8 T cell subtypes. Heatmap analysis of inhibitory and proinflammatory cytokine genes in PD-1+CD38hi and TeffCD8 T cells as determined by RNA-seq analysis. [Figure 1J]Differential regulation of CD8 T cell subtypes expressing PD1 and CD38 is shown. PD-1+CD38hi T cells have less promoter accessibility: ATAC-seq Tn5 nick site density in Teff and PD1+CD38hi T cell promoters. Log ratio ("minus") vs. mean expression ("MA") plot showing relative accessibility of macs2 peaks. Red indicates differential peak accessibility at FDR<0.05 (top). Heatmap showing row-scaled promoter accessibility for differentially regulated promoter-restricted peaks at FDR<0.05, |log2 FC|>1.2 (bottom). Pathway terms with FDR<0.05 and genes associated with these terms are shown on the right. [Figure 2A] Figure 1 shows metabolic signatures of PD1+CD38hiCD8 T cells. PCA plot, volcano plot, and heatmap for different metabolites in PD-1+CD38hi and TeffCD8 T cells. [Figure 2B] Metabolic signature of PD1+CD38hiCD8 T cells. PCA plot, volcano plot, and heatmap for different lipids in PD-1+CD38hi and TeffCD8 T cells. [Figure 2C] Metabolic characteristics of PD1+CD38hi CD8 T cells. The number of CD8 T cells positive for NBDG (glucose uptake rate) and BODIPY (FA uptake) and their respective expression levels (MFI) in PD-1+CD38hi cells compared with TeffCD8 T cells. Representative results from one of two experiments performed in triplicate are shown. Error bars indicate s.e.m. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). [Figure 2D]Metabolic characteristics of PD1+CD38hi CD8+ T cells. Numbers of CD8+ T cells positive for TMRMlo and TMRMhi (mitochondrial potential) and MitoFM (mitochondrial mass), as well as their respective expression levels (MFI), were shown in PD-1+CD38hi cells compared with TeffCD8+ T cells. Representative results from one of two experiments performed in triplicate are shown. Error bars indicate s.e.m. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). [Figure 2E] Figure 1 shows metabolic characteristics of PD1+CD38hiCD8 T cells. Larger mitochondria with disorganized cristae were found, as indicated by low cristae density, number, and score. [Figure 2F] Figure 1 shows metabolic profiles of PD1+CD38hiCD8 T cells. Figure 2 shows metabolic profiles of PD1+CD38hiCD8 T cells. DCFDA expression showing ROS levels in PD-1+CD38hi cells compared to Teff cells. [Figure 2G] Figure 1 shows metabolic profiles of PD1+CD38hiCD8 T cells. Figure 1 shows metabolic profiles of PD1+CD38hiCD8 T cells. Metabolic profiles of PD-1+CD38hi and TeffCD8+ T cells showing oxygen consumption rate (OCR, basal and maximal), spare respiratory capacity (SRC), and extracellular acidification rate (ECAR). Representative results from one of two experiments performed in triplicate are shown. Error bars indicate s.e.m. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P≦0.05, ****P≦0.0001). [Figure 3A]Figure 1 shows that PD-1+CD38hi CD8 T cells kill T cells in a contact-dependent manner mediated by CD38:CD31 interactions. Figure 2 shows the apoptosis assay scheme for checking the ability of PD-1+CD38hi cells to induce apoptosis and loss of viability in target pMel-1 CD8 T cells in a contact-dependent or contact-independent manner using a transwell chamber assay. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by one-way ANOVA (****P≦0.0001, ns, not significant). [Figure 3B] Figure 1 shows that PD-1+CD38hi CD8 T cells kill T cells in a contact-dependent manner mediated by CD38:CD31 interactions. Figure 1 shows the apoptosis assay scheme for checking the ability of PD-1+CD38hi cells to induce apoptosis and loss of viability of target pMel-1 CD8 T cells in a contact-dependent or -independent manner using FACS micrographs and statistical analysis of the frequency of Annexin V-positive target cells when incubated in contact with or isolated from PD-1+CD38hi cells (M: membrane). Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by one-way ANOVA (****P ≤ 0.0001, ns, not significant). [Figure 3C]Figure 1 shows that PD-1+CD38hi CD8 T cells kill T cells in a contact-dependent manner mediated by CD38:CD31 interactions. Figure 1 shows a schematic of an apoptosis assay to check the ability of PD-1+CD38hi cells to induce apoptosis and loss of viability of target pMel-1 CD8 T cells in a contact-dependent or -independent manner using FACS micrographs and statistical analysis of the frequency of viable Annexin V-negative target cells when incubated in contact with or isolated from PD-1+CD38hi cells (M: membrane). Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by one-way ANOVA (****P ≤ 0.0001, ns, not significant). [Figure 3D] We show that PD-1+CD38hiCD8 T cells kill T cells in a contact-dependent manner mediated by CD38:CD31 interactions. Target CD8 T cells from the spleens of pMel-1 mice and target CD8 and CD4 T cells from the spleens of C57BL / 6 mice were incubated with PD1+CD38hiCD8 T cells, and the number of live target cells was assessed by viability assay (live / dead staining). [Figure 3E] Figure 1 shows that PD-1+CD38hiCD8 T cells kill T cells in a contact-dependent manner mediated by CD38:CD31 interactions. Schematic of adoptive transfer experiment and gating strategy. [Figure 3F] Figure 1 shows that PD-1+CD38hi CD8 T cells kill T cells in a contact-dependent manner mediated by CD38:CD31 interactions. Apoptosis levels (Annexin V MFI) and viable cell counts were measured in adoptively transferred target CD8 T cells isolated from the spleens of Rag1- / - mice seeded with PD-1+CD38hi cells 1 day after target cell transfer. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P ≤ 0.05, **P ≤ 0.01). [Figure 3G]Figure 1 shows that PD-1+CD38hi CD8 T cells kill T cells in a contact-dependent manner mediated by CD38:CD31 interactions. Apoptosis levels (Annexin V MFI) and viable cell counts in adoptively transferred target CD4 T cells isolated from the spleens of Rag1- / - mice seeded with PD-1+CD38hi cells 1 day after target cell transfer. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P ≤ 0.05, **P ≤ 0.01). [Figure 3H] Figure 1 shows that PD-1+CD38hiCD8 T cells kill T cells in a contact-dependent manner mediated by CD38:CD31 interactions. Schematic diagram of the isolation of PD-1+CD38hi and CD8+CD38lo cells to check the involvement of CD38:CD31 interactions in inducing apoptosis in target pMel-1CD8 T cells. In separate groups, either CD38 or CD31 was knocked down using specific siRNA, and anti-CD38 or anti-CD31 was also used to block the interaction between CD38 and CD31. [Figure 3I] Figure 1 shows that PD-1+CD38hiCD8 T cells kill T cells in a contact-dependent manner mediated by CD38:CD31 interactions. Annexin V levels or counts on target CD8 cells incubated with Tfrat killer cells in the presence or absence of anti-CD38 or CD38KD. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by one-way ANOVA (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; ns, non-significant). [Figure 3J]Figure 1 shows that PD-1+CD38hiCD8+ T cells kill T cells in a contact-dependent manner mediated by CD38:CD31 interactions. Annexin V levels or counts on target CD8+ T cells in the presence or absence of anti-CD31 or CD31KD CD8+ target cells. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by one-way ANOVA (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; ns, non-significant). [Figure 4-1] This shows that Tfrat cells kill target cells by transferring granzyme B (Gzm B), and that CD38:CD31 interactions induce degranulation of Gzm B in killer cells via the Zap70-PI3K-RAC-ERK pathway. (a) Annexin V expression in mouse target CD8 T cells after incubation with killer Tfrat cells in the presence or absence of a Gzm B inhibitor. (b) Schematic diagram for generating killer PD-1+CD38hi cells and confirming their ability to transfer Gzm B into target cells. (c-d) Expression and frequency of Gzm B+ in target CD8 T cells (c) and target CD4 T cells (d) after co-incubation with killer (Tfrat) cells. (e–f) Degranulation in Tfrat cells was estimated by CD107a expression (e) and MFI of Gzm B in target CD8 cells (f) after incubation of these two populations with or without anti-CD31 or anti-CD38. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by one-way ANOVA (**P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). [Figure 4-2]This shows that Tfrat cells kill by transferring granzyme B (Gzm B) to target cells, and CD38:CD31 interactions induce Gzm B degranulation in killer cells via the Zap70-PI3K-RAC-ERK pathway. (g) Schematic diagram of the determination of Gzm B transfer from killer Tfrat cells to target cells by immunofluorescence analysis. (h) Target (no nuclear staining) and Tfrat (nuclear staining) CD8 T cells stained for Gzm B. (i-j) Estimation of Gzm B transfer from killer Tfrat (blue) to target (unlabeled in "bright field") CD8 T cells by immunofluorescence analysis at various time points (5 min to 300 min). "Merge" shows Gzm B on a dark field for better visualization (i), and (j) shows a higher magnification image over 180 min. [Figure 4-3] This figure shows that Tfrat cells kill target cells by transferring granzyme B (Gzm B), and that CD38:CD31 interaction induces Gzm B degranulation in killer cells via the Zap70-PI3K-RAC-ERK pathway. (k) Western blot analysis of various signaling molecules involved in granule migration in PD-1+CD38hi cells treated with recombinant (r)CD31. β-actin is shown as an internal control. On the right, densitometric analysis of various predicted signaling molecules is shown. (l) Flow cytometry of degranulation in killer Tfrat cells after incubation with rCD31 and inhibition of PI3K and ERK. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by one-way ANOVA (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). (m) Overview of the signaling pathway mediated by CD38 and CD31 involved in Gzm B production and granule trafficking in cells. [Figure 5A]Figure 1 shows that CD38 depletes NAD, leading to increased SIRT1-FOXO1-TCF7-mediated Gzm B production in killer Tfrat cells. Frequency and expression of Gzm B in Tfrat with and without CD38KD compared to Teff cells as determined by flow cytometry. [Figure 5B] Figure 1 shows that CD38 depletes NAD, leading to SIRT1-FOXO1-TCF7-mediated increased Gzm B production in killer Tfrat cells. NAD levels in Tfrat and Teff cells. [Figure 5C] CD38 depletes NAD, leading to increased SIRT1-FOXO1-TCF7-mediated Gzm B production in killer Tfrat cells. SIRT1 activity by fluorescence analysis (left) and expression by Western blot analysis (right, tubulin expression is shown as a control) in Tfrat and Teff cells. [Figure 5D] Figure 1 shows that CD38 depletes NAD, leading to increased SIRT1-FOXO1-TCF7-mediated Gzm B production in killer Tfrat cells. Expression of FOXO1 and TCF7 at the protein level (by Western blot analysis; lamin B is shown as a control) in Tfrat and Teff cells. Numbers on the blots indicate relative expression after densitometric analysis. Representative data from one of two experiments are shown. Error bars indicate sem. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). [Figure 5E]Figure 1 shows that CD38 depletes NAD, leading to increased SIRT1-FOXO1-TCF7-mediated Gzm B production in killer Tfrat cells. Expression of FOXO1 and TCF7 at the RNA level (by qRT-PCR analysis) in Tfrat and Teff cells. Numbers on the blots indicate relative expression after densitometric analysis. Representative data from one of two experiments are shown. Error bars indicate sem. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). [Figure 5F] Figure 1 shows that CD38 depletes NAD, leading to increased SIRT1-FOXO1-TCF7-mediated Gzm B production in killer Tfrat cells. Gzm B levels after NAD repletion in Tfrat compared with TeffCD8+ T cells. Representative data from one of two experiments are shown. Error bars indicate sem. Statistical analysis was performed by one-way ANOVA. (*P≦0.05, **P≦0.01, ***P≦0.001). [Figure 5G] Figure 1 shows that CD38 depletes NAD, leading to increased SIRT1-FOXO1-TCF7-mediated Gzm B production in killer Tfrat cells. Gzm B levels after addition of SIRT1 activators in Tfrat compared with Teff CD8+ T cells. Representative data from one of two experiments are shown. Error bars indicate s.e.m. Statistical analysis was performed by one-way ANOVA. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). [Figure 6-1]Figure 1 shows that PD-1+CD38hiCD8+ cells suppress autoimmune disease. (a) Number of PD1+CD38hiCD8+ T cells in the spleens of experimental autoimmune encephalomyelitis (EAE) mice compared to WT mice. (b) Frequency and levels of Gzm B+PD1+CD38hi and TeffCD8+ T cells in the spleens of EAE mice. (c) In vitro killing of pMel-1 target cells by PD1+CD38hiCD8+ T cells obtained from EAE mice with or without anti-CD38 compared to killing induced by Teff cells. [Figure 6-2] PD-1+CD38hiCD8+ cells suppress autoimmune disease. (d-f) Number of PD1+CD38hiCD8+ T cells in two cohorts of SLE patients (d-e) and MS patients (f) compared to healthy individuals. Frequency of Gzm B+PD1+CD38hi and TeffCD8+ T cells in SLE patients (g) and MS patients (h). [Figure 6-3] Figure 1 shows that PD-1+CD38hiCD8+ cells suppress autoimmune disease. (i) The ability of Tfrat cells (both isolated from SLE patients) compared with Teff cells to kill autologous target CD4 T cells in the presence or absence of anti-CD38, as determined by flow cytometric Annexin V staining and viability assays. Each symbol represents an independent patient. Error bars indicate sem. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P≦0.05, **P≦0.01, ns, non-significant). (j) Schematic diagram of EAE induction and infusion of PD1+CD38hiCD8+ T cells, as well as clinical score grading. (k) Clinical score of EAE and mouse survival. [Figure 6-4]PD-1+CD38hiCD8 cells suppress autoimmune disease. (l-n) Flow cytometric analysis of immune responses in various tissues (spleen, brain, and lymph nodes (LN)) from untreated EAE mice or EAE mice injected with Tfrat cells. Error bars indicate sem. Statistical analysis was performed by unpaired, one-tailed Student's t-test. The log-rank (Mantel-Cox) test was used to compare survival rates between the two groups (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). [Figure 7A] PD-1+CD38hiCD8+ T cells from patients with advanced COVID-19 kill T cells. Frequency of Tfrat cells in the blood of healthy individuals (controls), convalescent, and severe COVID-19 patients. [Figure 7B] Figure 1 shows that PD-1+CD38hiCD8+ T cells from patients with advanced COVID-19 kill T cells. Frequency of Tfrat cells in bronchoalveolar lavage fluid (BALF) from COVID-19 patients with severe / critical (S / C) disease compared with that of moderate (O) disease and healthy controls (HC). (Analysis was performed using publicly available single-cell RNA-seq data.) Each symbol corresponds to one individual. Error bars indicate s.e.m. Statistical analysis was performed by one-way ANOVA (*P ≤ 0.05, **P ≤ 0.01). [Figure 7C] We show that PD-1+CD38hiCD8+ T cells from patients with advanced COVID-19 kill T cells. [Figure 7D] Figure 1 shows that PD-1+CD38hiCD8+ T cells from patients with advanced COVID-19 kill T cells. MFI of Gzm B in Tfrat and Teff cells from peripheral blood of patients with severe COVID-19 estimated by flow cytometry. [Figure 7E]PD-1+CD38hiCD8+ T cells derived from patients with advanced COVID-19 demonstrate T cell killing. The ability of Tfrat to kill autologous CD4 T cells compared with Teff isolated from patients with severe COVID-19, as determined by Annexin V staining by flow cytometry. Each line represents an independent patient. Error bars indicate sem. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P ≤ 0.05, **P ≤ 0.01, ns, non-significant). [Figure 8A] In vitro gating strategy is shown. [Figure 8B] Figure 1 shows the percentage of IFN-γ, CD40L, and CD69 IL-2-treated PD1+ CD38hi and TeffCD8 T cells as determined by flow cytometry. Representative data from one of two experiments are shown. Error bars indicate sem. Statistical analysis was performed by one-way ANOVA (***P≦0.001, ****P≦0.0001). [Figure 8C] In vivo gating strategy is shown. [Figure 8D] Volcano plot showing DEGs between IL-treated PD1+CD38hi cells and TeffCD8 T cells by RNA-seq analysis. [Figure 8E] Heatmap showing the top 50 DEGs overall from RNA-seq analysis. IL-treated, PD1+CD38hi and TeffCD8 T cells. [Figure 8F] Figure 1 shows exhaustion- and effector-like DEGs in IL-2-treated PD-1+CD38hi and TeffCD8 T cells by RNA-seq analysis. [Figure 8G] Volcano plot shows differentially open promoters with |log2FC|>1.2 by ATAC-seq analysis. [Figure 9A] Figure 1 shows the results of apoptosis experiments: Induction of apoptosis in target CD8 T cells after incubation with PD1+CD38hi or TeffCD8+ T cells, as determined by caspase 3 / 7 staining. [Figure 9B] Figure 1 shows the results of apoptosis experiments: induction of apoptosis in target CD8 T cells after incubation with PD1+CD38hi or TeffCD8+ T cells by Annexin V staining in the presence or absence of anti-CD38. [Figure 9C] Figure 1 shows the results of apoptosis experiments: induction of apoptosis in target CD8 T cells after incubation with PD1+CD38hi or TeffCD8+ T cells by Annexin V staining in the presence or absence of CD31KD in target cells. [Figure 10A] Results using mouse models of EAE and SLE, and patients with SLE and COVID-19 are shown. Gating strategy for splenocytes from EAE and WT mice. [Figure 10B] Results are shown using mouse models of EAE and SLE, and patients with SLE and COVID-19. The percentage of PD1+CD38hi cells among CD8 T cells at 9 months was analyzed in the spleens of SLE mice with severe disease (Sle1Tg7 and Sle1TLR9KO) compared with mice with mild SLE disease (Sle1) or TLR7KO (Sle1TLR7KO). Error bars: SEM. (*P≦0.05, **P≦0.01). [Figure 10C] Results are shown using mouse models of EAE and SLE, and patients with SLE and COVID-19. Gating strategy for cells from SLE patients. [Figure 10D] Results are shown using mouse models of EAE and SLE, as well as patients with SLE and COVID-19. The ability of Tfrat cells (both isolated from SLE patients) compared with Teff cells to kill autologous target CD4 T cells in the presence or absence of anti-CD38, as determined by flow cytometric Annexin V staining and viability assays. Each line represents an independent patient. Error bars indicate sem. Statistical analysis was performed by unpaired, one-tailed Student's t-test (*P ≤ 0.05). e. Gating strategy for endogenous and infused cells in EAE mice. [Figure 10E] Results are shown using mouse models of EAE and SLE, as well as patients with SLE and COVID-19. Gating strategies for endogenous and infused cells in EAE mice are shown. A schematic diagram of EAE induction and infusion of induced regulatory T cells (iTregs) is shown on the top panel. EAE clinical scores and mouse survival rates are shown on the bottom panel. Error bars indicate sem. Statistical analysis of EAE scores was performed using a one-tailed unpaired Student's t-test. The log-rank (Mantel-Cox) test was used to compare survival rates between the two groups (**P ≤ 0.01, ****P ≤ 0.0001). [Figure 10F] Results are shown using mouse models of EAE and SLE, and patients with SLE and COVID-19. [Figure 11A] Figure 1 shows that bradykinin (BK) induces PD1+CD38hiCD8+ cells in a CREB-1-dependent manner. The frequency of PD1+CD38hiCD8+ cells induced after BK treatment of normal human CD8 T cells and mouse CD8 T cells at various concentrations was determined by flow cytometry. [Figure 11B] We show that bradykinin (BK) induces PD1+CD38hiCD8+ cells in a CREB-1-dependent manner. BK induces PD1+CD38hiCD8+ cells by activating CREB in CD8 T cells. [Figure 11C] We show that bradykinin (BK) induces PD1+CD38hiCD8+ cells in a CREB-1-dependent manner. CD38 depletion reduces the number of BK-induced PD1+CD38hiCD8+ T cells in the lungs of wild-type mice. [Figure 11D] We show that bradykinin (BK) induces PD1+CD38hiCD8+ cells in a CREB-1-dependent manner. [Figure 11E] We show that bradykinin (BK) induces PD1+CD38hiCD8+ cells in a CREB-1-dependent manner. Increased numbers of PD1+CD38hiCD8+ T cells were associated with severe lung pathology in BK-treated mice, similar to features of ARDS. [Figure 12A] Figure 1 shows that PD1+CD38hiCD8+ cells induce killing in dendritic cells (DCs), macrophages, natural killer (NK) cells, and endothelial cells (ECs). Scheme for estimating killing in DCs, macrophages, and NK cells from the spleen of WT mice treated with intravenous infusion of PD1+CD38hiCD8+ cells. [Figure 12B] Figure 1 shows that PD1+CD38hiCD8+ cells induce killing in dendritic cells (DCs), macrophages, natural killer (NK) cells, and endothelial cells (ECs). Gating strategies for different cell populations. [Figure 12C] Figure 1 shows that PD1+CD38hiCD8+ cells induce killing in dendritic cells (DCs), macrophages, natural killer (NK) cells, and endothelial cells (ECs). Estimation of frequency and respective annexin expression in DCs after treatment with PD1+CD38hiCD8+ cells as described in A. [Figure 12D] Figure 1 shows that PD1+CD38hiCD8+ cells induce killing in dendritic cells (DCs), macrophages, natural killer (NK) cells, and endothelial cells (ECs). Estimation of frequency and respective annexin expression in macrophages after treatment with PD1+CD38hiCD8+ cells as described in A. [Figure 12E] Figure 1 shows that PD1+CD38hiCD8+ cells induce killing in dendritic cells (DCs), macrophages, natural killer (NK) cells, and endothelial cells (ECs). Estimation of frequency and respective annexin expression in NK cells after treatment with PD1+CD38hiCD8+ cells as described in A. [Figure 12F] Figure 1 shows that PD1+CD38hiCD8+ cells induce killing in dendritic cells (DCs), macrophages, natural killer (NK) cells, and endothelial cells (ECs). CD31+ frequency on live C166 endothelial cells. [Figure 12G]Figure 1 shows that PD1+CD38hiCD8+ cells induce killing in dendritic cells (DCs), macrophages, natural killer (NK) cells, and endothelial cells (ECs). Killing of CD31+ cells by PD1+CD38hi and PD1+CD38lo (Teff) cells was estimated by caspase 3 / 7 staining by flow cytometry (G). Representative data from one of two experiments are shown. Error bars indicate s.e.m. [Figure 12H] Figure 1 shows that PD1+CD38hiCD8+ cells induce killing in dendritic cells (DCs), macrophages, natural killer (NK) cells, and endothelial cells (ECs). The ability of PD1+CD38hiCD8+ cells isolated from severe COVID-19 patients to kill HUVEC endothelial cells was determined by Annexin V staining by flow cytometry. Representative data from one of two experiments are shown. Error bars indicate sem. Statistical analysis was performed by unpaired, one-tailed Student's t-test. *P ≤ 0.05. DETAILED DESCRIPTION OF THE INVENTION

[0014] PD1 + CD38 hi CD8 + T cells form a distinct subtype of phenotypically and functionally novel CD8 T cells. These cells kill target cells expressing the CD31 receptor, including CD4 and CD8 T cells and endothelial cells. PD1 + CD38 hi CD8 + T cells kill cells by binding of CD38 on these cells to CD31 on target cells, resulting in degranulation of Gzm B in CD38+PD1+ CD8 T cells, their transfer to target cells, and induction of apoptosis in the target cells. These cells are highly expressed in advanced COVID-19, which is known to be severely lymphopenic. Therefore, CD38:CD31 interactions, e.g., PD1, in subjects are important for the treatment of T cells. + CD38 hi CD8+ Targeting the interaction of T cells with CD31-expressing cells is useful for treating acute respiratory distress syndrome (ARDS) and ARDS-associated infections, such as COVID-19.

[0015] As described herein, PD1 + CD38 hi CD8 + T cells produce high levels of both pro- and anti-inflammatory cytokines. These cells co-express both effector and exhaustion-related genes. Furthermore, they have a closed chromatin structure and yet highly express effector cytokine genes. These cells exhibit metabolic catastrophe, identified by upregulated metabolites and lipids. These cells also exhibit higher mitochondrial mass with leaky mitochondria, indicating high reactive oxygen species (ROS) production. Furthermore, PD1 + CD38 hi CD8 + T cells have promiscuous, contact-dependent cytotoxicity mediated by CD38:CD31 interactions and (Gzm B) transfer to target cells (CD8, CD4, dendritic cells, natural killer cells, endothelial cells, macrophages), leading to lymphopenia.

[0016] Furthermore, these cells have been induced in autoimmune disease models, such as experimental autoimmune encephalomyelitis (EAE) and SLE mouse models, and injecting these cells into mice with EAE rescues the mice and reverses their symptoms. Higher numbers of these cells have also been found in patients with autoimmune diseases. PD1 cells obtained from SLE patients + CD38 hi CD8 + T cells target autologous CD4 + It has been shown to have the ability to kill T cells. + CD38 hi CD8 + Because T cells have immunoprotective effects, these cells are useful in the treatment of autoimmune disorders.

[0017] PD-1 + CD38 hi CD8 + T cells PD-1 + CD38 hi CD8 + T cells are dysfunctional T cells that can be targeted to treat inflammation-induced respiratory disorders, such as COVID-19, ARDS, and other ARDS-associated infections (e.g., viral infections). + CD38 hi CD8 + T cells also express, for example, PD-1 + CD38 hi CD8 + by administering to the subject a population of T cells or by administering to the subject a population of PD-1 + CD38 hi CD8 + By inducing the production of T cells, e.g., PD-1 in the subject + CD38 hi CD8 + They may also be used to treat autoimmune disorders or infectious diseases in a subject by increasing the levels of T cells. As used herein, dysfunctional T cells are T cells that do not respond to repeated immune stimulation and / or fail to generate immunological memory.

[0018] As described herein, PD-1 + CD38 hi CD8 + T cells form a phenotypically and functionally distinct subtype of CD8 T cells that have the ability to kill target cells that express the CD31 receptor. Cell killing or cytotoxicity is mediated through the binding of CD38 to CD31 on target cells, e.g., leukocytes or endothelial cells. When CD38+ T cells bind to target cells, e.g., leukocytes, that express CD31, (Gzm B) binds to CD31-expressing target cells, e.g., leukocytes, and binds to CD31-expressing target cells, e.g., PD-1. + CD38 hi CD8 + T cells) to target cells and induce apoptosis in the target cells. +CD38 hi CD8 + T cells can be immunized with, for example, an agent that reduces or inhibits binding of CD38 to CD31, e.g., PD-1 + CD38 + CD8 + It can be targeted by reducing or inhibiting the binding of CD38 on T cells to CD31 on target cells. + CD38 hi CD8 + The number of T cells can be determined by, for example, detecting PD-1 in a subject. + CD38 hi CD8 + It can also be reduced in a subject by depleting T cells. + CD38 hi CD8 + Decreased CD38:CD31 interactions between T cells and CD31-expressing cells and / or PD-1 + CD38 hi CD8 + Reducing the number of T cells is useful in treating diseases associated with respiratory inflammation or distress.

[0019] Also, for example, PD-1 + CD38 hi CD8 + administering a population of T cells to a subject, or PD-1 + CD38 hi CD8 + Inducing the production of T cells, thereby + CD38 hi CD8 + Increasing the number of T cells has also been found to be useful in treating autoimmune disorders.

[0020] PD-1 + CD38 hi CD8 +Methods for generating T cells are known in the art. Antigens / peptides that suboptimally prime / activate T cells via their T cell receptor (TCR) or that block PD-1 on CD8+ T cells prior to antigenic stimulation (both suboptimal and optimal antigens) can be used to generate PD-1. + CD38 hi CD8 + For example, CD8+ T cells can be contacted with one or more suboptimal antigens to induce PD1 T cell induction. + CD38 hi CD8 + T cells can be produced. Exemplary antigens include, but are not limited to, ovalbumin peptides, such as Ova 257-264 (SIINFEKL) (SEQ ID NO: 1) or Ova-V, a low-affinity variant of Ova 257-264 (SIIGFEKL) (SEQ ID NO: 2). As used herein, optimal priming refers to antigen stimulation via the T cell receptor (TCR) required by T cells to fully proliferate, exert effector function, and differentiate into memory cells. In contrast, suboptimal priming refers to weaker antigen stimulation via the T cell receptor that is insufficient to maximize T cell proliferation and / or function, resulting in an increased number of dysfunctional T cells. For example, suboptimal priming reduces one or more functions of T cells by at least about 10%, 20%, 30%, 40%, 40%, 60%, 70%, 80%, 90%, or 100% compared to optimal antigen priming / activation. For example, T cells can be contacted with a suboptimal peptide or agent that has reduced affinity for or reduced binding to the TCR compared to a peptide or agent that optimizes T cell priming or activation, thereby expanding the population of T cells that exhibit reduced functional and / or proliferative capacity.

[0021] Blockade of PD-1 can include contacting CD8+ T cells with an agent that inhibits or prevents the interaction of PD-1 with its ligand(s). In some methods, the agent inhibits the PD-1 / PD-L1 and / or PD-1 / PD-L2 pathway (i.e., the interaction between PD-1 and PD-L1 and / or the interaction between PD-1 and PD-L2). In some methods, a PD-1 inhibitor (e.g., an anti-PD-1 antibody) or a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody) is used to generate a population of PD-1+CD38hiCD8+ T cells. It is understood that inhibition of the PD-1 / PD-L1 pathway and / or the PD-1 / PD-L2 pathway can also be referred to as PD-1 blockade.

[0022] Optionally, the CD8+ T cells are contacted with an agent that inhibits IL-2 and / or PD-1 (e.g., an anti-PD-1 antibody) to inhibit PD1 + CD38 hi CD8 + The production of T cells can be induced. Optionally, the T cells are contacted with IL-2 and an agent that inhibits PD-1. Optionally, PD1 + CD38 hi CD8 + Induction of T cells involves contacting CD8+ T cells with a suboptimal antigen and an agent that inhibits PD-1 (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody).

[0023] PD-1 + CD38 hi CD8 + It is understood that methods for producing T cells can include stimulating, activating (i.e., priming), and / or differentiating CD8+ T cells in vivo, ex vivo, or in vitro. Optionally, cells produced by these methods can be expressed, for example, as PD1 T cells. + CD38 hi CD8 + It can be further purified by fluorescence-activated cell sorting (FACS) for use in adoptive transfer of T cells. + CD38 hi CD8 +T cells can also be engineered ex vivo to reduce Gzm B production in the cells prior to administration to a subject. + CD38 hi CD8 + PD-1 produced by T cells or by any of the methods described herein + CD38 hi CD8 + Any population of T cells may be expanded prior to adoptive transfer.

[0024] Treatment methods Provided herein is a method for reducing cell apoptosis in a subject, comprising administering to the subject an effective amount of an agent that reduces the binding of target cells, such as CD31-expressing cells, to CD38-expressing cells in the subject. In some methods, the apoptosis (i.e., cytotoxicity or cell killing) of CD31-expressing leukocytes in the subject is reduced. In some methods, the apoptosis of CD31-expressing endothelial cells in the subject is reduced.

[0025] As used throughout, leukocytes are immune cells involved in protection from infections and foreign antigens. Leukocytes include, but are not limited to, T lymphocytes, dendritic cells, natural killer cells, and macrophages. As used herein, T cells or T lymphocytes refer to lymphocytes that express T cell receptor molecules. T cells include human alpha beta (αβ) T cells and human gamma delta (γδ) T cells. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subpopulations thereof. T cells can be CD4+, CD8+, or CD4+ and CD8+.

[0026] Provided herein is a method for reducing cellular apoptosis in a subject, the method comprising administering to the subject an effective amount of an agent that reduces binding of CD38-expressing cells to CD31-expressing leukocytes in the subject.

[0027] A method for reducing apoptosis of cells in a subject, comprising: inducing CD8+ T cells (e.g., PD1) directed against CD31-expressing leukocytes in the subject; + CD38 hi CD8 + Also provided are methods comprising administering to a subject an effective amount of an agent that reduces binding of CD31-expressing leukocytes (e.g., CD8+ T cells). In some methods, the CD31-expressing leukocytes are T cells, dendritic cells, natural killer T cells, or macrophages. In some methods, the CD31-expressing leukocytes are CD8+ T cells (e.g., PD1 T cells), dendritic cells, natural killer T cells, or macrophages. + CD38 hi CD8 + T cells) and one or more types of CD31-expressing leukocytes.

[0028] It is understood that the reduced binding between CD38-expressing cells and CD31-expressing leukocytes is due, at least in part, to binding of CD38 on the surface of the CD38-expressing cells to CD31 on the surface of the CD31-expressing cells. As used throughout, the reduced or decreased binding need not be complete, and the reduction may be observed in the absence of an agent that inhibits the interaction between CD38 and CD31 in a subject, or in the absence of PD-1 + CD38 hi CD8 + The reduction can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any percentage reduction therebetween, compared to binding in the absence of T cell depletion. Similarly, the reduction or inhibition of PD-1 (i.e., PD-1 blockade) need not be complete; the reduction can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any percentage reduction therebetween, compared to the level of PD-1 / PD-1 activity in the absence of PD-1 blockade.

[0029] Agents that can be used to reduce binding of CD38 to CD31 include, but are not limited to, chemicals, small or large molecules (organic or inorganic), proteins, peptides, or antibodies. In some methods, the antibody specifically binds to CD38, e.g., satuximab, daratumumab (Darzalex), and isatuximab (Sarclisa), to name a few. Additional anti-CD38 antibodies are described in U.S. Patent Nos. 8,362,211, 8,088,896, 8,263,746, and 8,153,765. In some methods, the antibody specifically binds to CD31. In some methods, the antibody specifically binds to PD1. + CD38 hi CD8 + Expression of CD38 on T cells can be reduced ex vivo, for example, by contacting the cells with an inhibitory RNA or a gene editing system before administering the cells to a subject.

[0030] As used herein, the term antibody encompasses, but is not limited to, all immunoglobulins (i.e., intact antibodies) of any class. Naturally occurring antibodies are usually heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V(H)) at one end followed by several constant domains. Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end, with the light chain constant domain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy-chain constant domains corresponding to the different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The term variable is used herein to describe certain portions of antibody domains that differ in sequence among antibodies and are used in relation to the binding and specificity of each particular antibody for its particular antigen. However, the variability is usually not evenly distributed throughout the variable domains of antibodies. This is typically concentrated in three segments called the complementarity-determining regions (CDRs) or hypervariable regions in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FRs).The variable domains of naturally occurring heavy and light chains each contain four FR regions, adopting a primarily β-sheet configuration, connected by three CDRs, which form loops that connect and in some cases form part of the β-sheet structure. The CDRs within each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity.

[0031] As used herein, an antigen-binding fragment of an antibody refers to one or more portions of an antibody that contain the antibody's complementarity-determining regions (CDRs) and, optionally, framework residues comprising the antibody variable region antigen recognition site, and that exhibit the ability to specifically bind to an antigen. Such fragments include Fab', F(ab'), Fv, single-chain Fv, and mutants and natural variants thereof, as well as fusion proteins comprising the antibody variable region antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor, or a receptor ligand).

[0032] In some methods, reduced binding of CD38 to CD31-expressing cells inhibits PD-1 + CD38 hi CD8 + Degranulation of (GzmB) in T cells is reduced in the subject. In some methods, reduced binding of CD38 to CD31-expressing cells reduces PD-1 expression in the subject. + CD38 hi CD8 + GzmB transfer from T cells to leukocytes is reduced. + CD38 hi CD8 + T cells are engineered ex vivo to reduce Gzm B prior to transplantation into a subject, eg, a subject with an autoimmune disorder.

[0033] PD-1 in subjects + CD38 hi CD8+ Also provided is a method of reducing leukocyte apoptosis in a subject, comprising reducing the level of T cells. + CD38 hi CD8 + The level of T cells, e.g., PD-1 + CD38 hi CD8 + by administering an agent that depletes T cells or by inhibiting PD-1 in the subject + CD38 hi CD8 + It can be reduced by inhibiting the induction of T cells. + CD38 hi CD8 + Agents that can be used to deplete T cells include, but are not limited to, chemicals, small or large molecules (organic or inorganic), proteins, peptides, or antibodies. Immunomodulatory agents that bind to CD38 and / or PD-1 can be used to deplete PD-1. + CD38 + CD8 + T cells can be depleted or PD-1 signaling can be prevented, inhibited, reduced, or blocked. In some methods, a bispecific antibody that binds to CD38 and PD-1 is used to target CD38. + PD-1 + This bispecific antibody is engineered to bind to CD38 and CD8 on the same cell. Binding of the antibody to CD38 and PD-1 on the target cell results in depletion of the target cell.

[0034] PD-1 + CD38 + CD8 + T cells can also be depleted using various ex vivo methods. For example, flow cytometry can be used. In these methods, immune cells are collected from a subject's biological specimen, such as blood, or from a tissue biopsy. For example, immune cells are labeled with fluorescent antibodies against CD38, CD8, and / or PD-1, or a combination thereof. CD38+ CD8 + T cells, CD38 + PD-1 + T cells, CD38 + PD-1 + CD8 + T cells, or a combination thereof, can be sorted from the population of cells by flow cytometry. The remaining population of cells is CD38 + PD-1 + CD8 + By administering the T cell population back to the subject, the CD38 + PD-1 + CD8 + The number of T cells can be reduced.

[0035] In some methods, PD-1 + CD38 + CD8 + T cells are depleted using magnetic sorting. In these methods, immune cells are collected from a subject's biological specimen, such as blood, or from a tissue biopsy. The immune cells are labeled with magnetic nanoparticles conjugated to antibodies against CD38, CD8, PD-1, or a combination thereof. CD38 + CD8 + T cells, CD38 + PD-1 + T cells, CD38 + PD-1 + CD8 + T cells, or a combination thereof, can be sorted from the population of cells using a magnetic cell sorter or column. The remaining population of cells is CD38 + PD-1 + CD8 + By administering the T cell population back to the subject, the CD38 + PD-1 + CD8 + The number of T cells can be reduced. In some methods, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage therebetween of cells are depleted from a sample from a subject.

[0036] In some methods, the subject has lung injury (e.g., lung injury caused by acute or chronic pulmonary inflammation), or ARDS. As used herein, ARDS is a respiratory condition in which the lungs suffer severe and extensive damage, reducing their ability to provide sufficient oxygen to the body's organs. This condition causes fluid to accumulate in the lungs, resulting in dangerously low blood oxygen levels. Conditions that cause ARDS include, but are not limited to, pneumonia, sepsis, chest trauma, lung transplantation, cardiopulmonary bypass, and viral infections of the lungs, including those caused by SARS-CoV-2, the coronavirus that causes COVID-19 infection. Other ARDS-associated infections include, but are not limited to, influenza virus, pneumonia, herpes simplex virus, other coronaviruses, adenovirus, measles virus, mycobacterial tuberculosis, and cytomegalovirus. See, e.g., Luyt et al. Presse Med 40(12):e561-568 (2011) and Lee Int. J. Mol. Sci. 18(2):388 (2011). Thus, PD-1 receptors on CD31-expressing cells (e.g., leukocytes or endothelial cells) + CD38 hi CD8 + It will be appreciated that any of the methods described herein, including reducing T cell binding, can be used to treat lung injury or acute respiratory distress syndrome in a subject. Lung injury or ARDS can also be caused by the reduction of PD-1 in a subject. + CD38 hi CD8 + It can also be treated by reducing the number of T cells.

[0037] 1. A method for treating an autoimmune disease in a subject, comprising administering to the subject an antibody against PD-1 + CD38 hi CD8 +Also provided are methods comprising administering a population of T cells. As used herein, an autoimmune disease is a disease in which the immune system is unable to distinguish between a subject's own cells and foreign cells, causing the immune system to mistakenly attack healthy cells in the body. Exemplary autoimmune diseases include, but are not limited to, inflammatory bowel disease, systemic lupus erythematosus, vasculitis, rheumatoid arthritis, type 1 diabetes, myasthenia gravis, multiple sclerosis, psoriasis, Graves' disease, Hashimoto's disease, Sjogren's syndrome, and scleroderma.

[0038] 1. A method for treating an infection in a subject, comprising administering to the subject an antibody against PD-1 + CD38 hi CD8 + Also provided are methods, comprising administering a group of T cells.In some methods, infectious disease is non-ARDS-related infectious disease.Infectious disease can be acute or chronic.Acute infectious disease is usually short-lasting infectious disease, while chronic infectious disease is a type of persistent infectious disease that eventually disappears.In the methods provided herein, the infectious disease to be treated can be caused by bacteria, virus, protozoa, helminth, fungal pathogen, parasitic pathogen or other microbial pathogen.

[0039] PD-1 + CD38 hi CD8 + In any of the methods comprising administering T cells to a subject, the method may further comprise administering to a subject a T cell that inhibits PD-1 + CD38 hi CD8 + PD-1 can be inhibited by contacting CD8+ T cells with a suboptimal antigen and / or a PD-1 inhibitor prior to administering the population of T cells to the subject. + CD38 hi CD8 + This may include generating a population of T cells. + CD38 hi CD8 +A suboptimal antigen that increases T cell production can be selected by those skilled in the art based on the disease or disorder. In some methods, the suboptimal antigen is a variant of an antigen expressed by a pathological agent (e.g., SARS-COV-2 in the case of COVID-19).

[0040] In some methods, PD-1 + CD38 hi CD8 + The T cells are expanded before being administered to the subject. + CD38 hi CD8 + CD8 used to generate T cells + T cells are either autologous or autologous CD8 + T cells (i.e., PD-1 + CD38 hi CD8 + T cells can be allogeneic or allogeneic (i.e., derived from the same subject as the T cells are being administered), allogeneic or allogeneic (i.e., derived from a donor subject of the same species), or xenogeneic (i.e., derived from a different species). + T cells are obtained from the peripheral blood cell composition of the donor and CD4 + Peripheral blood cell compositions such as T cells, natural killer cells, etc. may be isolated from the donor subject by depletion. Optionally, CD8 + The donor of the T cells is HLA-matched, partially HLA-matched, or haploidentical to the recipient. In some methods, CD8 T cells obtained from the subject + T cells can be cryopreserved before priming them with an antigen, e.g., a suboptimal antigen as described above. Optionally, PD-1 T cells can be produced using any of the in vitro or ex vivo methods described herein. + CD38 hi CD8 + The T cells are cryopreserved prior to expansion and / or administration to a subject.

[0041] Any of the treatment methods described herein may further include administering to the subject an effective amount of a second therapeutic agent. The second therapeutic agent may be selected from the group consisting of a chemotherapeutic agent, an adjuvant, an immunomodulatory agent, an anti-infective agent (e.g., an antiviral agent, an antibacterial agent, etc.), a vaccine, a booster, a pathogen antigen, or a combination thereof. In some methods, the immunomodulatory agent is an immunostimulatory agent. As used herein, an immunostimulatory agent is an agent that stimulates or activates an immune response. Stimulating or activating an immune response includes inhibiting a suppressive immune response. Examples of immunostimulatory agents include vaccines that can be used to stimulate an immune response.

[0042] In some methods, the immunomodulatory agent is an immunosuppressant. As used herein, an immunosuppressant is a drug that suppresses or inhibits immune response, for example, an immunosuppressant used to treat autoimmune disorders. Examples of immunosuppressants include, but are not limited to, calcineurin inhibitors (e.g., cyclosporine, tacrolimus), corticosteroids (e.g., methylprednisolone, dexamethasone, prednisolone), and cytotoxic immunosuppressants (e.g., azathioprine, chlorambucil, cyclophosphamide, mercaptopurine, methotrexate).

[0043] In methods for treating ARDS-associated COVID or COVID, the second therapeutic agent may be selected from the group consisting of nirmatrevlir, ritonavir, remdesivir, molnupiravir, and anti-SARS-CoV-2 monoclonal antibodies. As used herein, an immune response is the development in a recipient patient of a beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) response to a peptide. Such a response may be an active response induced by administration of an immunogen, or a passive response induced by administration of antibodies or primed T cells. A cellular immune response may be an antigen-specific CD4 + T helper cells and / or CD8 +It is elicited by the presentation of polypeptide epitopes in association with class I or class II molecules to activate cytotoxic T cells. This response may also include activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, and activation or recruitment of neutrophils or other innate immune components.

[0044] Combinations, e.g., PD-1 + CD38 hi CD8 + It is understood that compositions comprising T cells and a non-cellular therapeutic agent (e.g., an immunomodulatory agent, an anti-infective agent, a vaccine, etc.) described herein can be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines to the same subject), or sequentially (e.g., one of the compounds or agents is administered first, followed by the second). Any of the methods provided herein can further include surgery.

[0045] As used throughout, a subject may be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig). The term does not denote a particular age or sex. Thus, it is intended to encompass adult, newborn, and pediatric subjects, regardless of male or female. As used herein, patient or subject may be used interchangeably and may refer to a subject having a disorder or at risk of developing a disorder. The terms patient or subject include human and veterinary subjects. In any of the methods provided herein, the subject may be a subject diagnosed with a disease, such as a respiratory disorder, an infectious disease, or an autoimmune disease.

[0046] As used herein, the terms "treatment," "treating," or "treating" refer to a method of reducing one or more of the effects of a disorder, such as an autoimmune disorder, or one or more symptoms of the disorder, or a disease associated with respiratory distress in a subject. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of the disease or disorder. For example, a method for treating an autoimmune disorder is considered therapeutic if one or more symptoms of the autoimmune disorder are reduced by 10% in a subject compared to a control. Thus, a reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to the natural or control level. It is understood that treatment does not necessarily refer to a cure or complete elimination of the disorder or symptoms of the disorder.

[0047] As used herein, the term therapeutically effective amount or effective amount refers to a therapeutically effective amount of PD-1 as described herein. + CD38 hi CD8 + It refers to an amount of a composition comprising T cells, or cells differentiated therefrom, an immunomodulator, etc., that, when administered to a subject, is effective to treat a disease or disorder, either alone or in combination with additional agents, either in a single dose or over multiple doses. The appropriate dose can vary depending on various factors, including the particular cells or agent used and whether it is used concomitantly with other therapeutic agents. Other factors that affect the dose administered to a subject include, for example, the type or severity of the disease. For example, a subject with multiple sclerosis may receive a different dose of PD-1 than a subject with lupus. + CD38 hi CD8 + This may involve administering a composition comprising T cells or cells differentiated therefrom, and / or an immunotherapeutic agent.

[0048] PD-1 + CD38 hi CD8 +An effective amount of T cells or cells differentiated therefrom can be determined by one of skill in the art, and an exemplary amount for a mammal is about 0.1 x 10 per kg of body weight. 5 ~Approx. 8×10 9 Examples include cells.

[0049] An effective amount of any compound described herein (e.g., an immunomodulatory agent or any other non-cellular therapeutic agent described herein), or a pharmaceutically acceptable salt or prodrug thereof, can be determined by one of skill in the art, and exemplary dosages for mammals include about 0.5 to about 200 mg of active compound per kg of body weight per day, which may be administered in a single dose or in the form of individual divided doses, such as 1 to 4 times per day. Alternatively, the dosage may be about 0.5 to about 150 mg of active compound per kg of body weight per day, about 0.5 to about 100 mg of active compound per kg of body weight per day, about 0.5 to about 75 mg of active compound per kg of body weight per day, about 0.5 to about 50 mg of active compound per kg of body weight per day, about 0.5 to about 25 mg of active compound per kg of body weight per day, about 1 to about 20 mg of active compound per kg of body weight per day, about 1 to about 10 mg of active compound per kg of body weight per day, about 20 mg of active compound per kg of body weight per day, about 10 mg of active compound per kg of body weight per day, or about 5 mg of active compound per kg of body weight per day. Other factors that affect the dosage may include, for example, other medical disorders concurrently or previously affecting the subject, the subject's overall health, the subject's genetic makeup, dietary habits, time of administration, excretion rate, drug combinations, and any other additional therapeutic agents administered to the subject. It is also understood that the specific dosage and treatment regimen for any particular subject will vary according to the judgment of the treating physician. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

[0050] As used herein, administering or administration refers to administering a substance present outside the body (e.g., PD-1 as described herein). + CD38 hi CD8+ "T cells, cells differentiated therefrom, or any non-cellular therapeutic agent) into a subject, such as by mucosal, intradermal, intravenous, intratumoral, intramuscular, rectal, oral, subcutaneous delivery, and / or any other physical delivery method described herein or known in the art. When a disease or symptom thereof is being treated, administration of the agent typically occurs after the onset of the disease or symptom thereof. When a disease or symptom thereof is being prevented, administration of the agent typically occurs before the onset of the disease or symptom thereof.

[0051] A therapeutic agent described herein (e.g., a PD-1 + CD38 hi CD8 + T cells, cells differentiated therefrom, or any other non-cellular therapeutic agent (e.g., vaccine, adjuvant, immunotherapeutic, etc.) can be administered in a number of ways, depending on whether local or systemic treatment is desired and the area to be treated. The composition can be administered via any of several routes, including orally, parenterally, intramucosally, intravenously, intraperitoneally, intracerebroventricularly, intramuscularly, subcutaneously, intracavity, or transdermally. Administration can be achieved, for example, by topical administration, local infusion, injection, or using an implant. The implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition into the subject. See, for example, U.S. Patent Application Publication No. 20080241223, U.S. Patent Nos. 5,501,856, 4,863,457, and 3,710,795, and European Patent Nos. EP488401 and EP430539.

[0052] In some methods, non-cellular therapeutic agents such as small molecules, vaccines, immunotherapeutics, etc., can be delivered to a subject by, for example, implantable devices based on diffusive, erodible, or convective systems, osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmotic systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems. Nanoparticle delivery is also contemplated herein. Effective doses for any of the administration methods described herein can be estimated from dose-response curves derived from in vitro or animal model test systems.

[0053] The cells and compounds described herein can be formulated as pharmaceutical compositions. In some embodiments, the pharmaceutical composition can further comprise a carrier. The term carrier refers to a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, the carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. Such pharmaceutically acceptable carriers include sterile, biocompatible pharmaceutical carriers, including, but not limited to, saline, buffered saline, artificial cerebrospinal fluid, dextrose, and water.

[0054] PD-1 + CD38 hi CD8 + T cells, or cells differentiated therefrom, can be formulated as a pharmaceutical composition for parenteral administration. In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent as described herein. T cells are typically administered by parenteral injection in an aqueous solution. The formulation can also be in the form of a suspension or emulsion.

[0055] Depending on the intended mode of administration, pharmaceutical compositions containing the non-cellular therapeutic agents described herein may be in solid, semi-solid, or liquid dosage form, such as tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage forms suitable for single administration of precise dosage amounts. The compositions contain a therapeutically effective amount of an agent or derivative thereof described herein in combination with a pharmaceutically acceptable carrier and may additionally contain other medicinal agents, pharmaceutical agents, carriers, or diluents. Pharmaceutically acceptable means a material that is not biologically or otherwise undesirable and that can be administered to an individual together with the selected agent without causing unacceptable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition in which it is included.

[0056] Disclosed are materials, compositions, and components that can be used for, used in conjunction with, used in preparation for, or are products of the disclosed methods and compositions. When these and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even though specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed. For example, when a method is disclosed and discussed, and several modifications that can be made to one or more molecules included in the method are discussed, any and all combinations and permutations of this method and possible modifications are specifically contemplated unless specifically indicated otherwise. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including, but not limited to, steps in methods using the disclosed compositions. Thus, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method step or combination of method steps of the methods of the present disclosure, and that each such combination or subset of combinations is to be considered specifically contemplated and disclosed.

[0057] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entirety.

[0058] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims. [Example]

[0059] CD8 expressing PD1 and high levels of CD38 generated by SOP + T cells are a unique CD8 T cell subtype Previously, this research group reported that a novel subtype of CD8+ T cells was induced under suboptimal priming (SOP) conditions in vitro and in the tumor microenvironment (TME) (Verma et al. Nat. Immunol. 20, 1231-1243 (2019)). These cells express PD1 and high levels of CD38 (PD1+CD38hi) compared with T effector CD8+ T cells (Teff), which was found to play a role in resistance to anti-PD1 therapy. In the study described herein, we further characterized the cells to understand their mechanism of action. To this end, we used the OT1 mouse system to generate Teff CD8+ T cells using WT OVA peptide or PD1+CD38hi CD8+ T cells by SOP with the low-affinity OVA-V peptide (Verma et al.). Priming was performed under the same conditions for 48 hours (Figure 8A). We first compared the activation status and function of these two cell populations. PD1+CD38hiCD8+ T cells generated by SOP were found to be functionally impaired due to the expression of low levels of IFN-gamma, CD40L, and CD69 (Fig. 1a and Fig. 8b). Because the hypoactivation state of PD1+CD38hiCD8+ T cells is CD38-dependent, we knocked down (KD) CD38 in these cells and directly compared their activation and function with Teff cells. KD of CD38 was found to restore their activation and effector function, as measured by the number of IFN-gamma+, CD40L+, and CD69+ cells, to levels comparable to Teff cells (Fig. 1a). Interestingly, KD of CD38 also enhanced the recall activity of PD1+CD38hiCD8+ T cells after antigen rechallenge (Fig. 1b, c), although this was still lower than that of Teff cells (Fig. 1c).

[0060] PD1 + CD38 hi CD8 + To further understand the function of T cells, their cytokine profiles were analyzed using T eff Compared with PD1 cells. + CD38 hiWe found that CD8 T cells produced significantly higher levels of both pro-inflammatory (IL-1β, IL-23, IL-6) and anti-inflammatory (IL-10, TGF-β, IL-4, IDO) cytokines when isolated from the TME of TC-1 tumors in vitro and in vivo (Fig. 1d, e). Interestingly, despite their reduced activation and dysfunctional state, PD1 + CD38 hi CD8 T cells are eff These results suggest that SOP-driven PD1 expression in PD1 cells produced significantly higher levels of GzmB and perforin (Prf) compared with PD1 cells (Fig. 1d, e) (gating strategy is outlined in Fig. 8a, c). + CD38 hi The cells are T eff These findings suggest that PD1 T cells are a unique subset of CD8 T cells distinct from PD1 T cells. To support this, we characterized these cells at the transcriptome level by RNA sequencing. Principal component analysis (PCA) of the RNA sequencing data revealed that PD1 T cells are distinct from PD1 T cells. + CD38 hi CD8 T cells are eff The PD1 T cells obtained by SOP were found to have distinct transcriptional profiles when compared with both activated and non-activated (IL-2) CD8 T cells (Fig. 1f). + CD38 hi CD8 T cells and optimally primed T eff The difference between the cells was mainly explained by PC2, which is a factor + CD38 hi We show that CD8 T cells are unique populations that are not confined to a linear axis from unprimed to properly primed, which may indicate their individual status (Fig. 1f). + CD38 hi T effWhen compared to CD8 T cells, we found significant changes in 1,209 genes (411 upregulated and 798 downregulated) (p<0.001 & log2 fold change >±1.2) (Venn diagram in Figure 1g, Figure 8d). Because PC2 explains most of the differences between these two CD8 T cell populations, we examined the top 50 differentially expressed genes (DEGs) associated with this axis, as shown in Figure 1h. A heatmap of the overall top DEGs is shown in Figure 8e. Next, we analyzed PD1 + CD38 hi The signature genes that contribute to the transcriptional specificity of CD8 T cells were analyzed using the individual controls (i.e., naive (IL-2), T eff and PD1 + CD38 hi PD1 T cells) were identified by comparing genes that were up- or down-regulated in the PD1 T cells (Venn diagram in Figure 1g). Utilizing this gene signature, GO pathway enrichment analysis was performed to identify genes that were up- or down-regulated in the PD1 T cells (PD1 T cells). + CD38 hi CD8 cells and T eff We defined the difference between PD1 and PD2 cells (Fig. 1g). + CD38 hi CD8 T cells showed significant enrichment of upregulated genes in immune effector processes, despite enrichment of downregulated genes in T cell activation and differentiation pathways (Fig. 1g). Furthermore, comparison of effector and exhaustion gene expression at the single gene level revealed that T eff Compared with cells, PD1 + CD38 hi Many DEGs were revealed in T (Fig. 8f). However, no overall enrichment at the gene set level was observed (p<0.01). Furthermore, cytokine production and regulation pathways were also found to be enriched from both up- and down-regulated genes, suggesting that cytokine regulation and subsequent expression are involved in T eff Compared with PD1 + CD38 hiConsistent with these observations and consistent with the protein data above (Fig. 1d, e), expression of both pro-inflammatory cytokines (Il1b, Il6, Il23) and anti-inflammatory cytokines (Il10, tgfb, Ido, Il10) was significantly increased in PD1-positive CD8 T cells. + CD38 hi Furthermore, based on the protein levels, the expression of Gzm B and Prf1 was upregulated simultaneously in properly activated T cells (Fig. 1i). eff These data suggest that PD1 + CD38 hi This strongly suggests that CD8 T cells exist in a novel transcriptional state, further demonstrating that these cells are a unique subset of CD8 T cells.

[0061] To understand this differential gene expression, we investigated PD1 + CD38 hi CD8 + T cells eff ATAC sequence analysis was performed by comparing PD1 and PD2 cells. Data shown in log ratio ("minus") versus mean expression ("MA") plots, heat maps, and volcano plots indicate that PD1 + CD38 hi In CD8 T cells, T eff Compared to cells, the overall promoter-restricted peaks were demonstrated to be less accessible (Fig. 1j and Fig. 8g; FDR < 0.05). + CD38 hi Cells vs. T eff Only 10 promoters were accessible in PD1 cells, and 147 promoters were inaccessible (heatmap in Fig. 1j and Fig. 8g; FDR<0.05, |log2FC|>1.2). + CD38 hi Among the more accessible promoters in cells were Gzmk and T cell receptor gamma (Trgj1, Trgj4) promoters 1 and 4 (heatmap in Figure 1j and Figure 8g). + CD38hi Inaccessible promoters in cells were enriched for pathways including cytokine and inflammatory responses, cytokine-cytokine receptor interactions, and JAK-STAT signaling (Fig. 1j heatmap). These data suggest that PD1 suppresses many T cell functions while amplifying a small subset. + CD38 hi Taken together, the sequencing data support a model of specific regulation in PD1 cells. + CD38 hi CD8 + We demonstrate a closed chromatin structure in T cells and suggest that this may coincide with their reduced activation and differentiation state observed at the transcriptional level.

[0062] In summary, high expression (total and active) of functionally contrasting pro- and anti-inflammatory cytokines at both the RNA and protein levels, differential epigenetic modifications, increased expression of cytotoxic proteins, and expression of effector function-related genes despite a low activation and differentiation state are key factors in the development of PD1. + CD38 hi CD8 + T cells transform from naive to T eff This strongly supports the existence of a novel and distinct cellular state in the cellular paradigm from previously documented cellular states. + CD38 hi CD8 + Although T cells express exhaustion-associated genes, they, unlike terminally exhausted T cells, exhibit the ability to recall after antigen rechallenge, further supporting that these cells represent a unique cellular phenotype.

[0063] PD1 + CD38 hi Characterization of CD8 T cell metabolism Since unique CD8 T cell subtypes have been identified, and since it is well established that different T cell subtypes are primarily dependent on specific metabolic pathways (glycolysis, lipids, oxidative phosphorylation), we performed deep metabolomics (N = 156) and lipidomics (N = 615) analyses to investigate the role of PD1 + CD38 hi We assessed whether CD8 T cells were also metabolically distinct. Indeed, PCA scores, volcano plots, and heat maps showed that PD1 + CD38 hi CD8 T on cells eff PD1 cells showed distinct metabolic patterns with significant differences in 63 metabolites and 162 lipids compared to normal cells (Fig. 2a, b). Interestingly, unsupervised analysis revealed that all of the top 50 metabolites, including AMP, lactate, aspartate, glutamine, and alanine, were significantly different from normal cells. + CD38 hi The results showed that the uptake of both glucose (NBDG) and fatty acids (FA) (BODIPY staining) was significantly increased in CD8 T cells (Fig. 2a, b). Similarly, all but two of the top 50 lipids (lyso-phosphatidylinositol (LPI) and free fatty acids (FFA)) were significantly upregulated in these cells (Fig. 2a, b). The above data indicate that these cells are metabolically overactive. Based on metabolic overactivity, both glucose (NBDG) and fatty acid (FA) uptake (BODIPY staining) were significantly increased in CD8 T cells. eff PD1 compared to cells + CD38 hi It was found to be significantly higher in CD8 T cells (Fig. 2c). Interestingly, however, despite the increased mitochondrial mass being correlated with hyperactivity, these PD1 + CD38 hi The cells exhibited mitochondrial dysfunction, as seen in a significant decrease in mitochondrial potential (Fig. 2d). Larger mitochondria were found with disorganized cristae, as indicated by low cristae density, number, and score (Fig. 2e), and significantly increased ROS production (Fig. 2f), suggesting that PD1 + CD38 hi Mitochondria in CD8 T cells effThis further demonstrates that PD1 is structurally and functionally incompatible with other cells. + CD38 hi CD8 + T cells had significantly lower oxygen consumption rates (OCR) / maximal and basal respiration and spare respiratory capacity (SRC), and the extracellular acidification rate (ECAR) was not significantly changed (Fig. 2g), indicating lower energy production. Collectively, findings from deep metabolomics and lipidomics, as well as metabolic assays, suggest that PD1 T cells are more likely to be involved in the development of PD1 T cells than PD1 T cells. + CD38 hi This strongly suggests that CD8 T cells are metabolically distressed, utilizing all energy substrates.

[0064] PD1 + CD38 hi CD8 T cells are fratricidal cells that kill in a contact-dependent manner As outlined above, the PD1 generated by the SOP + CD38 hi is T eff Even compared to PD1 (Thy1.2) mice, these cells are dysfunctional, underactivated, and metabolically distressed CD8 T cells, and these cells have high levels of Gzm B and Prf1. Therefore, it was hypothesized that these cells may have cytotoxic potential. To test this hypothesis, PD1 (Thy1.2) mice derived from OT1 mice were used. + CD38 hi We examined the ability of PD1 CD8 T cells to induce apoptosis in target pMel-1 (Thy1.1) CD8 T cells in vitro (Fig. 3a). + CD38 hi Co-culture of CD8 T cells with target pMel-1 CD8 T cells was found to strongly induce apoptotic killing, as indicated by Annexin V binding and caspase 3 / 7 staining, associated with a significant decrease in the number of viable Annexin V-negative pMel-1 target cells (Fig. 3b, c and Fig. 9a). On the other hand, as expected, CD8 T eff PD1 does not induce such killing. + CD38 hiThe killing ability of CD8 T cells was also tested by viability assay (live / dead cell staining), which showed that 90% of pMel-1-targeted CD8 T cells were killed (Fig. 3d), which is consistent with the PD1 + CD38 hi This further supports the killing ability of CD8 T cells. + CD38 hi We also tested the ability of CD8 T cells to kill wild-type (WT) CD8 and CD4 target T cells obtained from the spleens of C57BL / 6 mice. Indeed, PD1 T cells were significantly reduced in number of viable target T cells, as shown by the significant reduction in the number of viable target T cells in Figure 3d. + CD38 hi CD8 T cells were found to kill both CD8 and CD4 T cells from WT mice. + CD38 hi CD8 T cells have been demonstrated to be fratricidal T cells. + CD38 hi To assess the mechanism of CD8 T cell-mediated killing, we tested whether this killing was contact-dependent. + CD38 hi CD8 T cells were mixed with target pMel-1 CD8 T cells or separated by a plasma membrane using a transwell chamber, and target cells were stained for Annexin V and tested for viability (Figure 3a). Indeed, this killing occurred in a contact-dependent manner, with PD1 + CD38 hi We found that PD1 induces apoptosis (Annexin V) in target cells when mixed in suspension, but not when the cells are separated from the plasma membrane (Fig. 3b, c). + CD38 hi It is shown that CD8 T cells exhibit their fratricidal activity in a contact-dependent manner.

[0065] PD1 + CD38 hiTo further support the fratricidal killing of CD8 T cells, we tested whether these cells could kill autologous CD4 and CD8 T cells in an in vivo mouse setting. - / - PD1 generated from OT1 mice 24 hours after injection of target cells (pMel-1-CD8 (Thy1.1) and OTII-CD4 T cells) into mice. + CD38 hi CD8 T cells were transferred into mice (Fig. 3e). + CD38 hi CD8 T cells express Rag1 - / - Upon transfer into mice, apoptosis was induced in both CD8 and CD4 target T cells, as indicated by a significant decrease in the percentage of viable target cells and an increase in Annexin V MFI on these cells (Fig. 3f, g). This further supports the fratricidal ability of these cells to kill other T cells both in vitro and in vivo. The above data support the role of PD1 + CD38 hi CD8 induces contact-dependent apoptosis in gp100-specific pMel-1 CD8 T cells and WT autologous CD8 and CD4 T cells. frat ) and demonstrates antigen-independent fratricidal activity.

[0066] T frat kills target cells via CD38:CD31 interactions As outlined above, T frat T cells induce contact-dependent killing of target T cells (Figures 3a-c). frat We determined the mechanism by which CD38 induces contact-dependent apoptosis in target T cells. Because these cells express high levels of CD38, we hypothesized that CD38-mediated signaling may be essential for this contact-dependent killing. To test this hypothesis, we used FACS-sorted T cells as outlined in Figure 3h. fratCells were co-incubated with target pMel-1 CD8 T cells with or without blocking CD38 antibodies. frat cells induced apoptosis in pMel-1-targeted CD8 T cells, whereas T eff However, blocking CD38 did not induce T frat The induction of apoptosis mediated by T cells was prevented (Fig. 3i, left), but as expected, T eff We found that blocking CD38 on T cells had no effect on target cell apoptosis (Fig. 9b). Similarly, genetic knockdown of CD38 also inhibited the apoptosis of target cells. frat These results suggest that T frat These findings strongly suggest that cells induce a contact-dependent fratricidal effect via CD38.

[0067] CD31 is CD38 3 It is known to be a natural ligand of T frat It was hypothesized that CD38 may induce apoptosis on target T cells through the interaction between CD38 and its non-substrate ligand, CD31. To test this, we performed killing assays in the presence or absence of CD31-blocking antibodies, as described above. Blocking CD31 prevented apoptosis in target cells, whereas blocking T cells inhibited apoptosis. frat To further support the role of CD31, we performed genetic knockdown of CD31 on target cells, and found that the absence of CD31 inhibited the proliferation of CD8 T cells (Fig. 3j, left). frat We also found that the fratricidal activity of the cells (apoptosis due to Annexin V binding) was prevented, resulting in an increase in the number of viable target CD8 T cells (Figure 3j, right, and Figure 9c). These data support the conclusion that T frat We demonstrate that the cells induce apoptosis in target cells through direct CD38:CD31 interactions between the two cell populations.

[0068] T by transferring Gzm B into target cells frat Cell killing Next, T frat We investigated the mechanism by which T cells induce apoptosis in target T cells via binding of CD38 to CD31. frat Because these cells produce high levels of Gzm B (Fig. 1d, e, i), we tested whether the contact-dependent apoptosis mediated by these cells is Gzm B-dependent. Target pMel-1 CD8 T cells were cultured in the presence of the Gzm B inhibitor Z-AAD-CMK4. frat We found that incubation with T cells completely abrogated target cell apoptosis, as indicated by Annexin V binding (Fig. 4a), demonstrating a direct role for Gzm B in inducing apoptosis in target T cells. frat We examined whether T cells induce killing via direct transfer of Gzm B into target cells. frat We incubated the cells with target pMel-1 CD8 T cells or target OTII CD4 T cells, and determined the changes in Gzm B levels in target cells before and after incubation (Figure 4b). We found a significant increase in the intracellular levels of Gzm B in target cells (1.5-fold and 4-fold in CD8 and CD4 targets, respectively), as well as a 60% to 90% increase in the frequency of Gzm B+ target cells in CD8 and <1% to 80% increase in CD4 target cells (Figure 4c, d).

[0069] The above data suggest that increased levels of this Gzm B in target cells may contribute to the frat This may be due to the induction of degranulation of Gzm B from T cells and its transfer into target cells. frat Cells were incubated with pMel-1 CD8 target cells and degranulation was observed in T frat The level of CD107α in target cells was estimated by the level of CD107α in target cells. fratWe found a significant increase in the expression level of CD107α in T cells (Fig. 4e, f). Importantly, degranulation in killer cells was CD38:CD31-mediated, because blocking the CD38:CD31 interaction with anti-CD31 or anti-CD38 inhibited T cell degranulation. frat Degranulation in the target cells was completely inhibited, and + ) because the increase in Gzm B in cells is completely lost (Fig. 4e, f). frat To prove that T cells directly transfer T cells and cause their apoptosis, DAPI staining was performed. frat Dynamic immunofluorescence analysis was performed by labeling Gzm B in cells and incubating them with target pMel-1 CD8 T cells (bright field, unstained) as described above (Fig. 4g, h). As shown in Fig. 4i, target cells upregulate T cells in a time-dependent manner, starting from a 60-min co-culture of the two cell populations. frat Figure 4j shows that T cells showed enhanced uptake of labeled Gzm B. frat High magnification shows the uptake of Gzm B by target cells after 3 hours of co-culture with T cells. These results suggest that CD38:CD31 interactions result in T frat It is conclusively shown that cellular degranulation occurs, leading to uptake of Gzm B and killing of the target cell.

[0070] CD38:CD31 interaction mediates T cell proliferation via the Zap70-PI3K-RAC-ERK pathway. frat Induces degranulation of Gzm B in cells. As shown above, T frat T cell-induced target cell killing is mediated by CD38:CD31 interactions, which lead to degranulation and T frat This leads to the transfer of Gzm B from CD38 to target cells (Fig. 4e-j). Next, we elucidated the molecular mechanism by which CD38:CD31 ligation leads to Gzm B degranulation. Ligation of CD38 with CD31 increases the level of activated Zap70, leading to T fratWe investigated whether this treatment leads to enhanced PI3K-RAC-ERK-mediated degranulation in T cells. frat The protein and phosphoprotein levels of Zap70, PI3K, RAC, and ERK in cells were measured using Western blotting after incubation with recombinant CD31 (rCD31) protein. frat We found that the expression of phosphorylated Zap70, PI3K, RAC, and ERK increased in killer cells (Fig. 4k). To further support the involvement of this pathway in degranulation, we examined the degranulation of Gzm B resulting from CD38:CD31 binding, as measured by CD107α levels, in the presence or absence of PI3K and ERK inhibitors. Degranulation was inhibited in the presence of PI3K or ERK inhibitors (Fig. 4l). Thus, these results clearly demonstrate that CD38:CD31 interaction leads to the induction of degranulation in killer cells through activation of Zap70-PI3K-RAC-ERK signaling (Fig. 4m).

[0071] T frat Gzm B production in cells is induced by CD38-mediated NAD depletion through regulation of the SIRT1-FOXO1-TCF7 pathway. T frat The cells are T eff Because T cells have high levels of Gzm B compared with T cells, which is important for their fratricidal function, we investigated the mechanism by which these cells accumulate high levels of Gzm B and the potential role of CD38 in this process. frat CD38 was knocked down in T cells, and this KD resulted in a significant reduction in the level and frequency of cells expressing Gzm B, which still eff The KD of CD38 was incomplete, which may explain the 50% reduction in Gzm B. Therefore, this suggests that CD38 is a key regulator of T frat This indicates that it is essential for the accumulation of Gzm B in cells.

[0072] CD38 is an ectoenzyme that utilizes NAD as a substrate ligand, and NAD is required for the activity of SIRT1, a histone deacetylase that leads to the acetylation and activation of FOXO1. Activated FOXO1 then downregulates the production of Gzm B directly or via TCF7. Thus, T frat We hypothesized that high CD38 expression on T cells leads to NAD depletion, resulting in decreased SIRT1 activity, FOXO1, and TCF7 levels, and therefore enhanced Gzm B expression. frat The expression and activity levels of these molecules in T eff As shown in Figure 5b, T frat Cells were incubated at 0 hours and T eff Interestingly, NAD levels are reduced in T cells compared to T cells. frat In cells, it is further reduced, while in T eff In T cells, it was increased after 24 hours of stimulation with the relevant antigen (Fig. 5b). frat CD38-mediated NAD depletion in T cells eff This resulted in a decrease in SIRT1 activity without any changes in protein levels (Fig. 5c) or protein and RNA levels of FOXO1 and TCF7 (Fig. 5d, e) compared to T cells. To further support the link between this pathway and Gzm B accumulation, we investigated the effect of T frat Replenishing NAD or activating SIRT1 in T. truncatulae using the specific activator SRT1720 HCl resulted in a decrease in Gzm B expression (Fig. 5f, g). frat We show that CD38 in cells mediates the high expression of Gzm B by depleting NAD and then downregulating the SIRT1-FOXO1-TCF7 axis (Fig. 4m).

[0073] T frat is induced in autoimmune diseases and exhibits an immunoprotective role. T fratis generated by SOP, and autoimmune diseases (AIDs) are associated with the induction of T cells against suboptimal self-antigens, so T frat It was hypothesized that PD1 could be induced in AID. + CD38 hi The levels of CD8 T cells were examined in a mouse model of experimental autoimmune encephalomyelitis (EAE) (Fig. 10a). Interestingly, EAE mice had significantly higher levels of PD1 T cells compared to WT mice. + CD38 hi We observed an approximately three-fold increase in CD8 T cells (Fig. 6a). These cells were obtained from T cells obtained from the spleens of EAE mice. eff These PD1 cells express higher levels of Gzm B compared to PD1 cells (Fig. 6b). + CD38 hi CD8 T cells are actually T frat To demonstrate that pMel-1 is a PD1 receptor, we tested its ability to induce a contact-dependent fratricidal effect on target CD8 T cells. + CD38 hi CD8 T cells were isolated from EAE mice 6 days after disease induction and co-cultured with pMel-1 target CD8 T cells to test their fratricidal potential. Interestingly, these cells were indeed fratricidal and induced target CD8 T cell killing in a CD38-dependent manner, as killing was reversed by CD38 blockade (Fig. 6c). Similarly, high levels of PD1 + CD38 hi CD8 T cells were also found in SLE mice with severe disease (Sle1Tg7 and Sle1TLR9KO) compared to mice with mild SLE disease (Sle1) (FIG. 10b).

[0074] To support our findings in human AID, we analyzed PD1 expression in three cohorts of AID patients, one cohort of multiple sclerosis (MS), and two cohorts of systemic lupus erythematosus (SLE). + CD38 hi The number of CD8 T cells (gating strategy shown in Figure 10c) was analyzed. Patients with any of these AIDs had significantly higher PD1 T cell counts compared to healthy individuals. +CD38 hi There were high numbers of CD8 T cells (Fig. 6d-f). As expected, these cells expressed PD1 T cells from patients with SLE (Fig. 6g) and MS (Fig. 6h). + CD38 hi CD8 T cell subpopulation, T eff These cells have higher amounts of Gzm B compared to T cells. frat To determine whether PD1 act as a PD-1 receptor agonist, we isolated PD1 receptor agonists from PBMCs of SLE patients. + CD38 hi The ability of CD8 T cells to induce contact-dependent killing in autologous CD4 T cells as targets was tested. + CD38 hi When mixed with CD8 T cells, we found a significant increase in apoptosis of autologous CD4 T cells, as measured by Annexin V binding, accompanied by a significant decrease in their viability (Fig. 6i and Fig. 10d). We also found that this killing was mediated by CD38, as its blockade with anti-CD38 significantly reduced the killing of autologous CD4 T cells (Fig. 6i and Fig. 10d). The above data support the conclusion that these PD1 induced in AID patients + CD38 hi CD8 T cells actually frat , demonstrating that this may explain the lymphopenia in these patients.

[0075] Second, the studies described herein demonstrated that these cells play an immunosuppressive role, suggesting that T cells induced under AID conditions frat It was hypothesized that T may play an immunoprotective role in AID. To test this hypothesis, T was administered to mice with EAE. frat We examined whether infusion of T cells inhibited the autoimmune response and the outcome of EAE (Fig. 6j). Interestingly, T cells prepared in vitro as described above frat Injection of AID significantly reduced the clinical score and increased the survival rate of EAE mice (Fig. 6k). This suppression of AID was comparable to that exhibited by Tregs (Fig. 10e). Second, this suppression of AID was associated with the suppression of activated T cells, T fratTo examine whether cell-mediated killing was mediated by EAE in untreated EAE mice and T frat T in various tissues from EAE mice injected with cells frat The numbers of total CD4, and MOG-specific CD4 T cells were examined.

[0076] Studies have shown that T frat Importantly, both the total number of CD4 T cells and antigen-specific (MOG) CD4 T cells increased significantly (Fig. 6l, m). frat The spleen of EAE mice receiving cells, as well as the target tissues of the brain and mesenteric lymph nodes, were significantly reduced compared to untreated EAE mice (Fig. 6l-n). fratが It clearly demonstrates an inhibitory role on the immune response and reverses AID.

[0077] Patients with advanced COVID-19 are frat Large numbers of PD1 act as PD-1 receptors and kill autologous T cells + CD38 hi Has CD8 T cells. Patients with advanced COVID-19 have high numbers of circulating PD1 + CD38 hi CD8 + It has been reported that up to 30-60% of all CD8 T cells in peripheral blood are CD8 T cells.

[0078] Patients with advanced COVID-19 (n=11) had significantly higher numbers of PD1 in their blood compared to convalescent patients (n=5) or healthy individuals (n=11). + CD38 hi CD8 + We found that PD1 T cells were present (Figures 7a and 10f). To investigate this further, we examined public single-cell RNA sequencing data from bronchoalveolar lavage fluid (BALF) samples from COVID-19 patients. Indeed, PD1 T cells were significantly more abundant in BALF samples from COVID-19 patients with severe / critical disease compared to those with moderate disease. + CD38 hiIncreased numbers of CD8 T cells were found, which were not observed in healthy individuals (Fig. 7b). + CD38 hi CD8 + T cells are T frat To confirm the phenotypic similarity between these two groups, we performed subsetting and reclustering of BALF lymphocytes from previously reported data. Unbiased clustering demonstrated that CD38 is a signature marker for the three CD8 clusters 1, 2, and 3. Further subsetting based on CD38- and PDCD1-expressing CD8 T cells defined these cells as indeed belonging to the same clusters 1, 2, or 3 (cluster 10 was represented by only one patient) (Fig. 7b). As described above, T frat and T eff When cells were colored based on the expression of the signature gene set of T, cluster 2 was frat cluster 3 is most transcriptionally similar to T eff Therefore, COVID-19 patients were found to have the most similarity to mouse T frat PD1 with a similar gene signature to cells + CD38 hi Based on the findings of high numbers of CD8 T cells, PD1 in COVID-19 patients + CD38 hi CD8 + T cells are T frat This may also explain the severe lymphopenia observed in these patients. To demonstrate this, we used PD1 antibodies obtained from patients with severe COVID-19. + CD38 hi and T eff Gzm B levels were measured in the CD8 subpopulation, and PD1 + CD38 hi CD8 T cells are eff We found that these cells had higher levels of Gzm B compared to the patients' own CD4 T cells (Fig. 7d). We then determined whether these cells had the ability to kill the patients' own lymphocytes. Therefore, we investigated the effect of PD1 on the patients' own CD4 T cells.+ CD38 hi The killing effect of CD8 T cells was examined. + CD38 hi We found that incubation of CD8 T cells resulted in the induction of apoptosis in autologous target CD4 T cells, as measured by Annexin V staining in host target lymphocytes (Fig. 7e). Thus, these significantly higher numbers of PD1 T cells in severe COVID-19 patients may be due to the increased expression of PD1 T cells. + CD38 hi CD8 + T cells are T frat cells, which may provide a plausible explanation for the lymphopenia observed in these patients.

[0079] Bradykinin inhibits PD-1 in a CREB-1-dependent manner + CD38 hi CD8 + Induce cells Patients with advanced COVID-19 and acute respiratory distress syndrome (ARDS) are characterized by severe lymphopenia and thrombosis of unknown etiology. Due to lymphopenia, the proportions of T cells, innate lymphoid cells, and natural killer (NK) cells are significantly lower in severe cases compared with healthy donors. Furthermore, COVID-19 patients are characterized by a strong pro-inflammatory environment in multiple tissues. One of the pro-inflammatory factors found to be upregulated in these patients is bradykinin (BDK), also known as BDK storm. BDK is a potent vasopressor that induces hypotension and vasodilation, and is enhanced by angiotensin produced by angiotensin-converting enzyme 2 (ACE2), the entry point for the SARS-CoV-2 virus. Interestingly, BDK potently activates cAMP response element-binding protein (CREB-1), which subsequently upregulates CD38 upon binding to the CD38 promoter. Based on these observations, BDKstorm appears to be a potential target for PD1 + CD38 hi CD8 +This may result in an increase in the number of T cells, and these cells may be one of the contributing factors to the pathophysiological state observed in COVID-19 patients and any ARDS-related state. To test this hypothesis, we treated either human or mouse CD8 T cells with various concentrations of recombinant BDK. Interestingly, this treatment increased the number of PD1+CD38 T cells. hi This resulted in the generation of CD8 T cells (Figure 11A). To further understand the BDK-mediated molecular mechanism of induction of these cells, the levels of phospho-CREB (p-CREB), a transcription factor with a binding element in the CD38 gene, were estimated in BDK-treated CD8 T cells. BDK signaling increased p-CREB expression (Figure 11B). + CD38 hi CD8 + This induction of T cells was inhibited when either the BDK receptor or CREB was knocked down using specific siRNA (Figure 11C). Thus, these data suggest that BDK mediates the activation of PD1. + CD38 hi CD8 + This shows that T cells are induced in a p-CREB-dependent manner.

[0080] BDK inhibits PD1 under in vitro conditions + CD38 hi CD8 + Because BDK mediates the induction of T cells, it can induce these cells in the lungs of mice (or under in vivo conditions), leading to ARDS (and its maintenance). Indeed, administration of BDK to wild-type mice significantly increased the number of these killer cells in the lungs (Fig. 1D), which decreased after CD38 depletion (Fig. 11E). Notably, this PD1 + CD38 hi CD8 +The increase in T cell numbers was accompanied by severe lung pathology in BDK-treated mice. While control tissue showed plain lung parenchyma with intact alveolar spaces, lungs from BDK-treated mice showed loss of open alveolar spaces with focal alveolar wall thickening consistent with alveolar damage (AD) (Figure 11F). Septa were thickened with a dense interstitial mixed inflammatory infiltrate consistent with interstitial pneumonia. Some alveolar walls showed focal pink material suggestive of hyaline deposition. Overall, these findings are consistent with features encountered in ARDS (proliferative phase).

[0081] PD1 + CD38 hi CD8 + T cells kill other target cells in a CD31-dependent manner PD1 + CD38 hi CD8 + Because T cells kill effector T cells via CD38:CD31 interactions, we next assessed whether these cells could kill other target cells that express CD31, such as myeloid cells (dendritic cells (DCs), macrophages), natural killer (NK), and endothelial cells (EC). + CD38 hi CD8 + We first estimated killing in DCs, macrophages, and NK cells derived from the spleen of WT mice treated with intravenous injection of PD1 cells (Figure 2A-B). + CD38 hi CD8 + Myeloid populations isolated from mice receiving PD1 T cells exhibited higher apoptosis compared to control mice (Figure 2C-E). + CD38 hi CD8 + We estimated the subsequent killing of ECs by T cells. C166 mice, derived from an EC line that highly expresses CD31 (Fig. 12F), showed increased apoptosis when incubated with killer cells (Fig. 12G). Finally, PD1 T cells derived from COVID-19 patients were used to investigate the role of ECs in the killing of ECs. + CD38 hi CD8 +We performed studies to determine whether T cells can also kill ECs. To this end, we used human umbilical vein endothelial cells (HUVECs) and indeed found that enhanced killing of these cells was mediated by PD1 T cells derived from patients with advanced COVID-19. + CD38 hi CD8 + These results were derived from PD1 T cells (Fig. 12H). + CD38 hi CD8 + We show that T cells kill myeloid and endothelial cells, providing a plausible explanation for the thrombosis observed in COVID-19 patients and ARDS.

[0082] Human samples and processing PBMC samples from two cohorts of SLE patients and healthy donors (n = 12) and 14 were collected at NCI (patients, n = 12; healthy donors, n = 14) and Singapore (patients, n = 35; healthy donors, n = 25). PBMC samples from MS patients (n = 14) and five healthy donors were obtained from the Center of Multiple Sclerosis and Autoimmune Neurology at Mayo Clinic, Rochester, Minnesota. PBMC samples (collected during the acute phase of the disease) from COVID-19 patients (convalescent (n = 5) and severe (n = 10)) were collected at Providence Portland Medical Center (PPMC). Frozen PBMCs from COVID-19 patients were shipped on dry ice to Georgetown University, where experiments were performed on SLE and MS patients at the respective facilities. PBMCs from healthy donors (n = 8) were also purchased from Hemacare BioResearch Products & Services (Los Angeles, CA) (catalog number M009C-2) for comparison with COVID-19 patient samples. PBMCs were thawed and stained with the LIVE / DEAD Fixable Near-Infrared Dead Cell Stain Kit (Invitrogen, Waltham, MA; catalog number L10119), followed by immunofluorescence of the following surface markers: CD8, PD-1, and CD38. 1Cells were stained with a cocktail of antibodies against PD-1 and PD-2 at a concentration of 1:200. Cells were fixed and permeabilized in fixation buffer and then stained with appropriately labeled Gzm B antibodies (1:100). Control staining was performed on each sample using an isotype control antibody to determine marker positivity. Stained cells were acquired on a BD LSR Fortessa (Franklin Lakes, NJ). Acquired samples were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR). For the coculture assay, PBMCs from the NCI SLE patient cohort and severe COVID-19 patients were stained with the above cocktail of antibodies and then analyzed for PD-1 and PD-2 in separate tubes on a FACS Aria™ III (BD Biosciences). + CD38 hi CD8 T cells, T eff , and FACS sorting of CD4 T cells was performed. Co-culture experiments were performed using the sorted cell populations as detailed below.

[0083] The study was conducted in accordance with the protocol, Good Clinical Practice, and the Declaration of Helsinki. The protocol and all amendments were approved by the appropriate institutional review board or ethics committee at each institution. All patients provided written informed consent.

[0084] mouse C57BL / 6J (B6) and Rag1 - / - Female mice (4-6 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME) or Charles River Laboratories (Charleston, SC). 2 Autologous pMel-1 mice ((B6.Cg-Thy1a / Cy Tg(TcraTcrb)8Rest / J) harboring a rearranged TCR transgene (Vβ13) specific for the murine homolog of β13 (pmel-17), expressing a transgenic TCR specific for ovalbumin residues 257-264 in the context of H-2Kb, express a CD8 +OT I mice (C57BL / 6-Tg(TcraTcrb)1100Mjb / Crl) harboring a transgenic TCR specific for ovalbumin residues 323–339 on T cells and CD4 in the context of I-Ab. + OT-II mice (B6.Cg-Tg(TcraTcrb)425Cbn / J) bearing T cell-positive mice were used as outlined in various experiments. In addition, B6 mice bearing lupus-susceptible regions, Sle1 (B6.Sle1; defined by microsatellite markers D1Mit17, D1Mit113, and D1Mit202), TLR9-deficient Sle1 mice (B6.Sle1TLR9KO), and conditional BAC Tg7 mice (Sle1Tg7) were bred at the Biological Resource Centre (Singapore). The derivation and generation of these mice have been previously described. 3~6 All mice were maintained under specific pathogen-free conditions. All procedures were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) animal protocol approved by Georgetown University and an A*STAR IACUC-approved protocol (#161176) that conforms to NIH guidelines.

[0085] vaccine Various cell types were activated with their respective cognate peptides. gp100 was purchased from AnaSpec. 25~33 The nonamer peptide (KVPRNQDWL (SEQ ID NO: 4)) was injected onto magnetically enriched CD8 + Used for in vitro activation of T cells 7、8 On the other hand, CD8 cells derived from OT I mice + T cells respond to OVA 257~264 (SIINFEKL (SEQ ID NO: 1); Catalog No.: AS-60193-1; AnaSpec Inc.), or OVA 257~264 , also known as OVA-V (SIIGFEKL (SEQ ID NO: 1); Cat. No.: AS-64384; AnaSpec Inc.). 2Target CD4 from OTII mice + T cells were stimulated with H-2b-restricted Ova class II epitopes (OVA) 323~339 (ISQAVHAAHAEINEAGR (SEQ ID NO: 3); Catalog No. AS-27024; AnaSpec Inc., Fremont, CA). The purity of the enriched cells was >90%.

[0086] Antibodies and reagents Fluorescently labeled anti-mouse antibodies used for flow cytometry were obtained from BD Biosciences, eBioscience (San Diego, CA), BioLegend (San Diego, CA), and ThermoFisher Scientific. Antibodies used for Western blotting were obtained from Cell Signaling Technology (Danvers, MA). Antibodies used for cell activation included anti-mouse CD3 (5 μg ml -1 , clone 145-2C11, catalog number 553057, BD Biosciences), and CD28 (2.5 μg ml -1 The CD38 and CD31 blocking antibodies used were CD38 (clone 90, Rat IgG2a, ThermoFisher Inc.) and anti-mouse CD31 (clone 390, reference number 16-0311-85, eBiosciences), as well as isotype control antibodies (Rat IgG2a, K for anti-CD38 and anti-CD31). 35~55 / IA b Tetramer was obtained from MBL International Corp. (Woburn, Mass.).

[0087] CD8 + The enrichment kit (Miltenyi Biotec, Germany) was used according to the manufacturer's instructions. The LIVE / DEAD Fixable Near-Infrared Dead Cell Stain Kit (catalog number L34976) was obtained from ThermoFisher Scientific.

[0088] Recombinant CD31 (rCD31, 3628-PC-050, R&D), Gzm B inhibitor (Z-AAD-CMK), PI3K inhibitor, ERK1 / 2 inhibitor (SCH772984), and SIRT1 activator (SRT1720HCl) were obtained from Selleckchem (Houston, TX). NAD levels were measured using an NAD / NADH quantification kit (catalog number MAK037, Sigma-Aldrich, St. Louis, MO), and SIRT1 activity was measured using a SIRT1 activity assay kit (fluorimetric, catalog number ab156065, Abcam, Waltham, MA). Primers and reagents for RT-PCR were purchased from Applied Biosystems (Foster City, CA). Myelin oligodendrocyte glycoprotein (MOG) was also measured. 35~55 , catalog no. EK-2110), complete Freund's adjuvant (CFA, catalog no. F588, Sigma), pertussis toxin (PTX, catalog no. BT-0105), and methylated bovine serum albumin (mBSA, catalog no. DS0162, EK-0133) were from Hooke Laboratories (Lawrence, MA).

[0089] For metabolomics and lipidomics, all liquid chromatography-mass spectrometry (LC-MS) grade solvents, including acetonitrile and water, were purchased from Fisher Optima grade (Fisher Scientific). High-purity formic acid (99%) was purchased from Thermo-Scientific. Debrisoquine and 4-nitrobenzoic acid were purchased from Sigma-Aldrich. EquiSPLASH® LIPIDOMIX® quantitative mass spectrometry internal standards, as well as 15:0-18:1-d7-PA, C15 ceramide-d7 (d18:1-d7 / 15:0), and 18:1 Chol (D7) ester, were purchased from Avanti polar lipids. Internal standards for free fatty acids (FFA), dihydroceramide (DCER), hexosylceramide (HCER), and lactosylceramide (LCER) were purchased from Sciex (Toronto, CA) as Lipidyzer platform kits.

[0090] siRNA for CD38 and CD31 For knockdown of CD38 and CD31, OTI (killer T cells) were cultured in 1% FBS medium. frat cells) and pMel-1 CD8 + T cells (target cells) were incubated overnight with CD38 (catalog no. 4390771) and CD31 (catalog no. AM16708) siRNA (10 μM; ThermoFisher Scientific). The siRNA was prepared using Lipofectamine® RNAiMAX reagent and OPTI-MEM® (both from ThermoFisher Scientific) according to the manufacturer's recommendations. The next day, medium containing Ova, Ova-V, or gp100 (target cells, 1 μM) was added to the wells for cell activation. After 48 hours of incubation at 37°C, the cells were harvested.

[0091] EAE mouse model Experimental autoimmune encephalomyelitis (EAE) model in mice was performed using a previously reported method. 9、10In short, MOG 35~55 (200 μg / mouse) was mixed with an equal volume of complete Freund's adjuvant (CFA). B6 mice were inoculated subcutaneously with a total volume of 100 μl / mouse on day 0. Pertussis toxin (PTX) 400 ng / 200 μl / mouse was injected intraperitoneally on days 0 and 2. All animals were randomly assigned to each experimental group. Mice were observed daily for any signs of distress. PD1 expression in EAE mice compared to WT untreated mice + CD38 hi To measure the number of CD8 T cells, spleens were harvested from these mice on day 8 after EAE induction. CD8 T cells were stained for PD1, CD38, and Gzm B and measured by flow cytometry. + CD38 hi To examine the fratricidal properties of CD8 T cells, FACS-sorted PD1 T cells derived from splenocytes of EAE mice on day 6 after disease induction were used, as detailed below. + CD38 hi or T eff Co-culture experiments were performed using CD8 T cells.

[0092] For ACT experiments, mice were injected with 1 million FACS-sorted PD1 cells on days 8 and 10 after EAE induction. + CD38 hi CD8 T cells or FoxP3 + CD4 regulatory (Treg) cells were administered. To this end, Tregs were generated from MACS-sorted CD4 T cells derived from B6 mouse splenocytes activated for 72 hours in T cell medium containing IL-2 (100 IU / mL), plate-bound anti-CD3 (5 μg / mL), and soluble anti-CD28 (2.5 μg / mL) antibodies, as well as TGF-β (2.5 ng / mL). Induced EAE symptoms were graded as previously reported. 9、11 For immune response experiments, mice were sacrificed 2 days after the second cell injection, and spleen, mesenteric lymph node, and brain tissues were harvested. Single cell suspensions were prepared and analyzed by flow cytometry for various markers (CD8, CD4, PD1, CD38, and MOG). 35~55 ) were processed to estimate

[0093] SLE mouse model Single cell suspensions from the spleens of aged mice (6–9 months old) were prepared as previously described. 12 Splenocytes were resuspended in PBS containing 1% fetal bovine serum (staining buffer) and then 20% 2.4G2 hybridoma supernatant was used to block nonspecific Fc binding. They were incubated with a master mix of antibodies to capture CD45+ leukocytes, CD3+CD4+ and CD8+ T cells, and CD38 and PD1 populations, along with LIVE / DEAD fixable dye (Biolegend), on ice for 30 minutes. For intracellular TLR7 staining, the BD Cytofix / Cytoperm kit was used according to the manufacturer's instructions. Samples were analyzed using a BD FACSymphony A5.2 with Flowjo 10.9.0. Antibodies were purchased from BD Biosciences, ThermoFisher Scientific, and Biolegend.

[0094] Tumor implantation and treatment of mice The TC-1 tumor cell line, generated from lung epithelial cells immortalized with HPV16 E6 and E7 and the h-ras oncogene, was kindly provided by Dr. TC Wu, Johns Hopkins University. 13 Cell lines were routinely tested for the absence of any mycoplasma contamination by microscopic evaluation and PCR-based methods. Cells were cultured and tumors were implanted into B6 mice as previously reported. 7、8 To evaluate the expression of cytokines and cytolytic molecules, tumors were cultured at a volume of 1.5 cm after implantation (18-20 days). 3 Tumors were harvested from untreated mice at the time point. Samples were processed using the gentleMACS separator and solid tumor homogenization protocol as recommended by the manufacturer (Miltenyi Biotec). Single-cell homogenates from tumor samples were stained with the appropriate antibodies described above, and the expression of various markers was examined by flow cytometry.

[0095] Cellular activation, treatment, and recall responses Primary mouse CD8 + T cells and human CD8 T cells were isolated by fluorescence-activated cell sorting (FACS) or, in some cases, by negative selection using magnetic beads (Miltenyi Biotec) and cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 10 mM HEPES, and 55 μM β-mercaptoethanol. The purity of all cell populations was greater than 95%.

[0096] PD1 + CD38 hi and T eff CD8 T cells were generated by a method previously reported by the inventors. 2 Briefly, MACS-purified (>90%) OT1-CD8 (CD45.1) T cells from mouse spleens were activated with either low-affinity OVA-V peptide or high-affinity OVA peptide (1 μM each) for 48 h in T cell medium supplemented with 30 international units (IU) of IL-2, followed by PD1 activation, respectively. + CD38 hi and PD1 + CD38 lo T cells were purified by FACS. Various cell surface and intracellular markers, as well as levels of pro- and anti-inflammatory cytokines and cytolytic molecules, were analyzed by flow cytometry, as described in the next section. In some experiments, CD38 was knockdown using siRNA for 24 hours before various marker analysis. Additionally, in some experiments, either NAD (1 mM) or SIRT1 activators (1 μM or 2 μM) were added to T cells. frat The cells were added for 24 hours, and these cells and T eff The levels of Gzm B in T cells were measured by flow cytometry. To determine the role of CD38:CD31 interactions and signaling in Gzm B degranulation, T frat Cells were cultured with rCD31 (1 or 5 μg) with or without PI3K or ERK1 / 2 inhibitors (100 nM). After 24 hours, T fratDegranulation of Gzm B in cells was measured by CD107α staining, and the levels of various signaling molecules were measured by flow cytometry.

[0097] To estimate antigen recall responses, FACS-sorted T eff and PD1 + CD38 hi Cells were re-challenged overnight with the respective peptide (OVA or OVA-V), and the expression of IFN-γ, CD40L, and CD69 was estimated by flow cytometry. In some experiments, CD38 was expressed as PD1 + CD38 hi After 24 hours of KD in CD8 T cells, various markers were analyzed.

[0098] For BK studies, mouse spleen-derived CD8 T cells were activated with high-affinity OVA peptides (1 μM each), and human-derived CD8 T cells were incubated with anti-human CD3 (5 μg ml ) in T cell medium (RPMI 1640 medium, 10% FBS, 2 mM glutamine, 10 mM HEPES, and 55 μM β-mercaptoethanol) supplemented with 30 international units (IU) of IL-2. -1 , clone OKT3, catalog number 317302; BioLegend) and CD28 (2.5 μg ml -1 In some groups, recombinant BK was added at various concentrations (mouse: 50 nM–1000 nM, human: 3–15 μM) for 48 hours, followed by PD1 activation. + CD38 hi CD8 T cells were analyzed by FACS.

[0099] Flow cytometry analysis Flow cytometric analysis of lymphocytes of mouse and human origin yields 1–2 × 10 per sample. 6Cells were stained with the LIVE / DEAD Fixable Near-Infrared Dead Cell Stain Kit (Invitrogen; Catalog No. L10119) followed by fixation and permeabilization. All surface and intracellular markers were simultaneously stained in fixation and permeabilization buffer (per buffer) according to the manufacturer's recommendations. For IFN-γ and Gzm B staining, the BD Biosciences Cytofix / Cytoperm (Cat. No. 51-2090KZ) and BD Biosciences Perm / Wash (Cat. No. 51-2091KZ) buffer sets were used according to the manufacturer's instructions. In some experiments, variously treated cells were processed for Annexin V staining to examine the degree of apoptosis. For this, harvested cells were stained for Annexin V in Annexin Binding Buffer (ABB) (BD Biosciences) for 30 minutes at 4°C. After staining, samples were washed once in ABB and finally suspended, and FACS was acquired in ABB, as previously reported. 14 For caspase 3 / 7 staining, a Green Flow Cytometry Assay Kit (Thermo Fisher catalog number C10427) was used according to the manufacturer's recommendations.

[0100] MitoFM, a green fluorescent mitochondrial dye that localizes to mitochondria, and TMRM, a cell-permeant cationic red-orange fluorescent dye that is readily sequestered by active mitochondria and has been used to estimate mitochondrial potential, were used as previously described. 15 Glucose uptake assay was performed using 2-NBDG 8 All reagents were used according to the manufacturer's instructions. In some experiments, PD1 + CD38 hi and T eff Lipid profiling of CD8 T cells was performed by BODIPY uptake 8 For this purpose, PD1 + CD38 hi and T effCD8 T cells were activated with Ova-V and Ova, respectively, for 48 hours, followed by surface staining as described above. Cells were then washed and lysed with 0.5 μg ml Ova in PBS. -1 The cells were stained for 15 minutes at 20°C, then washed and finally suspended in PBS. + CD38 hi and T eff CD8 T cells were similarly stained for DCFDA and analyzed by flow cytometry.

[0101] Data acquisition was performed on a FACS Calibur or LSR Fortessa platform (BD Biosciences), and results were analyzed with FlowJo software.

[0102] Co-culture (in vitro cell killing) experiments PD1 + CD38 hi and T eff CD8 T cells were cultured in a 10 mL aliquot of gp100 peptide (10 per mL). 6 FACS-sorted Thy1.1 target pMel-1 CD8 activated with 1 μM per cell + The cells were co-cultured overnight with T cells (killer-to-target cell ratio 1:1). To examine contact-dependent killing, a separation membrane (Corning Costar) was used, and PD1 + CD38 hi CD8 T cells were placed in the upper chamber on the membrane and treated with pMel-1CD8 + Target T cells were placed in the lower chamber. After overnight incubation, cells were harvested and apoptosis in target cells was estimated by Annexin V staining. In some experiments, T cells were incubated for 24 hours. fratApoptosis was measured by Annexin V staining after blocking or knockdown of CD38 with anti-CD38 in target pMel-1 CD8 T cells and blocking or knockdown of CD31 with anti-CD31 in target pMel-1 CD8 T cells. In some experiments, Gzm Bi was added to the co-culture for 24 hours, followed by Annexin V staining. In addition to Annexin V staining, the levels of Gzm B and T in target pMel-1 CD8 or CD4 T cells after co-culture with or without anti-CD38 / CD31 were also measured. frat The levels of CD107α in cells were measured by flow cytometry. In vitro cell killing experiments were also performed using PD1 + CD38 hi This was performed after co-culture of CD8 T cells with target CD8 T cells obtained from wild-type B6 mice. Target cell viability was assessed by FACS analysis using fixable Live / Dead staining.

[0103] FACS-sorted PD1 from EAE mice + CD38 hi or T eff CD8 T cells were co-cultured overnight with target pMel-1 CD8 T cells. In some experiments, anti-CD38 was added to T cells during co-culture with target CD8 T cells. frat Similarly, FACS-sorted PD1 cells isolated from PBMCs of SLE and COVID-19 patients were added to the cells. + CD38 hi or T eff CD8 T cells were co-cultured with autologous target CD4 T cells with or without anti-CD38. Apoptosis induction by Annexin V staining and cell viability by live / dead staining were measured in target T cells by flow cytometry.

[0104] PD1 + CD38 hiTo examine endothelial cell killing by CD8 T cells, we used C166, a murine endothelial cell line that expresses high levels of CD31. First, CD31 expression on these cells was determined by flow cytometry using anti-mouse CD31 (clone JC / 70A; catalog: MA5-13188; eBiosciences) along with an isotype control antibody (mouse IgG1a, K, MA1-10406, eBiosciences). Next, we investigated the killing of PD1 T cells by flow cytometry. + CD38 hi CD8 T cells were co-cultured with these cells, and apoptosis was determined by flow cytometry in target endothelial cells by caspase 3 / 7 staining using the Green Flow Cytometry Assay Kit (Thermo Fisher catalog number C10427) according to the manufacturer's recommendations. Additionally, FACS-sorted PD1 cells isolated from PBMCs of COVID-19 patients were analyzed. + CD38 hi CD8 T cells were co-cultured with target human umbilical vein endothelial cells (HUVEC) for 24 hours, followed by measurement of apoptosis by Annexin V staining in the target endothelial cells by flow cytometry.

[0105] Adoptive cell transfer and in vivo killing experiments gp-100 (1 μM)-activated pMel-1 CD8 T cells or Ova-activated (1 μM) OTII-CD4 target T cells were treated with Rag1 - / - Mice were intravenously injected with OT1 mice (T1 mice) generated as described above. One day later, frat )-derived PD1 + CD38 hi CD8 T cells were transferred intravenously into these mice. Forty-eight hours later, spleens were harvested and the level of apoptosis in target cells was estimated by Annexin V staining of CD90.1CD8 or CD4 target T cells.

[0106] In another series of experiments, PD1 generated as described above + CD38 hiCD8 T cells were intravenously transferred into C57BL / 6J (B6) mice. 48 hours later, spleens were harvested, and the levels of apoptosis in target cells expressing CD31 (dendritic cells (DCs), macrophages CD11b+, F4 / 80+), natural killer (NK1.1), and endothelial cells (ECs) were estimated by Annexin V staining.

[0107] PD1 in vivo + CD38 hi BK-mediated induction of CD8 T cells BK inhibits PD1 under in vivo conditions + CD38 hi To investigate whether BK mediates the induction of CD8 T cells, C57BL / 6J (B6) mice were intravenously injected with BK (Sigma-Aldrich, 60 mg / kg, once daily) for 3 days. 48 hours later, the spleens were harvested, and CD8 T cells were stained for PD1 and CD38 and measured by flow cytometry. In a separate experiment, these mice were intraperitoneally administered two doses of anti-CD38 (100 μg / mouse) followed by intravenous injection of BK (60 mg / kg, once daily) for 3 days. 48 hours later, the mice were sacrificed, and their spleens and lungs were harvested for pathological evaluation. Furthermore, single-cell suspensions were prepared and processed for estimation of different markers (CD8, CD4, PD1, and CD38) and measured by flow cytometry.

[0108] RNA-seq analysis CD8 T cells were treated with IL-2 alone or activated with Ova-V or Ova peptide for 48 hours. frat and T effCells were FACS-sorted, and total RNA was extracted using TRIzol reagent (Invitrogen) and lysed in RNase-free water, followed by RNA sequencing. RNA sequencing was performed by Maryland Genomics, Institute for Genome Sciences, UMSOM. Paired-end Illumina libraries were mapped to the mouse reference, Ensembl release GRCm38.102, using HiSat2 v2.1.0 with default mismatch parameters. Read counts for each annotated gene were calculated using HTSeq. The DESeq2 Bioconductor package (v1.5.24) was used to estimate variance, normalize read counts by library size, generate counts per million for each gene, and determine differentially expressed genes between different cell types.

[0109] RNA-seq and pathway analysis: After filtering low-expressing genes and normalizing counts per million using the DESeq2 package, differential gene expression was performed using the Limma package (v3.54.2) against paired controls between sample conditions under a hierarchical testing scheme with an FDR < 0.001. Pathway enrichment analysis using the clusterProfiler package (v4.6.2) considered genes with an adjusted P value < 0.001 and a log fold change > 1.2, evaluated against pathways retrieved from the GO Biological Processes annotation set (https: / / geneontology.org) with an FDR < 0.01. After filtering out genes upregulated across all conditions, PD1 + CD38 hi CD8 +The T cell signature was considered to be all DE upregulated genes specific to the OVAV:OVA control and the upregulated genes OVAV:OVA ∩ OVAV:IL2. The appropriate activation signature was derived by capturing the upregulated genes specific to the OVA:IL2 control and the upregulated genes OVA:IL2:OVAV:IL2, since these genes were not upregulated in the OVAV:OVA condition.

[0110] Single-cell RNA-seq analysis from COVID-19 patients Using publicly available single-cell RNA sequencing data from bronchoalveolar lavage fluid (BALF) of COVID-19 patients 18 The integration and reclustering of samples on T cells was achieved using the code provided in the publication. CD8 T cell subsetting was achieved by obtaining cells with CD8A, CD8B, and CD3E expression above 0. T frat Cells were similarly subsetted with the addition of CD38 and PDCD1 genes. Plotting and subsetting functions were accomplished using the Seurat package or ggPlot2. 19~23 .

[0111] ATAC sequence analysis ATAC Sequencing Sample Preparation: Samples for ATAC sequencing were prepared in parallel with the preparation of samples for RNA sequencing. ATAC sequence analysis was performed by Maryland Genomics, Institute for Genome Sciences, UMSOM. Paired-end Illumina libraries were mapped to the mouse reference, Ensembl release GRCm38.102, using HiSat2 v2.1.0 with default mismatch parameters. 24 Peak calling was performed using MACS v2. 25 Peak areas with significant MACS p-values ​​(FDR<0.001) were retained. Differential accessibility was performed using the DiffBind v3 R package. 26Differentially accessible regions with an FDR ≤ 0.05 were used for downstream analysis. eff and PD1 + CD38 hi ATAC sequencing Tn5 nick site density in T cell promoters was analyzed to determine relative promoter accessibility in the two cell populations. This is represented by a plot of log ratio ("minus") versus mean expression ("MA"), showing the relative accessibility of macs2 peaks. Differences in peak accessibility were indicated by FDR < 0.05. Differentially open promoters with |log2FC| > 1.2 are shown by a volcano plot. Line-scaled promoter accessibility of peaks restricted to differentially regulated promoters with FDR < 0.05 and |log2FC| > 1.2 is shown by a heatmap with path terms with FDR < 0.05, and genes associated with these terms are indicated in the heatmap.

[0112] Metabolic assays FACS-sorted PD1 cells for metabolic characterization + CD38 hi CD8 + T cells and T eff Cells were subjected to a mitochondrial stress test (SeaHorse Bioscience) performed according to the manufacturer's specifications. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using an XFp flux analyzer (Seahorse Bioscience). For all assays, 160,000 cells per mL were plated in culture plates using Cell-Tak (BD Biosciences). OCR and ECAR were measured in unbuffered DMEM (Agilent Biotechnologies) supplemented with 10 mM D-glucose (Sigma-Aldrich), 10 mM L-glutamine, and 2.5 mM pyruvate as indicated. Spare respiratory capacity (SRC) was calculated according to a previously published method. 28 .

[0113] Metabolomics and Lipidomics Multiple reaction monitoring mass spectrometry (MRM) was used to quantify endogenous metabolites and lipids (21 classes of lipid molecules) using a triple quadrupole mass spectrometer (QTRAP 5500 LC-MS / MS System, SCIEX) operating in MRM mode. As previously reported, approximately 5 million PD1 molecules were detected in each group. + CD38 hi and T eff Cells were processed for deep metabolomics and lipidomics 8 .

[0114] Scanning electron microscope (SEM) Selected PD1 + CD38 hi and T effCells were fixed in 1% paraformaldehyde and 2.5% glutaraldehyde in 0.12 M sodium cacodylate buffer (pH 7.4). Fixed cells were embedded in 4% agarose and postfixed in 1% osmium tetroxide (OsO4) in 0.12 M sodium cacodylate buffer (in the dark) for 1 hour. Cells were then dehydrated through graded ethanol-propylene oxide (Embed-812; Electron Microscopy Sciences) and infiltrated for 90 min, 90 min, and overnight in 2:1, 1:1, and 1:2 propylene oxide / Epon mixtures (Embed-812; Electron Microscopy Sciences) respectively, and finally in 100% Epon overnight. The beam capsule was cured at 60°C for 48 hours before cutting. Ultrathin sections (120 nm) were cut using a Leica EM UC7 ultramicrotome (Leica Microsystems) equipped with a diamond knife. For SEM imaging in a Helios NanoLab 660 FIBSEM (ThermoFisher), sections were mounted in silicon wafers and carbon-taped to aluminum stubs. To maximize collection of backscattered electrons, we used a concentric detector in immersion mode with a working distance of 4 μm, landing at 2 kV and 0.10 nA. High-resolution tile images of each target cell were performed using 80,000x magnification (dwell time: 5 ms, resolution: 3072 × 2048) and a pixel size of 1.6862 (MAPS 3.22, ThermoFisher). Mitochondria were traced and manually counted on the images, and cristae morphology was examined and quantified using ImageJ V1.53a software according to the method by Lam et al. 29 .

[0115] Estimation of NAD levels and SIRT1 activity FACS-sorted T cells after 0 and 24 hours of culture in IL-2 frat and T eff The levels of NAD in the cell lysates of the cells were estimated using an NAD / NADH Quantitation Kit according to the manufacturer's instructions. SIRT1 activity in these cells was measured using FACS-sorted T frat and Teff The activity was measured on cells by fluorometry (360 / 460 nm fluorescence) using a SIRT1 activity assay kit.

[0116] Quantitative PCR using reverse transcription analysis T were FACS-sorted using TRIzol reagent (Invitrogen). frat and T eff Total RNA was extracted from cells and dissolved in RNase-free water. 1 μg of total RNA was subjected to single-stranded complementary DNA synthesis using the iScript cDNA Synthesis Kit (Bio-Rad). qrtPCR was performed using Applied Biosystems Taqman® Gene Expression Assays, and the gene expression of Foxo1 (Mm00490671_m1) and Tcf-7 (Mm00493445_m1) was detected using the Applied Biosystems primer pair (catalog no. 4331182). Expression data were obtained using the Applied Biosystems StepOnePlus Real-Time PCR System and normalized to the geometric mean of the Applied Biosystems™ Mouse Actb (actin, beta) Endogenous Control (catalog no. 4352933E).

[0117] Western blot FACS-sorted T frat and T eff The cells were activated as described above. + T cells and human CD8 +T cells were isolated and activated as described above, and in some groups, recombinant BK was added at various concentrations (mouse: 50 nM–1000 nM, human: 3–15 μM) for 48 hours. Cell lysates (RIPA buffer + 1% phosphatase inhibitor + 1% protease inhibitor) were prepared after various treatments. Protein concentrations in the cell lysates were determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Protein (30–40 μg) was loaded onto Novex 4–12% Tris-Glycin Mini Gels (ThermoFisher Scientific) and subsequently transferred to a nitrocellulose membrane. The membrane was blocked with 3% BSA in Tris buffer and subsequently probed overnight by Western blot using antibodies against SIRT1, Foxo1, TCF-1, phospho-CREB (Ser133) rabbit mAb (Cat. No. 9198), and CREB (Cat. No. 9197S). Blots were developed with the appropriate (anti-rabbit or anti-mouse) HRP-conjugated secondary antibody. Tubulin and lamin B levels were used as controls. Band density analysis was performed using LI-COR software (Image Studio Lite v.5.0) (https: / / www.licor.com / bio / image-studio-lite / ).

[0118] IF microscopy FACS-sorted PD1 generated above + CD38 hi CD8 T cells were treated with DAPI and Gzm B-specific staining agent according to the manufacturer's instructions. After washing away the free staining agent, the stained killer T cells were frat The cells were mixed with unstained target pMel-1 CD8 T cells at a 5:1 (killer:target) ratio and placed on CellTak-coated (22.5 μg / ml) slides. The transfer of Gzm B into target cells was followed in a time-dependent manner (5–300 min) using a Leica microscope (LEICA SP8).

[0119] statistical analysis All summary statistics (mean, SD, SEM, significant differences between groups) were calculated using GraphPad Prism v.6.0 or Excel, as appropriate. Statistical significance between groups was determined by unpaired one-tailed Student's t-test or one-way analysis of variance (ANOVA) (P ≤ 0.05 was considered statistically significant). Survival rates of various groups were compared using the log-rank (Mantel-Cox) test in GraphPad Prism.

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Claims

1. 1. A method for reducing apoptosis of cells in a subject, the method comprising reducing binding of CD38-expressing cells to CD31-expressing cells in the subject.

2. The method of claim 1, wherein the binding is reduced by administering an agent that reduces binding of CD38-expressing cells to CD31-expressing cells in the subject.

3. Binding is indicative of PD-1 in the subject. ＋ CD38 ｈｉ CD8 ＋ The method of claim 1, wherein the reduction is by reducing the number of T cells.

4. PD-1 ＋ CD38 ｈｉ CD8 ＋ The number of T cells (a) detecting ex vivo PD-1 in a biological sample derived from the subject; ＋ CD38 ｈｉ CD8 ＋ reducing the number of T cells; (b) administering to said subject the population of remaining cells.

5. The method according to any one of claims 1 to 4, wherein the CD31-expressing cells are leukocytes or endothelial cells.

6. The method of claim 5, wherein the CD31-expressing leukocytes are selected from the group consisting of T lymphocytes, dendritic cells, natural killer cells, and macrophages.

7. The method of claim 6, wherein the CD31-expressing leukocytes are T lymphocytes.

8. The method of claim 7, wherein the T lymphocytes are CD4+ T cells or CD8+ T cells.

9. The CD38-expressing cells are CD8 ＋ The method according to any one of claims 1 to 8, wherein the cells are T cells.

10. The CD8 ＋ T cells express PD-1 ＋ CD38 ｈｉ CD8 ＋ The method of claim 9, wherein the cell is a T cell.

11. The method according to any one of claims 2 to 10, wherein the agent is an antibody that specifically binds to CD31.

12. The method according to any one of claims 2 to 10, wherein the agent is an antibody that specifically binds to CD38.

13. The agent inhibits PD-1 in the subject. ＋ CD38 ｈｉ CD8 ＋ The method of any one of claims 2 to 12, wherein the level of T cells is reduced.

14. PD-1 ＋ CD38 ｈｉ CD8 ＋ The method of any one of claims 1 to 13, wherein degranulation of granzyme B (GzmB) in T cells is reduced in the subject.

15. PD-1 in the subject ＋ CD38 ｈｉ CD8 ＋ 15. The method of claim 14, wherein the transfer of GzmB from T cells to leukocytes is reduced.

16. The method of any one of claims 1 to 15, wherein the subject has acute respiratory distress syndrome (ARDS).

17. 17. The method of claim 16, wherein the subject has an infection associated with ARDS.

18. 18. The method of claim 17, wherein the subject has COVID-19.

19. 1. A method of treating an autoimmune disease in a subject, comprising administering to the subject an antibody against PD-1 ＋ CD38 ｈｉ CD8 ＋ The method comprising administering a population of T cells.

20. Prior to administration to the subject, the PD-1 ＋ CD38 ｈｉ CD8 ＋ 20. The method of claim 19, further comprising generating a population of T cells.

21. The PD-1 ＋ CD38 ｈｉ CD8 ＋ Prior to administering the population of T cells to the subject, the population of T cells may be ＋ by contacting the CD8 T cells with a suboptimal antigen and / or a PD-1 inhibitor. ＋ 21. The method of claim 20, wherein T cells are generated.

22. The method of claim 21, wherein the PD-1 inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

23. The PD-1 ＋ CD38 ｈｉ CD8 ＋ 23. The method of any one of claims 20 to 22, wherein the population of T cells is expanded prior to administration to the subject.

24. The CD8 ＋ T cells are autologous CD8 ＋ The method according to any one of claims 19 to 23, wherein the cells are T cells.

25. The CD8 ＋ T cells are allogeneic CD8 ＋ The method according to any one of claims 19 to 23, wherein the cells are T cells.

26. 26. The method of any one of claims 1 to 25, further comprising administering a second therapeutic agent to the subject.

27. 27. The method of claim 26, wherein the second therapeutic agent is an immunomodulatory agent.

28. 28. The method of claim 27, wherein the immunomodulatory agent is an immunosuppressant.

29. 28. The method of claim 27, wherein the immunomodulatory agent is an immunostimulant.

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