A method and composition for treating cancer with an Anti-ly6e antibody to reprogram CD8+ t cells for cancer immunotherapy

Abstract

A cancer immunotherapy for treating cancers such as melanoma is presented. A method for treating cancer in a subject via administration of anti-LY6E antibody to the subject. The method can further include administering an IFNAR blockade to inhibit type-1 interferon (IFN) signaling, thereby enhancing the ant-tumoral cytotoxic activity of T cells. The IFNAR can include anti-IFNα. The method can also include administering anti-PD1 antibody prior to, commensurate, or after treatment with anti-LY6E. A composition for treating cancer and modulating immune responses in a subject is also included. The composition can include, among other things, the anti-LY6E antibody, the IFNAR blockade, and anti-PD1. The composition and method also present a mechanism to modulate immune response in a subject by preventing upregulation of a Ly6ahigh T-cell subpopulation in response to a stimulus such as cancer, UVB, or interferons.

Description

A METHOD AND COMPOSITION FOR TREATING CANCER WITH AN ANTI-LY6E ANTIBODY TO REPROGRAM CD8+ T CELLS FOR CANCER IMMUNOTHERAPYSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] Grant support was provided from the European Research Council under the European Union’s Horizon 2020 Research and Innovation Program (grant agreement no. 726225) and the Israel Science Foundation (no. 2017 / 20).STATEMENT OF PRIORITY

[0002] This application claims priority to U.S. Provisional App. No. 63 / 668,421 for a Composition and Method for Treating Cancer with an Anti-Ly6a Antibody to Reprogram Cd8+ T Cells for Cancer Immunotherapy.FIELD OF THE INVENTION

[0003] The present disclosure relates to cancer immunotherapy and more specifically method and composition for treating cancer in a subject through administration of antibodies such as anti-LY6E antibody to crosslink and reprogram CD8+ T cells for cancer immunotherapy.BACKGROUND

[0004] Sunlight has been used as a treatment for skin autoimmune diseases such as psoriasis and vitiligo for more than 3500 years. About 30 years ago, it was discovered that UVB inhibits the immune response to cancer in mice. The suppressive effects of UVB are mediated by alterations in immune cell phenotypes. Many mechanisms have been suggested to explain how UVB induces immunosuppression. Some, such as increases in platelet activation factor, cis-urocanic acid, and vitamin D levels, affect not only skin but also internal organs. Other mechanisms are more local such as the migration of UVB-exposed Langerhans cells from the skin to skin-draining lymph nodes (sDLNs). Importantly, in the context of cancer, exposure of mice to UVB before cancer cell injection inhibits the immune response to cancer via T cell modulation. However, how UVB exposure inhibits the T cell response to cancer is not well understood.

[0005] Cancer cells often express antigens that are recognized by the immune system; however, cancer cells are "self" cells, and some mechanisms that inhibit the immunesystem's response to self, including PD1 - and CTLA4-mediated signaling, are endogenous mechanisms that prevent an autoimmune response. Although immune checkpoint inhibitors have shown remarkable efficacy as therapeutic interventions in melanoma, about 50% of patients are treatment-resistant.

[0006] The disclosers reasoned that use of UVB, which inhibits autoimmunity as well as the immune response to cancer, could serve as a platform for investigation the regulation of the immune response to cancer, possibly leading to the identification of factors that could be targeted for development of new treatments for cancer.

[0007] The present disclosure demonstrates UVB exposure prior to melanoma cell injection enhances tumor growth and induces resistance to anti-PD1 antibody treatment in mice via inhibition of the CD8+ T cell response to tumors in the skin. This repressive effect on T cells was specific to skin and sDLNs and was not observed in internal immune organs. The disclosers discovered that UVB induced expression of Ly6a in T cells. Ly6a expression was also increased upon chronic exposure to type 1 interferon (IFN), which accelerates the exhaustion of tumor-infiltrating T cells. Moreover, antibodies against Ly6a enhanced the T cell response to tumor cells in vitro and in vivo. Taken together, the disclosure demonstrates that targeting LY6E can enhance the immune response to cancer.SUMMARY OF THE INVENTION

[0008] Therapeutic antibodies targeting inhibitory molecules on T cells have revolutionized cancer treatment, especially in melanoma and renal cell carcinomas. Yet, the overall response rate is approximately 50% and about 30% 5-years survival rate in patients with metastatic melanoma. Hence, it is of paramount importance to identify the molecules on T cells that could serve as an alternative treatment for non-responders or as an additional line of defense for relapsed tumors.

[0009] Recently, by studying the immunosuppressive effects of chronic UVB exposure, the disclosers found that Ly6a is highly expressed on exhausted T cells. Crosslinking Ly6a by targeting antibodies restored T cell cytotoxic activity and promoted significant tumor regression in both in vitro and in vivo models of cancer that had progressed onPD-1 immunotherapy. The present disclosure relates to a novel anti-LY6E antibody therapy that will have immediate translational potential in the field of cancer immunotherapy, particularly for patients who do not respond to currently available treatments.

[0010] T cell inhibitory mechanisms prevent autoimmune reactions, while cancer immunotherapy aims to remove these inhibitory signals. Chronic UV exposure attenuates autoimmunity through promotion of unknown immune-suppressive mechanisms. Here, the disclosers show that mice with subcutaneous melanoma are not responsive to anti-PD1 immunotherapy following chronic UV irradiation, given prior to tumor injection, due to the suppression of T cell killing ability in skin-draining lymph nodes. Using mass cytometry and single-cell RNA-sequencing analyses, the disclosers discovered that skin-specific, UV-induced suppression of T-cell killing activity is mediated by upregulation of Ly6ahighT-cells subpopulation. UVB exposure causes an increase in Ly6ahighT-cells. These cells exhibit reduced cytotoxic function compared to non Ly6ahighT-cells. Independently of the UVB effect, Ly6ahighT cells were induced by chronic type-1 interferon in the tumor microenvironment. Treatment with an anti-Ly6a antibody enhanced the anti-tumoral cytotoxic activity of T cells and reprogrammed their mitochondrial metabolism via the Erk / cMyc axis. Remarkably, treatment with anti-Ly6a antibody significantly inhibited tumor growth in mice resistant to anti-PD1 therapy. Applying the disclosers findings to humans provides new cancer immunotherapy treatments for patients including those with resistance to existing treatments.

[0011] Therefore, based on the foregoing and continuing description, the subject invention in its various embodiments may comprise one or more of the following features in any non-mutually-exclusive combination:

[0012] A method for treating cancer in a subject, comprising administering a therapeutically effective amount of anti-LY6E antibody to the subject;

[0013] A method for treating cancer in a subject, comprising administering an IFNAR blockade to the subject, wherein the IFNAR blockade inhibits type-1 interferon (IFN) signaling thereby enhancing the cytotoxic activity of CD8+ T cells;

[0014] A method for treating cancer in a subject, wherein the IFNAR blockade comprises an agent that inhibits binding of type I interferons to IFNAR1 or IFNAR2;

[0015] A method for treating cancer in a subject, wherein the IFNAR blockade comprises an antibody selected from the group consisting of an anti-IFNa antibody, an anti-IFNp antibody, and an anti-IFNAR antibody;

[0016] A method for treating cancer in a subject, wherein treatment with anti-LY6E antibody reverses type I interferon-induced T cell exhaustion;

[0017] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody promotes T cell cytotoxic activity and tumor regression in a subject whose cancer has failed to regress on anti-PD-1 therapy;

[0018] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody is administered after administration of an anti-PD1 antibody;

[0019] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody is administered prior to administration of an anti-PD1 antibody;

[0020] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody induces phosphorylation of Erk1 / 2 and nuclear translocation of Myc in CD8+ T cells, thereby reprogramming mitochondrial metabolism and enhancing anti-tumoral cytotoxic activity in the subject;

[0021] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody increases Granzyme B expression in CD8+ T cells;

[0022] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody increases expression of at least one of CD69, Lcp-1 , and Itgb2 in the subject’s CD8+ T cells;

[0023] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody decreases expression of at least one of Lef1 , Tcf7, and CD62L in the subject’s CD8+ T cells;

[0024] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody enhances mitochondrial metabolism of CD8+ T cells;

[0025] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody upregulates MYC expression in CD8+ T cells;

[0026] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody downregulates mT0RC2 signaling in CD8+ T cells;

[0027] A method for treating cancer in a subject, comprising administering an agent that inhibits LY6E induction by blocking type I interferon signaling;

[0028] A method for treating cancer in a subject, wherein the agent is selected from the group consisting of an anti-IFNa antibody, an anti-IFNp antibody, and an anti-IFNAR antibody;

[0029] A method for treating cancer in a subject, wherein the anti-LY6E antibody binds an epitope on LY6E thereby inducing crosslinking of LY6E in the subject’s CD8+ T cells;

[0030] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody activates tumor-infiltrating lymphocytes independently of T cell receptor (TCR) signaling;

[0031] A method for treating cancer in a subject, wherein the tumor-infiltrating lymphocytes include bystander CD8+ T cells that are not specific for tumor antigens;

[0032] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody increases expression of mitochondrion-associated proteins in the subject’s CD8+ T cells;

[0033] A method for treating cancer in a subject, wherein treatment with the anti-LY6E antibody inhibits expression of Rictor in the subject’s CD8+ T cells;

[0034] A method for treating cancer in a subject, wherein the cancer is selected from the group consisting of melanoma, colorectal cancer, and lung cancer;

[0035] A method for treating cancer in a subject, wherein the cancer is resistant to checkpoint inhibitor therapy;

[0036] A method for treating cancer in a subject, wherein the cancer is characterized by elevated LY6E expression on CD8+ T cells in the tumor microenvironment;

[0037] A method for treating cancer in a subject, wherein the anti-LY6E antibody enhances killing of tumor cells by CD8+ T cells;

[0038] A method for treating cancer in a subject, comprising administering an immune checkpoint inhibitor selected from the group consisting of an anti-PD1 antibody and anti- CTLA-4 antibody;

[0039] A method for treating cancer in a subject, wherein the anti-LY6E antibody is administered concurrently with chemotherapy;

[0040] A method for treating cancer in a subject, wherein the anti-LY6E antibody is administered after development of resistance to a PD-1 inhibitor;

[0041] A composition for treating cancer in a subject, the composition comprising an anti-LY6E antibody;

[0042] A method for identifying a subject likely to benefit from treatment with an anti- LY6E antibody, comprising detecting elevated LY6E expression on CD8+ T cells in a tumor sample from the subject, wherein the elevated expression is relative to LY6E expression on CD8+ T cells in non-tumor tissue from the same subject or from a healthy control;

[0043] A method for modulating immune response in a subject with elevated LY6E expression on T cells, comprising administering anti-LY6E antibody to the subject;

[0044] A method for modulating immune response in a subject with elevated LY6E expression on T cells, wherein the anti-LY6E antibody induces crosslinking of LY6E on CD8+ T cells thereby reprogramming the subject’s CD8+ T cells;

[0045] A method for modulating immune response in a subject with elevated LY6E expression on T cells, wherein the anti-LY6E antibody enhances mitochondrial metabolism of CD8+ T cells;

[0046] A method for modulating immune response in a subject with elevated LY6E expression on T cells, wherein the anti-LY6E antibody upregulates MYC expression in CD8+ T cells;

[0047] A method for modulating immune response in a subject with elevated LY6E expression on T cells, wherein the anti-LY6E antibody downregulates mTORC2 signaling in CD8+ T cells;

[0048] A method for modulating immune response in a subject with elevated LY6E expression on T cells, wherein the anti-LY6E antibody reverses exhaustion in tumorinfiltrating CD8+ T cells; and

[0049] A method for modulating immune response in a subject with elevated LY6E expression on T cells, wherein the anti-LY6E antibody promotes expansion of effector memory CD8+ T cells.DESCRIPTION OF THE DRAWINGS

[0050] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0051] Figure 1. Chronic UVB exposure suppresses sDLN (skin-draining lymph node) CD8+ T cell-mediated killing and induces resistance to melanoma immunotherapy, a. Experimental flowchart, b. Mean tumor diameter (left) and individual plots (right) of SC Ret melanoma tumors growth in UVB- and mock-treated mice (n=4). c. Average radiances (left) and data for individual UVB- and mock-treated mice (right) with Ret melanoma lung metastases (n=7). d. Experimental flowchart, e-f. B16-OVA melanoma cells were incubated with OT-I CD8+ T cells isolated from UVB- or mock-irradiated mice. e. Mean percentages of caspase 3 / 7+ melanoma cells based on Incucyte data (n=10) (left) and flow cytometry (n=3) (right), f. Representative images of caspase 3 / 7 staining, g. Mean percentages of Granzyme B expression in T cells isolated from sDLN of UVB- or mock-irradiated (control) mice (n=6). h-j. Mice treated with UVB or mock- irradiated were injected subcutaneously with Ret melanoma cells and treated with anti- PD1 antibody or IgG control. Arrows indicate time of antibody injections, h. Mean tumor diameters with arrow indicating time of antibody injection (n=6) (left), data for individual mice (middle), and representative images of the mice described (right), i. Flow cytometry analysis of tumor-infiltrating CD45+ cells (left), tumor-infiltrating CD8+ cells(middle), and CD8+ cells (right) in sDLNs. j. Flow cytometry analysis of percent of immune infiltrate cell types. Shown are means ± SEM from one experiment out of three performed. Statistical significance was determined by two-way ANOVA test with Tukey correction (panels b, c, h), one-way ANOVA test with Tukey correction (panels i and j), or two-tailed t test (panel e,g). * p<0.05, ** p<0.01 , *** p<0.001 , **** p<0.0001 , n.s. not significant. Error bars represent standard errors.

[0052] Figure 2. UVB induces a Ly6ahighT cell subpopulation in the skin drain lymph nodes, a. Representative UMAPs for CD4+ subset markers levels (red, high; blue, low) in sDLNs of UVB- or mock-irradiated mice. b. Cell density plots (left), FlowSOM clusters indicated by different colors (middle), and Ly6a levels (red, high; blue, low) in CD4+ clusters from sDLNs of UVB- or mock-irradiated mice. Arrows, colored by cluster, indicate significantly higher or lower levels in UVB-irradiated versus mock-irradiated mice. c. Left: Heatmap of median expression levels of the indicated markers in CD4+ clusters of sDLNs. Colors reflect the transformed ratio relative to the minimum expression of the indicated marker in CD4+ cells. Right: Percentage of each cluster from the total population of CD4+ cells. (n=4 per condition), d-f. Analyses for sDLNs CD8+ cells analysis as done for CD4+ T cells in a-c. g. Mean percent of Ly6ahighcells in various organs from UVB- and mock-treated mice (n=4). Shown is one representative experiment out of at least three independent experiments performed. Statistical significance was determined two-tailed t test. * p<0.05, ** p<0.01 , *** p<0.001 , **** p<0.0001 , n.s. not significant. Error bars represent standard errors.

[0053] Figure 3. Ly6ahighT cells are induced by type 1 IFN secreted by DCs following UVB. a. Single-cell RNA-seq analyses of sDLNs isolated from UVB- and mock- irradiated (control) mice. t-SNE projection of all 14,783 CD4+ T cells (upper panel) and all 14,020 CD8+ T cells (lower panel) with subsets indicated by colors and numbers, accompanied by t-SNE projections colored by the expression levels of Ly6a (purple) in each cell. b. Prediction of the active cytokines within the clusters that express high levels of Ly6a (CD4_4- upper panel; and CD8_5- lower panel). Red indicates significant level of the activation signature, c. Box plot (percentile 90- percentile 10) of Ly6a expression in CD4+ T cells (upper) and CD8+ T cells (lower) following chronic UVB ormock irradiation (control) treatments, d. Box plots of type 1 IFN signature of CD4+ T cells (upper) and CD8+ T cells (lower) following chronic UVB or mock irradiation (control) treatment, e. Heatmap of difference (and significance level in Ly6a expression in UVB-treated mice versus mock-irradiated controls in each cluster of CD4+ and CD8+ T cells, f. Representative flow cytometry images of Ly6a expression in splenic T cells activated ex vivo as indicated, g. Mean percentages of Ly6a expression in splenic T cells activated ex vivo as indicated (n=3). h. Representative UMAP plots of non-T and non-B cells in sDLNs from UVB- or mock-irradiated (control) mice. i. FlowSOM clusters in sDLNs from UVB- or mock-irradiated (control) mice. j. Left: Heatmap of median expression levels of the indicated markers in clusters of sDLNs. Colors reflect the transformed ratio relative to the minimum expression of the indicated marker in CD4+ cells. Right: Percentage of each cluster from the total innate cell population (n=4). k. Experimental flowchart. I. Mean percentages of Ly6a levels in Ly6aneg / low / CD4+ cells after 72 h of co-culture with spleen or sDLN CD11 b+ cells from UVB irradiated mice. Shown is one representative experiment out of at least three independent experiments performed. Statistical significance was determined by two-tailed t test (panels c-e, h, i) or one-way ANOVA test with Tukey correction (panel g). * p<0.05, ** p<0.01 , *** p<0.001 , n.s. not significant. Error bars represent standard errors.

[0054] Figure 4. Ly6ahighT cells are enriched in the tumor microenvironment independently of UVB exposure, a. Experimental flowchart, b. Left: Representative flow cytometry analyses of Ly6a expression levels in CD4+ (left) and CD8+ (right) T cells from inguinal lymph nodes, spleens, and tumors. Right: MFI of Ly6a normalized to isotype control (left) and percentage of Ly6ahighcells in total CD4+ or CD8+ compartments (right) (n=5). c. Violin plots of Ly6a expression in CD4+ (left) and CD8+ (right) cells in lymph nodes and tumors. Single-cell RNA-seq data from Davidson et al. d. Experimental flowchart: Melanoma metastases were isolated from mouse lungs, and CD45+ cells were isolated and analyzed using CyTOF. CD4+ cells and CD8+ cells are divided into three groups: CD62I+ (blue gate), Ly6ahigh(red gate), and Ly6alow (green gate), e. Heatmap of median expression levels of T cell markers in indicated cells. The colors reflect the transformed ratio relative to the minimum expression of the marker (n=4). Statistical significance was determined by one-way ANOVA test with Tukeycorrection (panel b) or two-tailed t test (panel c). * p<0.05, ** p<0.01 , *** p<0.001 , n.s. not significant. Error bars represent standard errors.

[0055] Figure 5. Anti-Ly6a antibody has a strong immunotherapeutic effect even in mice resistant to anti-PD1 antibody treatment, a. Experimental flowchart, b. Normalized mean numbers of B16F10 melanoma cells following co-culture with splenic CD8+ T cells from transgenic mice bearing gp100-reactive T cells in the presence of various clones of anti- Ly6a antibody or IgG control (n=10). c. Mean tumor diameters (left) and data from individual mice (right) treated with UVB or mock-irradiated (control) and injected subcutaneously with Ret melanoma cells then treated with anti-Ly6a antibody or IgG control. Arrow indicates time of antibody injection (n=6). d. Mean percentages of Granzyme B expression in tumor-infiltrating (left) or sDLN (right) CD45+ cells, e. Experimental flowchart, f. Mean tumor diameters (left) and data for individual mice (right) injected with Ret melanoma cells and treated with anti-IFNR1 , anti-Ly6a, or IgG (control) antibodies. Arrow indicates day of treatment. (n=6). g. Experimental flowchart, h. Mean tumor diameters (left) and individual data (right) for mice injected with Ret melanoma cells and treated with anti-PD1 , anti-PD-1 and anti-Ly6a, or IgG (control) antibodies (n=6). Arrows indicate days of treatment. Tumor growth was significantly reduced in the anti-PD-1 + anti-Ly6a group relative to the other groups. Shown is one experiment out of at least three performed. Statistical significance was determined by two-way ANOVA test with Tukey correction (panels b, c, f, h) or two-tailed t test (panel d); * p<0.05, ** p<0.01 , *** p<0.001 , **** p<0.0001 , n.s. not significant. Error bars represent standard errors.

[0056] Figure 6. Anti-Ly6a antibody treatment enhances CD8+ T cell activity and prevents loss of mitochondrial function through effect on cMyc-mediated signaling, a. Experimental flowchart, b. Principal component analysis of the proteins that significantly changed across indicated conditions (n=9). c. Heatmap of the average Z scores for each group. Selected proteins in cluster 1 and cluster s are indicated, d. GO terms significantly associated with proteins of cluster 1 . Shared colors indicate related GO terms, e. String protein networks of significantly changed proteins in all samples that are associated with the GO term "mitochondrion". Selected GO terms are indicated.Clusters are indicated by circle colors (red and blue), f. Representative flow cytometry (left) and mean percentages (right) ofTMRE expression in CD8+ cell (n=4). g. MFI ofTMRE expression in IFNa exhausted CD8+ T cells (n=3). h. Predicted upstream regulators of the significantly changed proteins in all samples. Arrows represent the predicted effect of anti-Ly6a crosslinking, i. Mean intensity (upper) and representative flow cytometry (lower) of phosphorylated Erk1 / 2 in CD8+ T cells incubated with anti- Ly6a antibodies (n=5). j. Representative confocal images (left) and mean percentages of T cells with nuclear c-Myc (right) following exhaustion with IFNa and incubation with anti-Ly6a antibodies (n=10). Shown is one representative experiment out of at least three independent experiments performed. Statistical significance was calculated using one way ANOVA with Tukey’s correction for multiple comparisons (panels f, g, i, j) or FDR correction with FDR <0.1 significant (panels c, d). **p<0.001 , **** p<0.00001. Error bars represent standard errors.

[0057] Figure 7. Chronic UVB exposure suppresses skin draining lymph node CD8+ T cell-mediated killing and induces resistance to melanoma immunotherapy, a. Representative ultrasound images of tumors in UVB- and mock-irradiated mice. The tumors are outlined with dashed lines and locations of depth measurements are marked by arrows. Enlargements show blood flow (in red or blue) in the tumor area. Scale bars, 1 cm. b. Tumor depths determined by ultrasound in UVB-treated and mock irradiated (control) mice (n=4 per group), c. Average radiance, reflective of lung metastasis development, in UVB-treated or mock irradiated (control) mice injected intravenously with Ret melanoma cells (0.5x106 cells, n=7 per group) on average (left) and in individual mice (right), d. Fold change in mRNA levels in mCherry+ melanoma cells from liver (n=5) and lymph nodes (LN) relative to mice not injected with tumor cells (n=4). e. Flow cytometry gating strategy to evaluate apoptosis of melanoma cells co-cultured with T cells, f. Percent CD4+ T cells relative to total cells in sDLN (left) and relative to CD45+ cells in the tumor (right). Significance was determined by two-tailed t test. * p<0.05, ** p<0.01 , *** p<0.001 , **** p<0.0001 , n.s. not significant. Error bars represent standard errors.

[0058] Figure 8. UVB induces a Ly6ahighT cell subpopulation in the skin drain lymph nodes, a. Left: The markers for each cell type analyzed. Right: Percentages of the indicated cell types from total live CD45+ cells based on supervised analysis of mass cytometry data. b. Median intensities of indicated markers in CD4+ (upper) and CD8+ (lower) cells, c. Heatmap of median expression levels of Ly6a in the indicated cell subtypes in sDLNs. Colors reflect the transformed ratio relative to the minimum expression of Ly6a in the indicated cells. (n=4 per condition), d. Representative UMAPs for CD4+ subset marker levels (red, high; blue, low) in cells from spleens, e. Cell density plots (left) and UMAPs of CD4+ clusters (indicated by different colors) generated by FlowSOM (right) from spleen CD4+ cells, f. Left: Heatmap of CD4+ FlowSOM clusters with colors indicative of the transformed ratio relative to the minimum expression of the indicated marker in CD4+ cells. Right: Percentage of each cluster from the total population of CD4+ cells in UVB-treated and mock-irradiated mice. g-i. Analysis of spleen CD8+ cells as in d to f. j. Supervised mass cytometry analysis of T cell subtypes that significantly increased upon UVB treatment in spleen (left) and sDLN (right) according to the unsupervised analysis: CD62I+ / CD44- / Ly6a+ / CD8+ (Ly6a+ naive CD8), CD62I- / CD44+ / Ly6a+ / CD8+ (Ly6a+ EM CD8), and CD62I- / CD44+ / Ly6a+ / Ly6c- / CD4+ (Ly6a+ EM CD4). k. Left: Representative trace of Ly6a median fluorescence intensity (MFI) in CD4+ and CD8+ cells from sDLN relative to isotype control MFI (diagonal stripes) in UVB-treated (UVB, orange) and mock-irradiated (control, gray) mice as determined by flow cytometry. Right: Averages of Ly6a versus isotope MFIs (n=5 per group). I. Left: Representative flow cytometry analyses of CD44+ / Ly6a+ / CD4+ cells from sDLN of UVB-treated and mock-treated (control) mice. Right: Means of percentages of CD44+ / Ly6a+ cells from total CD4+ cells (n=5 per group), m-n. Flow cytometry analysis of Ly6a levels in T cells from m) spleens (n=5 per group) and n) mesenteric lymph nodes (n=4 per group) of UVB-treated and mock-treated mice, o, Left: Percent of Ly6a+ / PD-1- and Ly6a+ / PD-1+ cells from total CD8+ cells from skin, sDLN, and spleen following UVB or mock irradiation (control) treatment (n=4 each group). Right: Representative image. Significance was determined by two-tailed t test. * p<0.05, ** p<0.01 , *** p<0.001 , n.s. not significant. Error bars represent standard errors.

[0059] Figure 9. Ly6ahighT-cells are induced by type 1 IFN secreted by DCs following UVB. a. t-SNE projections colored by the expression levels of Ly6a (upper) and type 1 IFN signature genes (lower) for UVB- and mock-irradiated (control) CD4+ cells (cluster 2 of clusters defined in Figure 3a) from single-cell RNA-seq data of sDLN T cells from UVB- and mock-irradiated mice. b. Volcano plot of the differently expressed genes in CD4+ cells (cluster 2) between UVB-treated and control mice. Ly6a is marked in red. c. Volcano plots of genes upregulated or downregulated in Ly6ahigh effector memory and exhausted cells. The x axis represents fold change; y axis represents Iog10 of adjusted p value. Representative transcription factors associated with gene groups are indicated. Data from Elyahu et al. 28. d. MFI of Ly6a on CD4+ (upper) and CD8+ (lower) T cells at 24 hours after treatment with the indicated cytokine or factor (n=3). e. Percentages of CD62L+ / Ly6alow, CD62L+ / Ly6ahigh, CD62L- / Ly6alow, and CD62L- / Ly6ahigh cells from CD4+ (left) and CD8+ (right) compartments after the indicated treatments, f. MFI versus that of isotype control of Ly6a on CD4+ (left) and CD8+ (right) T cells after the indicated treatments (n=3). g. UMAP of mass cytometry data analysis of B cells from sDLN of mice treated with UVB or mock irradiated (n=4 per group). Marker levels are indicated by color (red, high; blue, low), h. Cell density plots (left) and UMAPs of B cell clusters (indicated by different colors) generated by FlowSOM (right), i. Left: Heatmap of transformed ratio relative to the minimum expression of the indicated marker in B cells. Right: Percentage of each cluster from the total population of B cells in UVB- and mock- treated mice. j-o. Mass cytometry analyses of j-i) spleen B cells and m-o) spleen innate immune cells (or non-B, non-T cells) of mice treated with UVB (n=3) or mock irradiated (n=2) as in g-i. p. Supervised mass cytometry analysis of the myeloid cell subtypes that significantly increase upon UVB in the sDLN (left) and the spleen (right): CD11 b+ / Ly6g- / Ly6chigh / CXCR3+ cells (inflammatory monocytes), CD11 b+ / Ly6g- / MHC2high / CD11c+ cells (CD11 b+ DCs), and CD11 b+ / Ly6g+ / Ly6clow cells (granulocytes), q. Experimental flowchart: C57BL / 6 mice were irradiated with UVB or mock irradiated for 8 weeks (n=4 per group). CD11 b+ cells were isolated from sDLN and the skin and cultured for 24 hours. Conditioned media was collected and added to Ly6aneg / low / CD8+ cells from untreated mice, with or without anti-IFNR1 blocking antibody. After 72 hours of culturing with the conditioned media, the percentage of Ly6ahigh T cells was determined, r. Meanpercentages of Ly6ahigh / CD8+ cells in indicated samples. Significance was determined by two-tailed t test. * p<0.05, ** p<0.01 , *** p<0.001 , n.s. not significant. Error bars represent standard errors.

[0060] Figure 10. Ly6ahighT-cells are enriched in the tumor microenvironment independently to UVB exposure, a. Gating strategy for Figure 4a-b. b. Percent of DCs and macrophages that express Ly6a in the tumor microenvironment, spleen, and sDLN from Figure 4a. (n=4). c. B16 or B16-OVA tumor cells were subcutaneously injected into C57BL / 6 OT-1 mice (n=2 each group). After 16 days, the expression of Ly6a on TILs was measured by flow cytometry. Left: Representative trace of Ly6a median fluorescence intensity (MFI). Right: Averages of Ly6a versus isotope MFIs. d. C57BI / 6 (n=5) and OT-I mice (n=4) were injected subcutaneously with B16-OVA melanoma cells. Once tumors were palpable, the percentage of Ly6ahighin infiltrating CD8+ T cells was analyzed by staining with H-2Kb:OVA (SIINFEKL) tetramer (MBL, cat# TB-5001-4). Significance was determined by two-tailed t test (panels c, d) or by one-way ANOVA test with Tukey correction (panel b). * p<0.05, ** p<0.01 , *** p<0.001 , n.s. not significant. Error bars represent standard errors.

[0061] Figure 11. Anti-Ly6a antibody has a strong immunotherapeutic effect even in mice resistant to PD1 treatment, a. B16F10 melanoma cell killing assay following coculture with splenic CD8+ T cells that were either primed with gp100 or exhausted with IFNa, in the presence of anti-Ly6a antibody or lgG2 control (n=6). b. Tumor mean diameters in mice subcutaneously injected with MC38 tumor cells and treated with anti- Ly6a antibody clone W18174, anti-Ly6a clone D7, or lgG2a isotype control on day indicated by arrows (n=3 per group), c. Survival curves of mice following treatment with anti-Ly6a. According to the ethical guidelines, mice with tumors of 2 cm were sacrificed (n=4 in each group), d. Analyses of DC subtypes from the lymph nodes and tumor microenvironment following treatment with anti-Ly6a as in Figure 5e. Left: Gating strategy. Right: Percent of the DC subtype from total immune cells, e. Mean tumor diameters (left) individual mouse data (middle) and representative images (right) for mice treated with UVB or mock-irradiated (control) and injected subcutaneously with Ret melanoma cells then treated with anti-Ly6a antibody, anti-PD-1 or IgG control (n=6).Arrows indicate days of treatment. Significance was determined by two-way ANOVA test with Tukey correction (panels b and e) or by one-way ANOVA test with Tukey correction (panel a) or by two-tailed t test (panel d) or by log-rank test (panel c). * p<0.05, ** p<0.01 , *** p<0.001 , **** p<0.0001 , n.s. not significant. Error bars represent standard errors.

[0062] Figure 12. Anti-Ly6a antibody crosslinking enhances CD8+ T-cells activity and prevent loss of mitochondrial function that is associated with T-cells exhaustion via cMYC signaling, a. GO terms of biological processes significantly associated with proteins of cluster 5, which are downregulated in the presence of melanoma cells and anti-Ly6a and upregulated in cells treated with IgG in the absence of melanoma cells as described in Figure 5c. b. Percentage of proteins associated with ribosome or mitochondria based on cellular compartment GO terms for the proteins of cluster 1 and cluster 5 from Figure 4d. c. Western blot analysis of phosphorylated and total Zap70 protein at 10 minutes and 1 hour after activation of CD8+ T cells with anti-CD3 or anti- Ly6a antibody plus anti-CD3 antibody. Numbers under lanes are the intensity of phosphorylated Zap70 divided by the intensity of total Zap70. d. Venn diagram of overlap between proteins significantly upregulated by anti-Ly6a antibody treatment with those significantly upregulated upon anti-CD3 antibody plus anti-CD28 antibody treatment ("common"), and the genes that upregulated by anti-Ly6a antibody treatment but not significantly upregulated upon anti-CD3 antibody plus anti-CD28 antibody treatment ("unique"). Data from 43. Right: Percentage of proteins associated with ribosome or mitochondria based on cellular compartment GO terms for the “common” and “unique” proteins, e. GO terms for biological processes significantly associated with the "common" (left) and "unique" (right) proteins, f. String protein networks of the “common" (left) and "unique" (right) proteins.DETAILED DESCRIPTION

[0063] Chronic UVB exposure suppresses sDLN CD8+ T cell-mediated killing and induces resistance to melanoma immunotherapy.

[0064] Chronic exposure to UVB upregulates various mechanisms of immune suppression across an organism. Although the suppressive effects of UVB are beneficial during treatment of certain autoimmune disorders, the opposite may be true for cancer. UVB exposure before cancer cell injection inhibits the immune response to the cancer cells and enhances primary tumor growth, but how UVB influences tumor progression, specifically on metastases, has yet to be assessed. Thus, the disclosers evaluated the local and systemic immunosuppression mechanisms in response to melanoma induction in immunocompetent mice that had been chronically exposed to UVB. Mice were irradiated five times per week for 8 weeks with UVB exposure, then Ret melanoma cells were injected subcutaneously to induce skin tumors or intravenously to induce metastases. Local tumor growth and metastatic burden were measured over time (Fig. 1a). UVB-irradiated mice injected subcutaneously with melanoma cells had significantly higher local tumor growth rates than control mice that were mock irradiated (Figs. 1 b, 7a, and 7b).

[0065] Next, mice were intravenously injected with Ret melanoma cells that express mCherry and luciferase to allow tracking of lung metastases that develop independently of the primary tumor burden in the skin. Bioluminescence quantification indicated that, in contrast to the enhanced tumor growth in the skin, no differences in growth rates of lung metastases between the UVB-irradiated mice and the control group were observed when melanoma cells were injected intravenously (Fig. 1c and 7c). Similarly, quantification of levels of the mRNA encoding mCherry in micro-metastases in liver and inguinal and brachial lymph nodes showed no significant differences between UVB- treated and control mice (Fig. 7d). This indicates that UVB treatment prior to tumor initiation enhances local tumor growth but does not enhance metastasis.

[0066] To test the effect of UVB on T cell responses to melanoma, the disclosers used C57BL / 6J OT-1 mice, which were genetically engineered to recognize the OVA albumin antigen and thus respond to B16 melanoma cells that over express the OVA peptide (B16-OVA). Dorsally shaved C57BL / 6J OT-1 mice were chronically exposed to low- dose UVB irradiation or were mock-irradiated as controls (Fig. 1 d). Spleens and sDLNs are reflective of systemic and local effects of UVB, respectively. CD8+ T cells fromsDLNs and spleens of UVB-exposed and from control mice were co-cultured with B16- OVA cells for 24 hours. The disclosers found that UVB treatment significantly inhibited the ability of sDLN CD8+ T cells to kill tumor cells as shown by quantification of apoptotic melanoma cells in live imaging analyses and in flow cytometry analyses of DAPI+ / caspase 3 / 7+ melanoma cells (Fig. 1 e, f), but there was no significant difference in cell killing by spleen CD8+ T cells from UVB- and mock-irradiated mice (Fig. 1 e, f). In accordance with these data, there was a significant decrease in Granzyme B in sDLN CD8+ cells, but not in the spleen CD8+ cells, from mice following UVB treatment (Fig. lg)-

[0067] Given the reduction in CD8+ T cell activity due to UVB irradiation, the disclosers next examined whether UVB affected the efficacy of the immunotherapeutic anti-PD1 antibody. The disclosers found that anti-PD1 antibody treatment failed to inhibit tumor growth in UVB-irradiated mice, although it was effective in mock-irradiated controls (Fig. l h). Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) showed that UVB- treated mice had significantly fewer CD45+ immune cells and significantly lower percentages of CD8+ T cells relative to total cells and to CD45+ immune cells than control mice (Fig. 1 i, left panel; Fig. 7e and f). Thus, the lower percentage of CD8+ T cells in tumors of UVB-treated mice relative to controls is not a reflection of larger tumor size. Interestingly, examination of T cells from sDLNs showed the opposite trend: After anti-PD1 antibody treatment, the UVB-treated group had a higher percentage of CD8+ T cells relative to all sDLNs cells than the mock-irradiated group (Fig. 1 i, right panel). Examination of other UVB-relevant immune cells, including CD4+ T cells, dendritic cells (DCs), monocytes, and granulocytes, showed no significant differences in percentage relative to total cells in UVB-irradiated versus mock-treated mice after anti-PD1 antibody treatment (Fig. 1j). Hence, the disclosers hypothesize that UVB exposure prior to injection of tumor cells alters T cell phenotypes and inhibits the response of CD8+ T cells to melanoma even in the presence of anti-PD1 immunotherapy treatment.

[0068] UVB induces a Ly6ahighT cell subpopulation in the skin drain lymph nodes.

[0069] To dissect the effect of UVB on the immune system at the local and systemic levels, the disclosers performed a multi-parameter mass cytometry analysis. Thedisclosers used chronically UVB or mock irradiated dorsally shaved mice, isolated their spleen and sDLNs, and subjected single cells to mass cytometry analysis. The disclosers analyzed each cell type (Fig. 8a) using UMAP dimensionality reduction to provide an overview of various reconstituted populations and employed FlowSOM algorithms for unsupervised cells clustering. First, unsupervised analysis of CD4+ T cells of the sDLNs was performed, which revealed cell segregation according to known subsets of T cells including naive (CD62L+ / CD44-), effector and effector memory (CD44+ / CD62L), and central memory (CD62L+ / CD44+) T cells (Fig. 2a). Comparison between the UMAP density plots, which provides a topographic view of cells’ amount, of UVB and control sDLNs CD4+ cells from the sDLNs revealed that a subset of cells was present at higher levels in UVB-irradiated mice compared to mock-irradiated controls (Fig. 2b). This UVB-induced cell subset was also observed using FlowSOM, which showed that cluster L_CD4_6 was present at significantly higher frequency in the UVB- treatment group than the control group (control, 4.98%; UVB, 14.46%; P=0.0097), whereas cluster L_CD4_3 was present at significantly lower frequency (control, 7.13%; UVB, 2.63%; P=0.017; Fig. 2b, c). Cluster L_CD4_6 is a cluster of effector and effector memory cells that express high levels of Ly6a but not Ly6c, CD25, Tbet, or PD1 ; this expression pattern is unlike other effector and effector memory clusters L_CD4_4 and L_CD4_5 (Fig. 2c).

[0070] Next, the disclosers analyzed the CD8+cells from sDLNs. The percentages of cluster L_CD8_2, which are CD44- / CD62L+ / Ly6aintnaive CD8+T cells (control, 21.6%; UVB, 27.63%; P=0.04) and of cluster L_CD8_5 (effector memory CD447CD62L- / Ly6ahigh / CD8+T cells; control, 0.43%; UVB, 1.59%; P=0.023) were significantly higher upon UVB exposure than in controls (Fig. 2d-f). The increases in Ly6a+CD8+cells were observed upon UVB treatment independently of cell maturation status indicating that UVB exposure induces a general increase in Ly6a expression in CD8+T cells. Supervised examination of the expression levels of all markers tested showed that UVB specifically enhanced Ly6a expression in CD8+and CD4+T cells but did not increase expression of other T cells markers that were examined (Fig. 8b). Further, among all immune cells subtype identified by supervised mass cytometry analysis of sDLN, Ly6a expression was significantly increased only in T cells (Fig. 8c).

[0071] To validate the effect of UVB on Ly6a expression in T cells, B cells, and DCs in the skin, sDLN, and spleen, the disclosers repeated the experiment and subjected the indicated organs to flow cytometry analysis. The disclosers found that UVB irradiation significantly increased Ly6a expression on T cells and that this effect was observed in the skin and sDLN but not in the spleen (Fig. 2g). Mass cytometry analysis of CD4+ and CD8+T cell populations in the spleen also showed no significant differences in Ly6a expression or in the percentages of T cell clusters in UVB- or mock-treated mice (Fig. 8d-j). Further, flow cytometry analysis of cells isolated from the skin, sDLN, mesenteric lymph nodes, and spleen showed that the increase in Ly6a following UVB is specific to the skin area and not these internal organs (Fig 8.k-o). Taking together, the disclosers found that among all T cells markers that were examined, only Ly6a increases upon chronic UVB treatment, an increase which is specific to T cells in the skin area. These results, together with the evidence about UVB immunosuppressive effect on T cells immune response to tumor in the skin initiated further investigation into the role of Ly6a in T cells immunity and the mechanism behind the increase in Ly6a expression.

[0072] Ly6ahighT cells are induced by type 1 IFN secreted by DCs following UVB.

[0073] Ly6a is a marker of hematopoietic stem cells and of CD62L+ / CD44- stem-like memory CD8+ T cells24. Additionally, Ly6a levels are increased in CD4+ and CD8+ T cells in response to inflammatory cytokines. Ly6a is detected on the membranes of T cells and other immune cells, but the functional role and its ligand are unknown. Since Ly6a is a marker of T cells which is induced following UVB treatment, the disclosers used the presently disclosed experimental model to gain deeper insights into the function of Ly6a role. The disclosers conducted single-cell RNA sequencing of sDLN cells from mice following UVB or mock irradiation. To infer the evolving dynamics among different T cell subsets, the disclosers performed sub-clustering analyses of CD8 and CD4 T cells (Fig. 3a). For defining CD8 and CD4 T cell subclusters, the disclosers employed the differentially expressed genes within each cluster. The disclosers’ unsupervised clustering identified one cluster of CD4+ cells and one cluster of CD8+ cells that expressed high levels of Ly6a (cluster CD4_4 and cluster CD8_5, respectively; Fig. 3a). These clusters included the majority of the cells that expressedhigh levels of Ly6a in the mock-irradiated control mice and thus are cells that have high Ly6a levels independently of UVB treatment. Enrichment test for predicting the active cytokines within these clusters using Response Enrichment Analysis, showed a notable and distinct impact of the type 1 IFN response, both in CD4 and in CD8 cells (Fig. 3b). Notably, IFNa and I FNp were the only cytokines identified in this analysis (Fig. 3b). These findings suggest that Ly6a marks a subset of IFN-exposed CD4+ and CD8+ T cells.

[0074] In line with the disclosers mass cytometry and flow cytometry results, a significant increase in Ly6a expression in UVB-treated versus control mice was found (Fig. 3c). The increase in Ly6a expression following UVB was accompanied by an increase in expression of type 1 IFN signature genes (Fig. 3d). Notably, the increases in Ly6a expression after UVB treatment was observed in multiple clusters of CD4+ and CD8+ cells including clusters of naive / memory cells and regulatory cells (clusters 0, 1 , 2, and 5 of CD4+ cells and clusters 0, 1 , 2, 3, 4 in CD8+ cells; Fig. 3e). For example, in cluster CD4_2, (regulatory T cells) the significant increase in Ly6a expression following UVB was accompanied by type 1 IFN signature enhancement (Fig. 9a and b). The genetic association between Ly6a expression in T cells and type 1 IFN signature genes was validated using independent single-cell RNA sequencing data of T cells (Fig 9c). These data suggest that Ly6ahighT cells are a subset of type 1 interferon exposed cells, that respond to the chronic effects of UVB exposure and thus may be targeted to counteract UVB immunosuppression.

[0075] To better understand the mechanisms behind Ly6a induction in T cells, the disclosers tested the effects of various cytokines (IL-1 , IL-2, IL-6, IL-12, IFNa, IFNy, and TNFa) and T cell receptor (TCR) activation factors (anti-CD3 antibody, anti-CD28 antibody, lipopolysaccharide, and CL307) on Ly6a levels. The disclosers found that almost all tested cytokines and activation factors (except for IL-1 for CD8+ T cells) significantly increased Ly6a levels; IFNa had the strongest effect (Fig. 9d). Since during UVB exposure and in the tumor environment, the exposure to the inflammatory environment is continuous, the disclosers also examined the effect of chronic exposure to either IFNa or TCR activation. It was found that chronic TCR activation by chronicexposure to anti-CD3 antibody or to anti-CD3 antibody plus anti-CD28 antibody increased the number of CD62L- effector T cells (Fig. 3f, g; Fig 9e, f); however, most of these cells did not express Ly6a, and there was no increase in the percent of Ly6ahighcells in CD4+ or CD8+ effector and effector memory compartments (Fig. 3f, g; Fig. 9e). In contrast, upon chronic IFNa exposure, almost all cells expressed Ly6a, and the percentage of Ly6ahighcells was significantly increased in both CD4+ and CD8+ compartments (Fig. 3f, g; Fig. 9f). Chronic IFNa treatment, but not the other treatments, significantly increased Ly6a levels in CD62L- / CD4+ and in CD62L- / CD8+ compartments. Taken together, these data suggest that Ly6a is a marker of T cells found in a chronically inflamed environment such as results from UVB chronic exposure or at the tumor microenvironment, and that Ly6a expression is induced following exposure to type 1 IFN rather than due to continuous signaling through TCR.

[0076] To identify the cells that regulate type 1 IFN-mediated induction of Ly6a expression on T cells, the disclosers analyzed the effect of UVB on all immune cells in the sDLN of UVB- or mock-irradiated mice using mass cytometry. Analysis of B cells showed no significant differences in B cell clusters in the sDLNs or spleens between UVB-exposed and mock-irradiated mice (Fig. 9g-l). All other cell types, which were detected at lower percentages than T or B cells (Fig. 8a), were then analyzed in one group of "non T and non B cell", or "other immune cells" as the disclosers termed, other lymph node clusters (L_O). UMAP and FlowSOM analyses showed significantly higher frequencies of cluster L_O_2, which contains inflammatory monocytes (control, 5.91 %; UVB, 20.87%; P=0.031), and in cluster L_O_4, which contains dendritic cells (CD11c+ / MHC2high / CD8- / CD11 b+; control, 5.01 %; UVB, 13.78%; P=0.0048; Fig. 3h-j). These results show that UVB increases frequencies of monocytes in the skin and in sDLN due to secretion of inflammatory cytokines from the skin upon UVB exposure and DC migration from the skin to the sDLN.

[0077] The disclosers detected significantly higher levels of cluster S_O_4, which contains CD11 b+ / Ly6g+ / Ly6clow granulocytes, but not in inflammatory monocytes or DC subtypes, in spleens of UVB-treated mice compared to mock-treated mice (Fig. 9m- o). UVB induces neutrophil migration not only to the skin but also to internal organssuch as spleen, lung, and kidney. UVB irradiation did not have a significant effect on natural killer cell percentages or phenotypes in sDLNs or in spleen (Fig. 3h-j; Fig. 9m-o). In accordance with the unsupervised analyses, supervised analysis of the mass cytometry data showed that UVB exposure significantly increased the percentage of CD11 b+ DCs and inflammatory monocytes as a fraction of all CD45+ cells in the sDLNs (Fig. 9p) but not in the spleen, whereas granulocyte frequencies were significantly increased in the spleen but not in the sDLNs (Fig. 9p). This indicates that UVB has a skin-specific effect on adaptive immune cells such as T cells and also on the innate immune cells including DCs and monocytes.

[0078] To examine the potential role of UV-induced DCs in modulating Ly6ahighexpression in T cells, the disclosers isolated DCs from the skin, lymph nodes, and spleens of UVB-treated or mock-irradiated mice. Isolated cells were then co-cultured for 48 hours with T cells that expressed no or low levels of Ly6a sorted from the spleens of naive mice, followed by flow cytometry analysis of the percentages of CD4+ and CD8+ cells that express high levels of Ly6a (Fig. 3k). Significantly higher percentages of Ly6ahigh / CD8+cells were detected upon incubation with DCs from sDLNs or skin of UVB-exposed mice compared to DCs isolated from mock-irradiated (control) mice; levels in upon incubation with splenic DCs were similar in the two groups (Fig. 3I). This suggests that the skin-specific effect of UVB on T cell phenotype is mediated by UVB- induced migration of myeloid cells to the skin area. Blocking of IFNa in the secreted media of the isolated DCs inhibited DC-mediated induction of Ly6a expression on T cells (Fig 9. q, r). Taken jointly, the disclosers data demonstrates that chronic exposure of CD8+ T cells to type 1 IFN, predominantly secreted by UV-activated DCs, induces Ly6a expression.

[0079] Ly6ahighT cells are enriched in the tumor microenvironment independently of UVB exposure.

[0080] Next, the disclosers examined whether Ly6a expressing T cells are present in the immunosuppressed tumor environment. Significantly higher levels of Ly6a expression were detected on MC38 colon cancer TILs than on T cells of spleen or lymph nodes (Fig. 4a, b, and 10a). In contrast, Ly6a levels in B cells, DCs, and macrophages fromthe tumor microenvironment did not differ significantly from Ly6a levels in those cells isolated from other organs (Fig. 10b). Consistent with this, when the disclosers analyzed published single-cell RNA sequencing data of TILs compared to T-cells from lymph nodes, the disclosers found significantly higher levels of Ly6a in B16 melanoma TILs than in T cells from lymph nodes (Fig. 4c). Further, in the Ret melanoma metastasis mouse model, the disclosers found that Ly6ahighTILs were enriched in lung metastases, as shown by mass cytometry analysis (Fig. 4d). This demonstrates that the induction of Ly6a occurs in primary and metastatic tumors but not in adjacent lymph nodes. Further, the disclosers analyzed markers associated with Ly6ahighT cells in the melanoma lung metastases mass cytometry data and found that Ly6a was highly expressed on CD62L- / CD44+ / PD1+T cells (Fig. 4e), suggesting that these cells are effector or effector memory or exhausted subpopulations of T cells.

[0081] The increase in Ly6a expression in the tumor microenvironment may be due to TCR-dependent activation that occurs when T cells recognize tumor cells, in which case Ly6a represents a marker of activated or exhausted T cells. On the other hand, it is also possible that the high levels of Ly6a expression on TILs is related to the inflammatory environment induced by tumor cells and by myeloid cells that migrate to the tumor. To test these possibilities, C57BL / 6J OT-1 mice were subcutaneously injected with B16- OVA melanoma cells, which are recognized by the TCR of the OT-1 T cells, or with B16 melanoma cells that are not recognized by the T cells of these mice. There was no difference in the level of Ly6a in the TILs of B16 and B16-OVA tumors; in both groups of mice, levels of Ly6a were higher in tumors than in T cells of the spleen (Fig. 10c).

[0082] The disclosers further examined the Ly6a levels in TILs that did or did not express the OVA tetramer by challenging C57BI / 6 and OT-I mice with B16-OVA melanoma cells injected subcutaneously. Once tumors were palpable, the disclosers analyzed the infiltrating CD8+T cells for their expression of Ly6a and the OVA tetramer. The disclosers found that approximately 20% of tumor-infiltrating CD8+ T cells that were positive for the OVA tetramer expressed high levels of Ly6a. This is equivalent to the percentages of Ly6a+ / CD8+T cells that infiltrated tumor tumors in control mice (Fig.10c). This indicates that the increase in Ly6a is not restricted by the TCR specificity, butresults from the inflammatory environment of the tumor, supporting the disclosers hypothesis that Ly6a is a marker of T cells that have been exposed to an inflammatory or an immunosuppressive environment that could result from chronic UVB exposure or cancer.

[0083] Anti -Ly6a antibody has a strong immunotherapeutic effect even in mice resistant to anti-PD1 antibody treatment.

[0084] The disclosers findings that Ly6a is a marker of T cell in the tumor microenvironment, together with the finding that chronic UVB suppressive effect on T cells in response to cancer is associate to Ly6a increase, initiated further examination of the function of Ly6a in the T cell response to cancer. The disclosers first evaluated the effect of anti-Ly6a antibodies on CD8+ T cell killing ability in vitro. Since the antibody effect is highly dependent on the epitope bound by the antibody, the disclosers used different anti-Ly6a antibody clones. An in vitro killing assay was performed by coculturing CD8+ T cells from spleens of gp10025,28-35 TCR transgenic mice36 with B16 melanoma cells (Fig. 5a). The anti-Ly6a antibodies all significantly enhanced killing of melanoma cells; clones E13.161 and W18174A were considerably more active than clone D7 or clone REA422 (Fig. 5b). The disclosers found that IFNa treatment of T cells did not enhance T cell killing ability in vitro but that there was a significant additive effect when cells exposed to IFNa were also treated with anti-Ly6a antibody (Fig. 11a). This suggests that Ly6a treatment may have a strong effect on tumor-infiltrating T cells that express high levels of Ly6a.

[0085] Next, the disclosers examined the potential of anti-Ly6a-antibody as an immunotherapy treatment in vivo. For these experiments, the disclosers used an established in vivo model of cancer response to immunotherapy. The disclosers subcutaneously injected MC38 colon cancer cells or Ret melanoma cells into C57BL / 6J mice, followed by two intraperitoneal injections of anti-Ly6a antibody. Remarkably, injection of clone E13.161 significantly inhibited the growth of tumors and improved the survival of the treated mice (Fig. 11 b, c). Clone W18174A was also active, whereas clone D7, which binds to a different epitope of the Ly6a protein, had no inhibitory effecton tumor growth (Fig. 11 b). These data demonstrate that anti-Ly6a antibody has an anti-cancer effect that is epitope specific.

[0086] Next, the disclosers tested the potential of anti-Ly6a antibody in the context of UVB-induced tumor suppression in vivo. Mice were exposed to UVB or were mock irradiated for 8 weeks. Post irradiation, 2x105 Ret melanoma cells were injected subcutaneously. Once tumors were palpable, mice were injected intraperitoneally twice a week with anti-Ly6a antibody (clone E13.161-7) or with anti-lgG2a as a control. Treatment with anti-Ly6a antibody significantly inhibited tumor growth in mice that were exposed to UVB and in those that were mock irradiated (Fig. 5c). The inhibitory impact of the anti-Ly6a antibody on tumor growth after UVB treatment was associated with an elevation in activated CD8+ cells within the tumor microenvironment (Figs. 5d and 11 d). This was in sharp contrast to the effect of anti-PD1 antibody, which did not reduce tumor growth following chronic exposure to UVB (Fig. 1h and 11 e). Thus, anti-Ly6a treatment hinders the accelerating effect of UVB-induced immunosuppression on tumor growth.

[0087] Given that chronic exposure of CD8+ T cells to type 1 IFN induces Ly6a expression (Fig. 3), the disclosers next tested whether IFNAR blockade phenocopies anti-Ly6a antibody treatment. The disclosers challenged mice with Ret melanoma cells and the treated the mice with anti-IFNAR-1 or anti-Ly6a antibodies. Like anti-Ly6a antibody treatment, INFAR-1 blockade significantly inhibited tumor growth compared to control IgG injection (Fig. 5f).

[0088] Finaly, in mice with established Ret melanoma tumors, the anti-Ly6a antibody was more effective as a second-line treatment after partial response to anti-PD1 antibody than were additional cycles of anti-PD1 antibody (Fig. 5g). Taken jointly, the disclosers results show that anti-Ly6a antibody treatment enhances the immune response to cancer even in tumors resistant to anti-PD-1 antibody. This suggests that anti-Ly6a antibody treatment has unique immunotherapeutic effect that modulates T cell phenotype and relieves suppression of T cell killing ability.

[0089] Anti -Ly6a antibody treatment enhances CD8+ T cell activity and prevents loss of mitochondrial function through effect on cMyc-mediated signaling.

[0090] To evaluate the downstream effects of anti-Ly6a antibody treatment, the disclosers examined the effect of anti-Ly6a antibodies on CD8+ T cell activity in the presence and absence of tumor cells using a high-resolution mass spectrometry proteomic analysis. Spleen cells were isolated from gp10025,28-35 TCR transgenic mice and incubated for 3 days with IL-2, IFNa, and gp10025,28-35 peptide. Next, CD8+ T cells were isolated and incubated overnight with anti-Ly6a antibody (clone E13.161) or IgG with or without B16F10 melanoma cells. CD8+ T cells were then sorted and subjected to proteomic analysis (Fig. 6a). Of the 4,202 identified proteins, 588 were significantly different between groups. Principal component analysis indicated that the proteome profiles of CD8+ T cells after Ly6a crosslinking markedly differed from the profile of IgG-exposed cells both in the presence and in the absence of melanoma cells (Fig. 6b). Unsupervised clustering of the significantly different proteins revealed five clusters indicative of the effect of anti-Ly6a antibody on CD8+ T cells (Fig. 6c). The largest cluster, cluster 1 , includes proteins that were significantly upregulated upon anti- Ly6a antibody treatment compared to those treated with IgG. Among the 269 proteins of cluster 1 , the disclosers found T cell activation markers such as CD69, Lcp-1 , and Itgb2 (Fig. 4e). The next largest cluster, cluster 5, includes proteins with decreased expression upon anti-Ly6a antibody treatment such as the transcription factors Lef1 and Tcf7, known to be expressed in naive T cells, and CD62L (encoded by Sell), which is downregulated upon T cell activation (Fig. 6c). Further, Gene Ontology (GO) analysis indicated that cluster 1 proteins were enriched in factors involved in translation and ribosome biogenesis (Fig. 6d), two well-established indicators of T cell activation. Interestingly, the disclosers found that anti-Ly6a antibody treatment significantly increased the levels of multiple proteins associated with mitochondrial metabolism, especially ATP metabolic process and fatty acid oxidation (Fig. 6e). Enrichment of factors involved in mitochondrial functions such as ATP production by cellular respiration, mitochondrial gene expression and fatty acid metabolism was observed specifically in cluster 1 (Fig. 6e and Fig. 12a, b). Thus, treatment of T cells with the anti- Ly6a antibody induces two processes, although not necessarily opposed, their copresence is somewhat surprising. On one hand, treatment of T cells with the anti-Ly6a antibody induces the expression of classical markers of T cell activation, includingupregulation of ribosomal proteins and CD69 and downregulation of CD62L, (Fig. 6c). On the other hand, treatment with the anti-Ly6a antibody also caused increased mitochondrial activity (Fig. 6c-e and Fig. 12a, b), a metabolic process that is expected to decrease during T cell activation, which is associated with increased anaerobic glycolysis.

[0091] Examination mitochondrial activity analysis using tetra-methylrhodamine ester (TMRE), a dye sensitive to membrane potential that is indicative of the mitochondrial activity level, showed that in contrast to anti-CD3 antibody-activated T cells or exhausted T cells, anti-Ly6a antibody-treated cells had high mitochondrial activity (Fig. 6f). Further, incubation of CD8+ T cells with anti-Ly6a antibody rescued T cells from exhaustion and deactivated mitochondria (Fig. 6g). These findings support the hypothesis that anti-Ly6a antibody not only activates T cells to enhance their cytotoxicity but also reprograms T cell metabolism to favor mitochondrial activity.

[0092] Since the disclosers saw markers of activation without the expected decrease in mitochondrial function, the disclosers sought to identify the specific pathways that mediate the downstream effects of anti-Ly6a antibody. First, the disclosers test whether anti-Ly6a antibody directly impacted TCR-mediated signaling. The disclosers found no change in Zap70 phosphorylation in cells treated with both anti-CD3 antibody and anti- Ly6a antibody compared to anti-CD3 antibody treatment alone. This indicates that anti- Ly6a antibody does not directly influence signaling mediated by the TCR.

[0093] Next, the disclosers used Ingenuity Pathway Analysis to predict upstream regulators of the effect of the anti-Ly6a antibody on T cell function. The disclosers found that Myc, a transcription factor that upregulates multiple metabolic pathways upon T cell activation, was the most significant upstream regulator (Fig. 6h). Myc-deficient T cells have impaired proliferation upon TCR activation and rapidly differentiate toward an exhaustion phenotype in the tumor microenvironment due to mitochondrial defects. One of the copartners of Myc is Rictor, a subunit of the mTOR complex 2 (mT0RC2), which inhibits mitochondrial metabolic activity upon T cell activation. Rictor-deficient CD8+ cells have enhanced mitochondrial metabolic activity but their effector function is not inhibited. Rictor was predicted to be an upstream regulator of Ly6a (Fig. 6h). ChronicTCR activation may enhance Rictor activity leading to exhaustion via loss of mitochondrial activity. Anti-Ly6a antibody inhibited Rictor expression. Taken together, the disclosers proteomic analysis suggests that anti-Ly6a crosslinking reprograms metabolic pathways in CD8+ T-cells via Myc induction together with mTOR complex 2 inhibitions, which can enhance the metabolic fitness of CD8+ T-cells and prevent loss of mitochondrial activity that is associated with T-cells exhaustion. Further, incubation of CD8+ T cells with anti-Ly6a antibody induced phosphorylation of Erk1 / 2 and mobilization of Myc from the cytoplasm to the nucleus (Fig. 6i, j). These data exemplify the Erk-Myc axis in mitochondrial metabolism.

[0094] Intersection of the disclosers proteomic data with a proteomic analysis of T cells treated with anti-CD3 and anti-CD28 antibodies, revealed the unique effect of anti-Ly6a antibody on T cells: The anti-Ly6a antibody enhanced mitochondrial metabolism, which does not result from TCR activation (Fig. 12d-f). In summary, the disclosers results indicate that Ly6a increase in T cells following chronic UVB or in the tumor microenvironment, is not only a marker of chronic type 1 interferon inflammatory condition, but also a target that opposite T cells immunosuppression. The unique effect of anti Ly6a may use as a model for immunotherapy treatment that, independently to T cells activation via TCR, can enhance T cells metabolism and cancer cells killing ability.

[0095] Discussion

[0096] Although the suppressive effects of UVB on the immune response to cancer have been known for several decades, little research has been conducted on how UVB specifically impacts the killing ability of CD8+ T cells, and there have been no investigations into how UVB influence the response to immunotherapy. The disclosers discovered that UVB irradiation impaired the tumor killing ability of T cells from sDLN. Moreover, UVB reduced the frequency of CD8+ TILs during anti-PD1 immunotherapy and thus caused resistance to this treatment. The disclosers UVB model therefore provided an opportunity to identify factors that suppress the T cell response to cancer independently of PD1 -mediated signaling.

[0097] The disclosers comprehensive investigation of the effects of UVB on both systemic and local compartments of the immune system revealed three significanteffects that specifically influence sDLN immune cells. Two effects, the increase in DC percentage and the increase of inflammatory monocytes percentage, are recognized as UVB immunosuppressive and inflammatory mechanisms. The third effect, the upregulation of Ly6a expression in T cells, is a novel finding. The disclosers single-cell RNA sequencing analysis revealed that the upregulation of Ly6a expression following UVB exposure was accompanied by a significant enhancement of the type 1 IFN gene signature on CD4+ and CD8+ T cells. There is an association between UVB and type 1 IFN secretion in both mice and humans. However, the link between UVB-induced type 1 IFN gene expression and UVB-induced immunosuppression had not been studied. Induction of type 1 IFN expression by UVB exposure appears to promote an inflammatory response: For example, autoimmune symptoms of systemic lupus erythematosus worsen after UVB exposure. However, there is also evidence suggesting that chronic exposure to type 1 IFNs has the opposite effect, contributing to the suppression of T cell responses to viruses and cancer. The disclosers finding that Ly6a, which was induced by type 1 IFNs, can be targeted to inhibit UVB-induced immunosuppression and to enhance the immune response to cancer indicates that the inflammatory effect of UVB and the immunosuppressive effect of UVB are not distinct phenomena but rather mutually influence each other.

[0098] Independently of UVB treatment, the disclosers observed high Ly6a levels on T cells within the tumor microenvironment. These high levels resulted from chronic exposure to factors from the tumor microenvironment rather than from TCR activation. Thus, similar to the effect resulting from UVB exposure, type 1 IFNs secreted by cells of the tumor microenvironment contribute to the increase in Ly6a. Although IFNy and IL-27 have been shown to modulate Ly6a expression in parasitic protozoan models, the present data support type I interferons (IFNa and I FNp) as the dominant drivers of Ly6ahighT cell induction in the tumor microenvironment. The contribution of other cytokines, including IFNy and IL-27, is also contemplated. Type 1 IFN is an activator of T cells during acute viral infections; however, when chronically present, it suppresses the T cell response to viruses and tumors. Further, high levels of expression of IFN signature genes on T cells are correlated with lack of response to immunotherapy. The disclosers found that CD8+ T cells that are chronically exposed to IFNa can beactivated by antibodies against Ly6a, suggesting opportunities for development of new immunotherapy treatments.

[0099] Since the endogenous ligand of Ly6a is not known, it is currently not possible to fully analyze the biological role of Ly6a. Nonetheless, several of the disclosers findings strongly suggest that anti-Ly6a antibody does not block its interactions with a ligand. First, incubation of CD8+ T cells with the antibody induces expression of activation markers such as CD69 and ribosomal proteins and elevates the metabolic and mitochondrial activity of CD8+ T cells. Second, incubation of CD8+ T cells with anti- Ly6a antibody rescued T cells from exhaustion, deactivated mitochondria, induced phosphorylation of Erk1 / 2, and induced mobilization of Myc from the cytoplasm to the nucleus. These data suggest that the influence of anti-Ly6a antibody on CD8+ T cells does not depend on the presence of a ligand found on other cells of the immune system or on cancer cells.

[0100] The mechanism by which T cells are activated by anti-Ly6a antibody exemplifies the capacity to trigger T cell responses independently of TCR signaling. Recently, there is increasing evidence suggesting that even TILs not specifically targeting tumors can exhibit activity against cancer cells. Bystander activated T cells possess the ability to recognize and eliminate cancer cells in a manner reminiscent of innate immunity. The potential of an antibody targeting Ly6a to augment the immune response, even in cases of anti-PD1 resistance, is linked to this distinctive mechanism. The disclosers anticipate that the capability to activate TILs independently of TCR signaling will complement existing immunotherapies, offering a promising approach for treating patients with resistance to current immunotherapy treatments.

[0101] Methods

[0102] Mice

[0103] All animal experiments were performed in accordance with the guidelines of the Tel Aviv University Institutional Animal Care and Use Committee using approved protocols (IACUC permits: 01-20-033 and 01 -19-086). Animals were housed under specific pathogen-free conditions under 12 h dark / 12 h light conditions at 22 ± 1 °C and32-35% humidity with ad libitum water and food. C57BL / 6 mice aged 7-8 weeks old were purchased from Envigo™ and were allowed to acclimatize for one week after arrival. OT1 TCR C57BK / 6 CD45.1 + mice expressing the T cell receptor that recognizes ovalbumin peptide (amino acids 257-264) were a kind gift from Professor Steffen Jung (Weizmann Institute of Science). gp1OO mice expressing the transgenic Pmel 17 TCR36 were a kind gift from Professor Michal Lotem (The Hebrew University of Jerusalem).

[0104] UVB treatment

[0105] Seven to eight-week-old C57BL / 6J female were habituated for 7 days prior to experiment initiation and dorsally shaved 2 days prior radiation exposure. Mice were exposed to UVB 5 days / week for a period of 8 weeks in reverse light setting with a XX- 15 stand equipped with 15-W, 302-nm UVB bulbs (Ultraviolet Products™). The dose of UVB was increased gradually to mimic the protocol used for human UVB phototherapy53. Starting at an initial dose of 50 mJ / cm2, a sub-erythematic dose in this mouse strain54, the UVB dose was increased by 30% at each treatment reaching a final dose of 200 mJ / cm2. UVB emission was measured using a UVX radiometer (Ultra- Violet Products) equipped with a UVB-measuring head. Mock-irradiated mice were shaved and placed in the radiation chamber for equal amount of time. Mice were reshaved once a week if there were patches of fur regrowth.

[0106] In vivo tumor growth models

[0107] All the animal experiments were conducted according to the guidelines of the Tel Aviv University Institutional Animal Care and Use Committee (#01 -21 -046). For / n vivo tumor growth, cultured tumor cells suspended in PBS at 2x105 cell / 50 pl for MC38 and B16 cells, 1x106 cells / 50 pL for B16-ova cells (in extended data figure 4b) 2x105 cells / 50 pL for Ret melanoma cells in figure 1 experiments and 1x106 cells / 50 pL for Ret melanoma cells in figure 5 experiments (for partial resistance to anti-PD-1), and were mixed in a 1 :1 ratio with growth factor-reduced Matrigel (BD Biosciences™) for subcutaneous injection. An aliquot of 100 pl was injected subcutaneously into the shaved area on the dorsal side of the mouse. The height and width of the subcutaneous tumors were measured twice a week using calipers. Mice were sacrificed when the tumors reached 200 mm2. To induce lung metastases, ReTMChery+luciferase+melanomacells were suspended in PBS (5x105 cells / 100 pL), and an aliquot of 100 pl was injected into the tail vein. This line is widely used in melanoma models and induces spontaneous melanoma progression that resembles human melanoma in its progression, metastases potential, melanotic level, and immunogenicity. It results in activation of MAPK and eJun signaling, which are downstream of the Ret oncogene. Lung tumors were quantified twice a week by in vivo imaging beginning on day 7 after injection. For in vivo imaging, mice were anesthetized using 2.5% isoflurane and administered 150 mg / kg D-luciferin (Biovision™) by intraperitoneal injection. Ex vivo imaging of lungs and liver was performed after sacrifice and pulmonary artery perfusion. The average radiance (photons / s / cm2 / sr) was calculated using an I VIS Lumina III system (PerkinElmer™).

[0108] Survival curve

[0109] Mice were their subcutaneously injected as described before. When tumor reached 2cm length, mice were sacrificed as per the ethical approval. The scarified date was taken into survival curve analysis. Significance was determined using two-tailed long rank test.

[0110] / n vivo immunotherapy treatment

[0111] Mice were injected intraperitonealy with 200 pg of anti-PD1 (RMP1-14, BioXCell), 200 pg of rat lgG2a isotype control (BioXCell), or 100 pg anti-Ly6a (clone D7, E13.161 , or W18174A; Biolegend™) as indicated.

[0112] Cell culture

[0113] B16F10 melanoma cells, Ret melanoma cells, and MC38 colon cancer cells were cultured in RPMI (Biological Industries™), supplemented with 10% fetal bovine serum (FBS; Gibco) and 1 % penicillin / streptomycin / L-glutamine (Biological Industries). Generation of Ret melanoma cells, which stably express mCherry and luciferase, was conducted by transfecting the PLKO-mCherry-luc-puro plasmid (Addgene™) into the ReT-cells using the jetPEI DNA transfection reagent (Polypus). After 48 h, the stable clones were selected by culture in puromycin (Sigma-Aldrich™, 10 pg / ml). All cells were cultured at 37 °C with 5% CO2.

[0114] Primary cell isolation

[0115] Spleen and lymph node were homogenized in Hank’s balanced salt solution (Gibco) supplemented with 2% FBS and 5 mmol / L EDTA and were isolated through a 70-pm strainer (Coming). Tumors were digested in RPMI 1640 with 2 mg / mL collagenase IV, 2,000 U / mL DNase I (Sigma Aldrich) for 30 minutes at 37 °C and then homogenized and strained through a 70-pm strainer.

[0116] For killing assays, mononuclear layers were collected from splenocyte suspensions using Ficol Histopaque®-1077 (Sigma Aldrich), washed by adding 10 rnL of RPMI 1640 medium, and centrifuged at 700 xg for 10 minutes at 18 °C. The pellet of spleen mononuclear cells or the pellet of sDLN cells was re-suspended in MACS buffer (0.5% BSA, 2 mM EDTA in PBS). CD8+ T-cells enrichment was conducted using CD8 mouse magnetic MicroBeads (Miltenyi Biotec™) according to manufacturer's instructions. CD4+ and CD8+ cells were enriched by negative selection using Pan T- cells Isolation Kit II, mouse (Miltenyi Biotec). CD11 b+ cells were isolated using magnetic CD11 b MicroBeads and column (both from Miltenyi Biotec). Ly6anegative / low T-cells were sorted from spleen mononuclear cells using FACS (BD FACSAria III, BD Biosciences™).

[0117] / n vitro killing assay

[0118] Spleen and lymph node cells of OT-1 mice were isolated post 8 weeks of UVB radiation 5 days a week (Fig. 1) or spleen cells of gp100 mice (Fig. 5) were activated with OVA peptide or gp100 peptide and with IL-2 (100 lU / mL; PeproTech™) for 48 h. After activation CD8+ cells were isolated as described above and cultured for 48 h with IL-2 (200 lU / mL; PeproTech™). CD8+ T-cells were co-cultured with B16-OVA (4:1). Melanoma cells were also cultured alone. Cultures were treated with antibodies to Ly6a, clones D7, E13.161 , or W18174A (Biolegend™) or REA422 (Miltenyi Biotec™), Ly6e (EB10216, Everst Biotech™), or rat lgG2a isotype control (BioXCell™) at concentrations of 10 pg / ml or as indicated. The percentage of cells positive for caspase-3 / 7 (clone #C10423, Invitrogen; dilution used 1 :1000) was determined after 24 h. The number of melanoma cells was normalized to the number of melanoma cells in each well at time 0. Analysis was performed using the IncuCyte® Live Cell Analysis System (Sartorius).Samples were also analyzed after 24 h using flow cytometry (CytoFLEX™, Beckman Coulter™) for the percent of CD45- / CD8- / caspase-3 / 7+ / DAPI+ cells from total CD45- / CD8- cells in the wells.

[0119] Flow cytometry

[0120] Cells were analyzed using flow cytometry (CytoFLEX, Beckman Coulter) and sorted by FACS (BD FACSAria III, BD Biosciences). Datasets were analyzed using Kaluza software (Beckman Coulter). Intracellular flow cytometry as performed using Miltenyi Biotec fixation buffer and permeabilization buffer according to the manufacture protocol. The following antibodies were used: CD4-FITC, mouse (lgG2bK, Miltenyi Biotec), CD8-APC-VIO770 APC (REA734, Miltenyi Biotec), CD11 b-PE (REA592, Miltenyi Biotec), Ly6c-APC (REA796, Miltenyi Biotec), Ly6g-FITC (REA526, Miltenyi Biotec), CD3-VioBlue (BW264 / 56, Miltenyi Biotec), CD45 APC (REA737, Miltenyi Biotec), Ly6A-PE (REA422, Miltenyi Biotec), CD44 APC (REA664, Miltenyi Biotec), MHC2 APC (M5 / 114.15.2, Biolegend), CD11c (N418, Biolegend), FoxP3 Alexa fluor 647 (MF-14, Biolegend), CD25 BV650 (PC61 , Biolegend), REA control antibody- PE (isotype control, REA293, Miltenyi Biotec), CD69 (clone REA937) and CD62L (clone MEL14-H2.100).H-2Kb:OVA (SINFEKEL) tetramers conjugated to APC were purchased from MBL™ (Woburn, MA). Anti-phopho-Erk1 / 2 (clone 6B8B69, Biolegend).

[0121] Mass cytometry analysis

[0122] High-throughput mass cytometry analyses were performed. Briefly, sDLN cells or splenocytes were isolated, fixed, and permeabilized with 100 pl of 2% PFA in CyPBS and eBioscience perm buffer and then stained with metal-tagged antibodies to intracellular markers. All antibodies were conjugated using the Maxpar® reagent (Fluidigm). The antibodies used are described in Table S1. Rhodium and iridium intercalated were used to identify live / dead cells. Cells were washed twice with PBS, fixed in 1.6% formaldehyde (Sigma-Aldrich), and then washed with H2O. Data were acquired using a CyTOF mass cytometry system (Fluidigm™) and uploaded to the Cytobank web server. CD45+ live cells were analyzed, and the gated cells were segregated into subpopulation clusters by expression markers. Data analysis was performed using UMAP56 and FlowSOM57 algorithms for unsupervised analysis and atraditional flow cytometry gating strategy for supervised analysis on the Cytobank server. After removal of dead cells, CD45- cells, and low-quality events, the disclosers separately analyzed the major immune cell populations (T cells, B cells, and other cells). Each population was analyzed separately using only markers relevant for each cell type (Table S1). Results of one algorithm were not used as an influencing factor in the analysis by the other algorithm; however, the number of clusters in the FlowSOM analysis was determined by the number of separate populations observed after reducing the dimensions in the UMAP algorithm. There were 6 clusters for CD4+ T cells, 6 for CD8+ T cells, 5 for B cells, and 12 for all other cells.

[0123] / n vitro cytokine treatment

[0124] CD4+ and CD8+ T cells, isolated from C57BL / 6J mouse spleens, were treated with the 0.5 pg / mL anti-CD3 (clone 17A2, BioLegend), 0.5 pg / mL anti-CD28 (clone 37.51 , Biolegend), anti-LEAF (BioLegend), LPS (Invivogen), or CL307 (TLR7 agonist, Invivogen) or with IFNa, IFNy, IL-1 , IL-6, IL-12, TNFa (10 ng / mL; Miltenyi Biotec) or IL-2 (1000 lU / mL; PeproTech). After 24 h, cells were analyzed by flow cytometry for Ly6a.

[0125] US and MS imaging of tumor growth

[0126] Ultrasound measurements were performed using a portable ultrasound device (Mindray M9, Shenzhen Mindray Bio-medical Electronics™). The ultrasound transducer was placed on the tumor center, and the tumor depth measurement was performed. Doppler imaging was used to evaluate blood flow within the tumor area. Ex vivo imaging of lungs and liver was performed after sacrifice and pulmonary artery perfusion using D- luciferin. The bioluminescence signals were detected using an I VIS Lumina III system (PerkinElmer).

[0127] RN purification and quantitative RT-PCR

[0128] Quantitative RT-PCR was performed to quantify mCherry mRNA for detection micro metastases as described before. Lymph nodes and liver were homogenized, and total RNA was extracted using Trizol (Invitrogen™). cDNA was produced using the cDNA SuperMix kit (QuantaBio™) and then subjected to qRT-PCR using the Blue SYBR low ROX kit (PCR Biosystems™). mRNA levels were normalized to endogenousactinp. Naive lymph nodes and liver were used as negative controls. The qRT-PCR primers were mCherry forward, 5'-GAACGGCCACGAGTTCGAGA-3'; mCherry reverse, 5'-CTTGGAGCCGTACATGAACTGAGG-3'; actinp forward, 5'- CTGTCCCTGTATGCCTCTG-3'; and actinp reverse, 5'-ATGTCACGCACGATTTCC-3'. For the other genes the disclosers used these primers: 36B4 forward, 5'- GATGATCAAAGGGATGTGGC-3' and reverse, 5'-AGCTCGGCAACAGACTCTTC-3'.

[0129] RNA and single-cell RNA sequencing dataset analyses

[0130] Single-cell RNA sequencing data of CD4+ cells from spleens of young and old mice were used for analysis of Ly6a expression in CD4+ cell subsets. The disclosers used this dataset because of the deep separation of the CD4+ cells into seven different subsets. The read counts were download from https: / / singlecell.broadinstitute.org / single_cell, normalized as described in the original analysis. (The expression level of a gene was divided by total expression of all genes in that subset and multiplied by 10,000. This product was log-transformed (base e) after the addition of a pseudocount of 1). For analysis genes co-expressed with Ly6a in effector memory and exhausted CD4+ T cells, the disclosers segregated the cells defined in the metadata of the original analysis as effector memory (2181 cells) or exhausted (1841 cells) according to Ly6a expression levels and compared the expression level of each gene in Ly6aneg cells to Ly6ahigh cells (defined as level of Ly6a is more than the median expression of cells with any level of Ly6a.

[0131] Single cell RNA sequencing data from a human melanoma patient samples34,59 and from a triple negative breast cancer) were used to examine the expression of human Ly6 family genes. Normalized expression data from CD8+ cells from immunotherapy responders and non-responders were analyzed.

[0132] Proteomics

[0133] Samples were prepared by sorting 12,000 CD8 T-cells (>98% purity) into separate wells in a 96 wells plate (Eppendorf twin. tec® PCR Plates) containing 33 ul of lysis buffer [composed of 2% Sodium dodecyl sulfate (SDS), 20mM Tris(2-carboxyethyl) phosphine (TCEP), 80 mM Chloroacetamide (CAA)] and the total volume wascompleted with 100mM HEPES solution (pH=8.5). After sorting, the plates were spun to enable proper mixing between the cells and the lysis buffer and heated at 95°C for 5 minutes. Further sample preparation was based on the SP3 protocoled, which is suited for samples with low protein amounts. The first stage of the protocol was done using the Bravo Automated Liquid Handling Platform (Agilent™) and the second stage was done manually. Briefly, magnetic beads (SeraMag™ magnetic beads; Thermo Fisher Scientific™) were added to the samples followed by the addition of ethanol (final concentration 50%), and incubated to allow binding of the proteins to the beads. Beads were then washed twice with ethanol using a magnetic rack and incubated for overnight digestion with trypsin (Promega™). Thereafter, the samples were transferred to clean lobind tubes and fresh beads were added with the addition of acetonitrile (ACN) to promote the attachment of peptides to the beads. Samples were washed once more with ACN using a magnetic rack. Finally, to release the peptides from the beads, 2% DMSO (in water) was added. Samples were then collected in lobind tubes. ~3 pl of each sample were loaded directly into Evotips (Evosep™) for proteomic analysis. All reagents are LC-MS grade.

[0134] For all other proteome analyses, the disclosers used an EvoSep One liquid chromatography system and analyzed with a 40 SPD gradient. The disclosers used a 15 cm nanoflow UHPLC packed emitter column with nanoZero® technology, compatible with Bruker CaptiveSpray source. (Ion optics™). Mobile phases A and B were 0.1 % FA in water and 0.1 % FA in ACN, respectively. Mass spectrometric analysis was performed in a data-independent (dia) PASEF mode. Raw files were analyzed with DIA-NN62 (version 1.8) using default settings (e.g., 1 % precursor and protein FDR), except, enabling MBR, and quantification strategy to robust LC (high precision), and library generation to IDs, RT, and IM profiling using the Uniprot Mus musculus proteome. The DIA-NN protein group data output was filtered first for at least 70% in at least one group (out of 4), imputed, and then quantile normalized. Data were analyzed through the use of IPA (QIAGEN Inc., https: / / www.qiagenbioinformatics.com / products / ingenuity- pathway-analysis). using “upstream analysis” and using STRING analysis at https: / / stringdb.org / as indicated in the results.

[0135] Immunofluorescence of cells

[0136] For immunocytochemistry, cells were fixed with 1.8% paraformaldehyde (PFA) for 20 minutes at RT, and permeabilized with 0.05% Triton-X. Cells were then washed, blocked for 1 h with PBS containing 10% goat serum and 5% BSA and stained overnight with primary antibody to anti-MYC antibody (clone 9E10, Abeam) at 4°C. Cells were stained for nuclear staining with DAPI (vector laboratories™) and imaged under Zeiss800 confocal microscope and MYC nuclear localization was analyzed by Zen Blue™ software.

[0137] Single Cell RNA-sequencing

[0138] Library preparation

[0139] 10pL of sorted cells were stained with Trypan Blue and counted using a hemocytometer and optical microscope. Cells were then centrifuged at 300 G for 5 min, resuspended into sorting medium at concentration of 1000 cells I pL. Cells were encapsulated into droplets, and libraries were prepared using Chromium Single Cell 30 Reagent Kits v3 according to manufacturer’s protocol (10* Genomics). The generated single-cell RNA-seq libraries were sequenced using a 75 cycle NextSeq 500 high output V2 kit.

[0140] Droplet-based single-cell RNA-Seq data processing

[0141] FASTQ files were created using CellRanger mkfastq function (v7 xGenomics). Gene counts were obtained by aligning reads to the mm10 genome using CellRanger count function. To remove doublets and poor-quality cells, the disclosers excluded cells that: Contained more than 25% mitochondrially-derived transcripts. Contained less than 200 or more than 5,000 genes detected. Contained less than 200 or more than 25,000 detected molecules. The filtering process yielded 33,703 cells. Fthe disclosers samples of lymph nodes from control mice and 3 samples of lymph nodes of UV treated mice were merged and transcript counts were normalized and scaled using the NormalizeData ScaleData functions in Seurat 4.3.264 R package, which normalizes scales, and centers features in the dataset. For principal component analysis (PCA) and clustering, the disclosers used a log-transformed expression matrix. Variably expressedgenes were used to construct principal components (PCs) and PCs covering the highest variance in the dataset were selected. The selection of these PCs was based on an elbow plot.

[0142] Single-cell RNA-seq clustering

[0143] Clusters were calculated by the FindClusters function with a resolution between 0.25 and 0.35 and visualized using the t-SNE dimensionality reduction method. The FindClusters function in Seurat was used to identify clusters of cells by a shared nearest neighbor (SNN) modularity optimization-based clustering algorithm. First, the disclosers calculated the k-nearest neighbors and constructed the SNN graph. To generate tSNE plots of single cell profiles, the disclosers used the Seurat's RunTSNE function.

[0144] Genes differentially expressed between clusters

[0145] The FindAIIMarkers Seurat function was used to find marker genes that were differentially expressed between clusters. This function identifies differentially expressed genes between two groups of cells using a Wilcoxon Rank Sum test with limit testing chosen to detect genes that display an average of at least 0.25-fold difference (logscale) between the two groups of cells and genes that are detected in a minimum fraction of 0.25 cells in either of the two populations.

[0146] Visualization of single-cell data

[0147] To generate both CD4 and CD8 tSNE plots, the scores along the 13 or 15 significant PCs described above were used as input for the R implementation of tSNE, by the RunTSNE Seurat function. Specific gene expressions were visualized over the tSNE using FeaturePlot Seurat function and some genes were visualized using the VlnPlot Seurat function.

[0148] Single-cell gene signature scoring

[0149] Single-cell gene signature scoring was performed. Briefly, as an initial step, to remove bias towards highly expressed genes the data was scaled using z-score across each gene. For a given gene signature (list of genes), a cell-specific signature score was computed. Scores were computed by first sorting the normalized scaled gene expression values for each cell followed by summing up the indices (ranks) of thesignature genes. The same method was also applied to gene-signatures which includes both up-regulated and down-regulated genes. In this case, two ranking scores were obtained separately, and the down-regulated associated signature score was subtracted from the up-regulated generated signature score. A contour plot which takes into account only those cells that have a signature score above the indicated threshold was added on top of the tSNE space, in order to further emphasize the region of highly scored cells.

[0150] Statistical analyses

[0151] All statistical analyses were conducted in Prism (GraphPad Software, lnc.TM). For time-course experiments, significance was calculated using two-way ANOVA with Tukey’s correction for multiple hypotheses. For analysis of more than two groups, oneway ANOVA was used, with Tukey’s correction for multiple hypotheses. A t test was used for comparisons between two groups. For the analysis of RNA and single-cell RNA sequencing data, FDR correction for multiple comparison was used. All the statistical tests were two-tailed.

[0152] Additional Aspects and Definitions

[0153] It is to be understood that the aspects of the disclosure described herein are not limited to the specific embodiments presented and can vary. The terminology used in this disclosure is intended to describe particular examples and is not limiting, unless otherwise defined or clear from the context. Embodiments described herein can be combined with one another, as would be understood by a person of ordinary skill in the art, unless stated otherwise.

[0154] Unless explicitly stated or apparent from context, the term “or” as used herein is intended to be inclusive, meaning “and / or.” Likewise, the articles “a,” “an,” and “the” are intended to encompass both singular and plural referents unless the context clearly dictates otherwise.

[0155] In certain embodiments, the anti-LY6E antibody may be administered in combination with one or more immune checkpoint inhibitors. Suitable checkpoint inhibitors include, but are not limited to, antibodies that bind to PD-1 (e.g., nivolumab,pembrolizumab), or CTLA-4 (e.g., ipilimumab, tremelimumab). Co-administration of anti- LY6E antibodies with checkpoint inhibitors may enhance anti-tumor immune responses by synergistically promoting CD8+ T cell activation and reversing exhaustion in the tumor microenvironment. The agents may be administered simultaneously, sequentially, or in overlapping regimens, or before or after administration of anti-LY6E antibodies.

[0156] As used herein, a subject is considered to exhibit resistance to current cancer treatment if the subject's cancer fails to regress, continues to grow, or recurs following administration of one or more established therapeutic interventions, including but not limited to immune checkpoint inhibitors (such as anti-PD1 or anti-CTLA-4 antibodies), chemotherapy, targeted therapies, or radiation. Resistance can include both primary resistance, in which the subject does not respond to therapy from the outset, and acquired resistance, in which the subject initially responds but subsequently relapses or progresses despite continued treatment. In some embodiments, resistance can be identified by radiographic progression, stable disease without tumor shrinkage, or increased tumor burden following one or more treatments.

[0157] The treatment methods and compositions described herein can be applied to a wide range of cancer types in which activation of cytotoxic T cells contributes to antitumor efficacy. These include, but are not limited to, solid tumors such as melanoma, colorectal cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, renal cell carcinoma, ovarian cancer, prostate cancer, head and neck squamous cell carcinoma, and hepatocellular carcinoma, as well as hematologic malignancies such as leukemias and lymphomas. In various embodiments, the disclosed anti-LY6E antibody can be used in cancers where T cell activity is functionally relevant for tumor control, but is limited or suppressed by immunosuppressive factors in the tumor microenvironment. Such factors can include chronic type I interferon signaling, T cell exhaustion, expression of checkpoint ligands, and the presence of suppressive myeloid or stromal cells. In these contexts, treatment with the anti-LY6E antibody can restore or enhance T cell function, including cytotoxicity, mitochondrial metabolism, and expansion of memory-like T cell populations, thereby promoting tumor regression.

[0158] As used herein, the term “a therapeutically effective amount” refers to an amount of an anti-LY6E antibody (or composition comprising the same) that is sufficient to achieve a desired therapeutic effect in the subject being treated. In the context of cancer treatment, a therapeutically effective amount can refer to an amount that results in inhibition of tumor growth, reduction in tumor size, prevention of tumor progression, delay in disease recurrence, improvement in survival, enhancement of immune- mediated tumor clearance, reprogramming of exhausted T cells, or improvement in any clinical parameter associated with anti-tumor efficacy. The precise effective amount can vary depending on factors such as the type and stage of cancer, the route of administration, the subject's immune status, and whether the antibody is administered alone or in combination with other therapeutic agents.

[0159] In certain embodiments, the anti-LY6E antibody can be administered concurrently with chemotherapy to enhance therapeutic efficacy. Suitable chemotherapeutic agents can include, but are not limited to, alkylating agents (e.g., cyclophosphamide, temozolomide), antimetabolites (e.g., 5-fluorouracil, gemcitabine, pemetrexed), topoisomerase inhibitors (e.g., irinotecan, topotecan, doxorubicin), microtubule-targeting agents (e.g., paclitaxel, docetaxel, vincristine), and platinumbased compounds (e.g., cisplatin, carboplatin, oxaliplatin). The concurrent administration can be simultaneous or sequential, and can occur within the same treatment cycle or therapeutic regimen. In some embodiments, chemotherapy can sensitize tumors to T cell-mediated killing by increasing antigen release, reducing immunosuppressive cell populations, or modulating the tumor microenvironment. In such contexts, the anti-LY6E antibody can further enhance T cell cytotoxicity and mitochondrial metabolism, contributing to synergistic tumor clearance.

[0160] As used herein, the term “anti-LY6E antibody” refers to an antibody or antigenbinding fragment thereof that selectively binds to a LY6E polypeptide and is capable of inducing crosslinking of LY6E on the surface of T cells. In some embodiments, binding to a specific LY6E epitope can induce LY6E crosslinking, which in turn modulates T cell function. Anti-LY6E antibodies described herein can enhance cytotoxicity, mitochondrial metabolism, and effector function in CD8+ T cells through downstream signalinginitiated by LY6E crosslinking. The antibody can be a monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, fully human antibody, recombinant antibody, affinity-matured antibody, or multispecific antibody (e.g., bispecific or trispecific). The term further includes antigen-binding fragments such as Fab, F(ab')2, scFv, diabodies, triabodies, minibodies, nanobodies, and other engineered variants, provided they retain the ability to bind LY6E and modulate T cell activity. Binding interactions can involve non-covalent forces such as hydrogen bonding, hydrophobic interactions, and ionic bonds, and may exhibit a dissociation constant consistent with high-affinity antigen recognition. Antibodies described herein can be of any isotype or subclass. In certain preclinical embodiments, antibodies may target LY6A, the murine homolog of LY6E.

[0161] In some embodiments, the anti-LY6E antibody is administered once or twice every one, two, three, or four weeks. Follow-on doses can be administered at intervals determined based on the patient’s clinical response, tumor burden, age, weight, and physician judgment. In certain embodiments, multiple doses of the anti-LY6E antibody are administered, including at least three, four, five, six, or more doses over a period of two, four, six, eight, twelve, or twenty-four weeks, or over a period of one year or longer.

[0162] In various embodiments, the anti-LY6E antibody is administered by parenteral routes, such as intravenous infusion or subcutaneous injection. In some embodiments, the antibody is administered by intravenous infusion.

[0163] As used herein, the term “epitope” refers to a localized region of an antigen that is specifically recognized and bound by a binding molecule, such as an antibody. Epitopes can be linear, consisting of contiguous amino acid sequences, or conformational (also referred to as discontinuous or non-linear), in which the recognized amino acids are non-contiguous but brought together in three-dimensional space by the folding of the polypeptide. In some embodiments, antibody binding to a linear epitope can be independent of the antigen's secondary or tertiary structure, while in other embodiments, binding can require a specific conformation of the epitope.

[0164] As used herein, the terms “polypeptide,” “peptide,” and “protein” can be used interchangeably and refer to a polymer of amino acids of any length, which can be linearor branched and may include modified residues or non-amino acid components. Polypeptides may be naturally occurring, synthetically produced, or recombinantly expressed, and may include post-translational modifications such as glycosylation, disulfide bonding, lipidation, phosphorylation, or acetylation. In certain embodiments, the polypeptides disclosed herein comprise one or more domains of an antibody or are components of an antibody-based therapeutic.

[0165] As used herein, the terms “subject” and “patient” can be used interchangeably and refer to a mammal, including but not limited to a human, dog, cat, mouse, rat, nonhuman primate, or livestock animal. In some embodiments, the subject is a human diagnosed with or at risk for developing a condition or disease, such as cancer, for which treatment with an anti-LY6E antibody is indicated.

[0166] As used herein, the terms “cancer” or “cancer cell” refer to cells that exhibit characteristics distinguishing them from normal cells, including but not limited to: morphological abnormalities, dysregulated growth, increased mitotic activity, invasive or metastatic potential, anaplasia, and expression of tumor-associated antigens or markers. As used herein, the term encompasses malignant and pre-malignant conditions and includes, without limitation, melanomas, carcinomas, sarcomas, leukemias, lymphomas, and other neoplasms.

[0167] Although certain experimental studies described herein are performed using murine antibodies (e.g., anti-LY6A) in mouse models, the disclosure is not limited to murine antibodies, and the therapeutic antibodies provided by this disclosure can be used for human clinical use. The anti-LY6E antibodies described herein can be fully human or humanized antibodies that bind the human LY6E antigen. Mechanistic insights and therapeutic effects observed in murine systems using anti-LY6A antibodies are relevant to human applications, as LY6A is a functional homolog of LY6E and exhibits conserved expression and signaling features on T cells. Accordingly, murine in vivo and ex vivo results provide predictive and translational support for the clinical utility of anti-LY6E antibodies in human subjects.

[0168] As used herein, the term “T cells” includes both CD4+ and CD8+ T cell subsets unless otherwise specified. The treatment methods described herein can be applied tomodulate the function of either or both CD4+ and CD8+ T cells. In certain embodiments, treatment with an anti-LY6E antibody can be used to reprogram CD4+ T cells, CD8+ T cells, or both, including by enhancing mitochondrial metabolism, reversing exhaustion, increasing cytotoxic activity, or modulating expression of activation markers.

Claims

CLAIMS1 . A method for treating cancer in a subject, comprising: administering a therapeutically effective amount of anti-LY6E antibody to the subject.

2. The method of claim 1 , further comprising: administering an IFNAR blockade to the subject, wherein the IFNAR blockade inhibits type-1 interferon (IFN) signaling thereby enhancing the cytotoxic activity of T cells.

3. The method of claim 2, wherein the IFNAR blockade comprises an agent that inhibits binding of type I interferons to IFNAR1 or IFNAR2.

4. The method of claim 2, wherein the IFNAR blockade comprises an antibody selected from the group consisting of an anti-IFNa antibody, an anti-IFNp antibody, and an anti-IFNAR antibody.

5. The method of claim 1 , wherein treatment with anti-LY6E antibody reverses type I interferon-induced T cell exhaustion.

6. The method of claim 1 , wherein treatment with the anti-LY6E antibody promotes T cell cytotoxic activity and tumor regression in a subject whose cancer has failed to regress on anti-PD1 therapy.

7. The method of claim 1 , wherein treatment with the anti-LY6E antibody is administered after administration of an anti-PD1 antibody.

8. The method of claim 1 , wherein treatment with the anti-LY6E antibody is administered prior to administration of an anti-PD1 antibody.

9. The method of claim 1 , wherein treatment with the anti-LY6E antibody induces phosphorylation of Erk1 / 2 and nuclear translocation of Myc in T cells, thereby reprogramming mitochondrial metabolism and enhancing anti-tumoral cytotoxic activity in the subject.

10. The method of claim 1 , wherein treatment with the anti-LY6E antibody increases Granzyme B expression in T cells.11 . The method of claim 1 , wherein treatment with the anti-LY6E antibody increases expression of at least one of CD69, Lcp-1 , and Itgb2 in the subject’s T cells.

12. The method of claim 1 , wherein treatment with the anti-LY6E antibody decreases expression of at least one of Lef1 , Tcf7, and CD62L in the subject’s T cells.

13. The method of claim 1 , wherein treatment with the anti-LY6E antibody enhances mitochondrial metabolism of T cells.

14. The method of claim 1 , wherein treatment with the anti-LY6E antibody upregulates MYC expression in T cells.

15. The method of claim 1 , wherein treatment with the anti-LY6E antibody downregulates mT0RC2 signaling in T cells.

16. The method of claim 1 , further comprising: administering an agent that inhibits LY6E induction by blocking type I interferon signaling.

17. The method of claim 16, wherein the agent is selected from the group consisting of an anti-IFNa antibody, an anti-IFNp antibody, and an anti-IFNAR antibody.

18. The method of claim 1 , wherein the anti-LY6E antibody binds an epitope on LY6E thereby inducing crosslinking of LY6E in the subject’s T cells.

19. The method of claim 1 , wherein treatment with the anti-LY6E antibody activates tumor-infiltrating lymphocytes independently of T cell receptor (TCR) signaling.

20. The method of claim 19, wherein the tumor-infiltrating lymphocytes include bystander CD8+ T cells that are not specific for tumor antigens.21 . The method of claim 1 , wherein treatment with the anti-LY6E antibody increases expression of mitochondrion-associated proteins in the subject’s T cells.

22. The method of claim 1 , wherein treatment with the anti-LY6E antibody inhibits expression of Rictor in the subject’s T cells.

23. The method of claim 1 , wherein the cancer is selected from the group consisting of wherein the cancer is selected from the group consisting of solid tumors and hematologic malignancies.

24. The method of claim 1 , wherein the cancer is resistant to checkpoint inhibitor therapy.

25. The method of claim 1 , wherein the cancer is characterized by elevated LY6E expression on T cells in the tumor microenvironment.

26. The method of claim 1 , wherein the anti-LY6E antibody enhances killing of tumor cells by CD8+ T cells.

27. The method of claim 1 , further comprising: administering an immune checkpoint inhibitor selected from the group consisting of an anti-PD1 antibody and anti-CTLA-4 antibody.

28. The method of claim 1 , wherein the anti-LY6E antibody is administered concurrently with chemotherapy.

29. The method of claim 1 , wherein the anti-LY6E antibody is administered after development of resistance to a PD-1 inhibitor.

30. A composition for treating cancer in a subject, the composition comprising: an anti-LY6E antibody.31 . A method for identifying a subject likely to benefit from treatment with an anti- LY6E antibody, comprising: detecting elevated LY6E expression on T cells in a tumor sample from the subject, wherein the elevated expression is relative to LY6E expression on T cells in non-tumor tissue from the same subject or from a healthy control.

32. A method for modulating immune response in a subject with elevated LY6E expression on T cells, comprising: administering anti-LY6E antibody to the subject, wherein the anti-LY6E antibody induces crosslinking of LY6E on T cells thereby reprogramming the subject’s T cells.

33. The method of claim 32, wherein the anti-LY6E antibody enhances mitochondrial metabolism of T cells.

34. The method of claim 32, wherein the anti-LY6E antibody upregulates MYC expression in T cells.

35. The method of claim 32, wherein the anti-LY6E antibody downregulates mT0RC2 signaling in T cells.

36. The method of claim 32, wherein the anti-LY6E antibody reverses exhaustion in tumor-infiltrating T cells.

37. The method of claim 32, wherein the anti-LY6E antibody promotes expansion of effector memory T cells.

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