PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity

Abstract

CD8⁺ T cell exhaustion is a state of dysfunction acquired in chronic viral infection and cancer, characterized by the formation of Slamf6⁺ progenitor exhausted and Tim-3⁺ terminally exhausted subpopulations through unknown mechanisms. Here we establish the phosphatase PTPN2 as a new regulator of the differentiation of the terminally exhausted subpopulation that functions by attenuating type 1 interferon signaling. Deletion of Ptpn2 in CD8⁺ T cells increased the generation, proliferative capacity and cytotoxicity of Tim-3⁺ cells without altering Slamf6⁺ numbers during lymphocytic choriomeningitis virus clone 13 infection. Likewise, Ptpn2 deletion in CD8⁺ T cells enhanced Tim-3⁺ anti-tumor responses and improved tumor control. Deletion of Ptpn2 throughout the immune system resulted in MC38 tumor clearance and improved programmed cell death-1 checkpoint blockade responses to B16 tumors. Our results indicate that increasing the number of cytotoxic Tim-3⁺CD8⁺ T cells can promote effective anti-tumor immunity and implicate PTPN2 in immune cells as an attractive cancer immunotherapy target.

Figure 1:. Loss of Ptpn2 promotes the early proliferation of CD8⁺ T cells during LCMV Clone 13 infection

(a) Schematic of co-transfer experiment during LCMV Clone 13 infections. Chimeric mice were generated using the CHIME system. (b) TIDE assay on naïve CD8⁺ T cells for a control and Ptpn2-targeting sgRNA. Representative of four independent experiments, n = 1 mouse. (c) Representative flow cytometry plot of co-transferred control or Ptpn2-deleted P14 T cells in the spleen 8 days post LCMV Clone 13 infection. Representative of eight independent experiments, n ≥ 4 mice. (d-e) Frequency of CD45.1⁺ transferred cells (d) and number (e) of control or Ptpn2-deleted P14 T cells in the spleen 8, 15, 22, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. (f) Quantification of BrdU incorporation for co-transferred control and Ptpn2-deleted P14 T cells 8, 15, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 3 mice. (g) Representative flow cytometry plots of Granzyme B expression from splenic control or Ptpn2-deleted P14 CD8⁺ T cells co-transferred at day 8 post LCMV Clone 13 infection as in (c). Representative of two independent experiments, n ≥ 4 mice. (h) Quantification of (g) days 8, 15, 22, and 30 post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (d, e, f, h) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also Supplementary Figure 1.

Figure 2:. Deletion of Ptpn2 enhances formation of the Tim-3⁺ terminally exhausted subpopulation during LCMV Clone 13 infection

(a) Representative flow cytometry plots of Tim-3 and Slamf6 expression on control or Ptpn2-deleted P14 T cells in the spleen 8 days post LCMV Clone 13 infection. Representative of eight independent experiments, n ≥ 4 mice. (b) Ratio of Tim-3⁺/Slamf6⁺ control or Ptpn2-deleted P14 T cells in the spleen 8, 15, 22, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. (c) Number of Tim-3⁺ control or Ptpn2-deleted P14 T cells in the spleen 8, 15, 22, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. (d) Number of Slamf6⁺ control or Ptpn2-deleted P14 T cells in the spleen 8, 15, 22, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. (e) Quantification of Granzyme B expression for Tim-3⁺ control or Ptpn2-deleted P14 T cells in the spleen 8, 15, 22, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. (f) Quantification of BrdU incorporation for Tim-3⁺ control or Ptpn2-deleted P14 T cells in the spleen 8, 15, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 3 mice. (g) Quantification of Granzyme B expression for Slamf6⁺ control or Ptpn2-deleted P14 T cells in the spleen 8, 15, 22, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. (h) Quantification of BrdU incorporation for Slamf6⁺ control or Ptpn2-deleted P14 T cells in the spleen 8, 15, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 3 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (b-h) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also Supplementary Figure 2.

Figure 3:. Ptpn2 deletion promotes effector-skewed Slamf6⁺ and Tim-3⁺ subpopulations during LCMV infection

(a) tSNE projection of single-cell RNA-seq profiles from 7,027 control or Ptpn2-deleted P14⁺ CD8⁺ T cells responding to day 30 LCMV Clone 13 infection. Clusters are distinct colors. Representative of one experiment, n = 4 pooled mice. (b) Enrichment of gene signatures in the clusters. Representative of one experiment, n = 4 pooled mice. (c) Plots depicting the inter-cluster density for control or Ptpn2-deleted cells. Representative of one experiment, n = 4 pooled mice. (d) Quantification of the proportion of control or Ptpn2-deleted cells in each cluster. Error bars represent the 95% confidence interval and the dotted line represents the proportion of Ptpn2-deleted cells among all cells projected. Representative of one experiment, n = 4 pooled mice. (e-f) Signature enrichments of co-transferred control and Ptpn2-deleted cells from the (e) progenitor or (f) terminally exhausted clusters. Representative of one experiment, n = 4 pooled mice. (g-h) GSEA curves for significantly enriched signatures in (g) Slamf6⁺ cells and (h) Tim-3⁺ cells for bulk RNA-seq of co-transferred control and Ptpn2-deleted cells day 8 post LCMV Clone 13 infection. Representative of one experiment, n = 2 mice with n = 2 technical replicates per mouse. (i-j) Row-normalized heat map of effector-associated genes in (i) Slamf6⁺ cells and (j) Tim-3⁺ cells for bulk RNA-seq of co-transferred control and Ptpn2-deleted cells day 8 post LCMV Clone 13 infection. Representative of one experiment, n = 2 mice with n = 2 technical replicates per mouse. (k) Quantification of frequency of tumor cells killed when co-cultured with control or Ptpn2-deleted CD8⁺ T cells (2:1 effector: target ratio) isolated from day 8 LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (l) Quantification of frequency of tumor cells killed when co-cultured with Tim-3⁺ control or Tim-3⁺ Ptpn2-deleted CD8⁺ T cells (2:1 effector: target ratio) isolated from day 8 LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. Statistical significance was assessed by the two-sided Wilcoxon rank sum test (b, e, f), two-sided binomial test (d), two-sided Kolmogorov-Smirnov test (g-h), and two-sided Student’s paired t-test (k-l) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also Supplementary Figure 3.

Figure 4:. Ptpn2 deletion increases Tim-3⁺ cell differentiation and proliferation

(a) Quantification of frequencies of co-transferred control or Ptpn2-deleted CD8⁺ T cells day 4 post LCMV Clone 13 infection. Frequencies at day 4 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 5 mice. (b) Representative flow cytometry plots of Slamf6 and Tim-3 expression on splenic CD8⁺ T cells day 4 post LCMV Clone 13 infection for co-transferred control and Ptpn2-deleted cells. Representative of two independent experiments, n = 5 mice. (c) Quantification of Slamf6⁺ Tim-3^–, Slamf6⁺ Tim-3⁺, and Slamf6^– Tim-3⁺ subsets in (b). Representative of two independent experiments, n = 5 mice. (d) Quantification of Granzyme B expression of cells as in (a). Representative of two independent experiments, n = 5 mice. (e) Schematic of in vitro conversion and CTV proliferation assays using co-transferred control or Ptpn2-deleted CD8⁺ T cells isolated at day 8 post LCMV Clone 13 infection. (f) Quantification of Slamf6⁺ Tim-3^–, Slamf6⁺ Tim-3⁺, and Slamf6^– Tim-3⁺ subsets following in vitro stimulation (αCD3/CD28 with IL-2 and IFN-α) of control or Ptpn2-deleted CD8⁺ T cells isolated at day 8 post LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (g-h) Quantification of frequency of divisions following in vitro stimulation (αCD3/CD28 with IL-2) of (g) CTV-labeled Slamf6⁺ or (h) CTV-labeled Tim-3⁺, control or Ptpn2-deleted CD8⁺ T cells isolated at day 8 post LCMV Clone 13 infection. Representative of two independent experiments, n = 3 mice (g), 5 mice (h). (i) Schematic of in vivo persistence assay of Tim-3⁺ cells. (j) Quantification of number of recovered Tim-3⁺ control or Ptpn2-deleted cells in the spleen day 6 post LCMV Clone 13 infection, following the transfer of these cells which were previously isolated at day 8 post LCMV Clone 13 infection. Representative of two pooled experiments, n = 8 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (a, c-d, f-h) or two-sided Student’s unpaired t-test (j) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also Supplementary Figure 4.

Figure 5:. Ptpn2 deletion increases Tim-3⁺ cell differentiation through enhanced IFN-α signaling

(a-c) Quantification of (a) Slamf6⁺ Tim-3^–, (b) Slamf6⁺ Tim-3⁺, and (c) Slamf6^– Tim-3⁺ subsets following in vitro stimulation (αCD3/CD28) of control or Ptpn2-deleted naive CD8⁺ T cells in the presence of indicated cytokines or blocking antibodies. Representative of two pooled experiments, n ≥ 4 technical replicates. (d) Quantification of pSTAT1 expression of splenic CD8⁺ T cells day 6 post LCMV Clone 13 infection for co-transferred control and Ptpn2-deleted cells following ex vivo restimulation with IFN-α. Representative of two independent experiments, n = 5 biological replicates. (e-f) Quantification of pSTAT1 in (e) Slamf6⁺ or (f) Tim-3⁺ cells following ex vivo IFN-α restimulation of co-transferred control and Ptpn2-deleted cells as in (d). Representative of two independent experiments, n = 5 biological replicates. (g) Quantification of frequencies of co-transferred control and Ptpn2-deleted CD8⁺ T cells day 4 post LCMV Clone 13 infection following treatment with isotype (left graph) or IFNAR blocking antibody (right graph). Frequencies at day 4 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 3 biological replicates. (h) Quantification of Slamf6⁺ Tim-3^–, Slamf6⁺ Tim-3⁺, and Slamf6^– Tim-3⁺ subsets day 4 post LCMV Clone 13 infection in mice that received co-transferred control and Ptpn2-deleted P14 CD8⁺ T cells and were treated with isotype (left) or IFNAR blocking antibody (right). Representative of two independent experiments, n = 5 biological replicates. (i) Quantification of Slamf6⁺ Tim-3^–, Slamf6⁺ Tim-3⁺, and Slamf6^– Tim-3⁺ subsets day 4 post LCMV Clone 13 infection in mice that received co-transferred control and Ptpn2-deleted P14 CD8⁺ T cells and were treated with isotype (left) or IFN-γ neutralizing antibody (right). Representative of two independent experiments, n = 5 biological replicates. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-way ANOVA (a-c) or two-sided Student’s paired t-test (d-i) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also Supplementary Figure 5.

Figure 6:. Loss of Ptpn2 enhances Tim-3⁺ CD8⁺ T cell differentiation in tumors

(a) Representative flow cytometry plot of control or Ptpn2-deleted OT-1 T cells in the tumor 7 days post MC38-OVA injection. Representative of two independent experiments, n = 4 biological replicates. (b) Quantification of frequencies of co-transferred control or Ptpn2-deleted CD8⁺ T cells day 7 post MC38-OVA injection. Frequencies at day 7 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 4 biological replicates. (c-d) (c) GSEA TIL Slamf6⁺ vs. Tim-3⁺ Up top 50 and TIL Slamf6⁺ vs. Tim-3⁺ Down top 50 signature enrichment and (d) GSEA effector signatures for co-transferred control or Ptpn2-deleted OT-1 T cells in MC38-OVA tumors 7 days post injection. Representative of one experiment, n = 3 pooled mice and 2 technical replicates. (e) Quantification of Granzyme B expression in co-transferred OT-1 CD8⁺ T cells day 7 post MC38-OVA implantation in the tumor, draining lymph node, and spleen for control and Ptpn2-deleted co-transferred mix as in (a). Representative of two independent experiments, n ≥ 3 mice. (f) Schematic of adoptive transfer of either control or Ptpn2-deleted naive OT-1 CD8⁺ T cells separately to mice challenged with B16-OVA one day post transfer of T cells. (g) Tumor growth curves for B16-OVA tumors following transfer of naive OT-1 control or Ptpn2-deleted CD8⁺ T cells separately into WT recipients that were implanted with B16-OVA cells. Representative of two independent experiments, n = 8 mice. (h) Survival curves of mice in (g). Representative of two independent experiments, n = 8 mice. (i) Quantification of Slamf6⁺ Tim-3^–, Slamf6⁺ Tim-3⁺, and Slamf6^– Tim-3⁺ subsets day 9 post B16-OVA implantation in the tumor for co-transferred control and Ptpn2-deleted cells as in (a). Representative of two independent experiments, n = 7 mice. Bar graphs represent mean and error bars represent standard deviation (except for g where error bars represent standard error). Statistical significance was assessed by two-sided Student’s paired t-test (b, e, i), two-sided Kolmogorov-Smirnov test (c, d), two-way ANOVA (g), or two-sided log-rank Mantel-Cox test (h) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also Supplementary Figure 6.

Figure 7:. Deletion of Ptpn2 enhances CD8⁺ T cell responses to tumors and checkpoint blockade efficacy

(a) Schematic for MC38-WT tumor challenge in chimeric mice where approximately 50% of immune cells express a control sgRNA or a Ptpn2-targeting sgRNA. (b) Tumor growth curves for control or Ptpn2-deleted chimeric mice following 1 × 10⁶ cell MC38-WT challenge. Representative of two independent experiments, n ≥ 8 mice. (c-d) Quantification of (c) frequency and (d) number of Slamf6⁺ Tim-3^–, Slamf6⁺ Tim-3⁺, and Slamf6^– Tim-3⁺ subsets in CD8⁺ T cells infiltrating day 9 MC38 tumors in control or Ptpn2-deleted bone marrow chimeras. Representative of two independent experiments, n ≥ 9 mice. (e-f) Quantification of (e) frequency and (f) number of Granzyme B-expressing CD8⁺ T cells infiltrating day 9 MC38 tumors implanted in control or Ptpn2-deleted bone marrow chimeras. Representative of two independent experiments, n ≥ 9 mice. (g) Quantification of frequency of Granzyme B⁺ CD8⁺ T cells from the blood of control or Ptpn2-deleted bone marrow chimeras day 14 post MC38 tumor implantation, pregated on CD8β⁺ Vex⁺ cells. Representative of two independent experiments, n = 5 mice. (h) Tumor growth curves for mice as in (a) challenged with 1 × 10⁶ MC38-WT tumor cells following treatment with CD8-depleting antibody or isotype control. Representative of two independent experiments, n ≥ 9 mice. (i) Tumor growth curves for control or Ptpn2-deleted bone marrow chimeras challenged with 1 × 10⁶ B16 tumor cells treated with GVAX (green triangles) on days 1, 4 and αPD-1 (black triangles) on days 12, 14, 16, 18, 20, 22, 24, and 26. Representative of two independent experiments, n ≥ 9 mice. (j) Quantification of frequency of Granzyme B⁺ CD8⁺ T cells from the blood of chimeras in (i) day 14 post B16 tumor implantation, pregated on CD8β⁺ Vex⁺ cells. . Representative of two independent experiments, n = 5 mice. Bar graphs represent mean and error bars represent standard deviation (except for b, h, i where error bars represent standard error). Statistical significance was assessed by two-way ANOVA (b-d, h-i), or two-sided Student’s unpaired t-test (e-g, j) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also Supplementary Figure 7.