Sex-Biased T-cell Exhaustion Drives Differential Immune Responses in Glioblastoma

Abstract

Sex differences in glioblastoma (GBM) incidence and outcome are well recognized, and emerging evidence suggests that these extend to genetic/epigenetic and cellular differences, including immune responses. However, the mechanisms driving immunologic sex differences are not fully understood. Here, we demonstrate that T cells play a critical role in driving GBM sex differences. Male mice exhibited accelerated tumor growth, with decreased frequency and increased exhaustion of CD8+ T cells in the tumor. Furthermore, a higher frequency of progenitor exhausted T cells was found in males, with improved responsiveness to anti-PD-1 treatment. Moreover, increased T-cell exhaustion was observed in male GBM patients. Bone marrow chimera and adoptive transfer models indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, partially mediated by the X chromosome inactivation escape gene Kdm6a. These findings demonstrate that sex-biased predetermined behavior of T cells is critical for inducing sex differences in GBM progression and immunotherapy response.

Significance: Immunotherapies in patients with GBM have been unsuccessful due to a variety of factors, including the highly immunosuppressive tumor microenvironment in GBM. This study demonstrates that sex-biased T-cell behaviors are predominantly intrinsically regulated, further suggesting sex-specific approaches can be leveraged to potentially improve the therapeutic efficacy of immunotherapy in GBM. See related commentary by Alspach, p. 1966. This article is featured in Selected Articles from This Issue, p. 1949.

Figure 1.

T cells drive sex differences in GBM survival. A, Survival analysis was performed after intracranial injection of the mouse GBM cell line SB28 in immunocompetent B6 mice (15,000 cells/injection) and immune-deficient NSG (10,000 cells/injection) and RAG1^−/− mice (15,000 cells/injection). Median survival days and number of animals are indicated in the graph. Data combined from two to three independent experiments. Statistical significance was determined by log-rank test, considering P < 0.05 to be significant. B, Frequency of CD45^hi immune cells and CD3⁺ T cells from the tumor-bearing left hemisphere of SB28-injected mice or the left hemisphere of the sham-injected group on days 8 and 15. Data are shown as mean ± SD from two independent experiments. n = 10 for SB28-bearing mice and n = 3–5 for sham-injected mice. One-way ANOVA with the Tukey multiple comparisons test was performed to determine statistical significance (*, P < 0.05; ***, P < 0.001). C, Frequency of OVA-specific CD8⁺ T cells was measured using tetramer (Tet) antibody from the tumor-bearing left hemisphere of SB28-OVA (25,000 cells/mouse)–injected B6 wild-type mice on day 14 after tumor implantation. Data are shown as mean ± SD from two independent experiments. n = 9–10/group. *, P < 0.05 was determined by an unpaired t test. D, Kaplan–Meier curves depicting survival of SB28-bearing male and female mice treated with anti-CD8–depleting antibody. Log-rank test was performed to determine statistical significance (*, P < 0.05; **, P < 0.01). d, days; F, female; M, male.

Figure 2.

More male CD8⁺ T cells are functionally exhausted and skewed toward a progenitor exhausted T-cell phenotype. Tumor-infiltrating T cells were analyzed on day 15 after implantation of SB28 tumor cells. A, Frequency of T-cell subsets in CD45^hi immune cells. Data are combined from two independent experiments. n = 11–12 for the SB28-injected group and n = 4 for the sham-injected group. B, Inhibitory receptor expression in CD8⁺ and CD4⁺Foxp3⁻ effector T cells (Teff). Data are combined from two independent experiments. n = 10–12 for the SB28-injected group and n = 4 for the sham-injected group. C, Intracellular cytokine expression in CD8⁺ and CD4⁺Foxp3⁻ effector T cells was measured after ex vivo stimulation. Data are combined from two independent experiments. n = 7–10 for the SB28-injected group and n = 3 for the sham-injected group. D, Exhausted T-cell subsets in CD8⁺ T cells: TEX (CD8⁺CD44⁺PD-1⁺TCF1⁻TIM3⁺), PEX (CD8⁺CD44⁺PD-1⁺TCF1⁺TIM3⁻), and EFF (CD8⁺CD44⁺TCF1⁻TIM3⁻). Data are combined from two independent experiments. n = 9–10 for the SB28-injected group and n = 4 for the sham-injected group. E, TOX expression in each CD8⁺ T-cell subset. gMFI, geometric mean fluorescence intensity. Intracellular expression of granzyme B (F) and IFNγ⁺TNF⁺ (G) in each CD8⁺ T-cell subset after ex vivo stimulation. Data are combined from two independent experiments. n = 5–7 for SB28-injected group. Two-way ANOVA with the Tukey multiple comparisons test (A–C and E–G) or unpaired Student t test (D) was performed (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

Males are more responsive to anti–PD-1 therapy. A, Schematics depicting treatment regimen for anti–PD-1 and immune profiling. B, Kaplan–Meier curves depicting survival of male and female SB28-bearing mice treated with anti–PD-1 or isotype antibodies (10 mg/kg) starting from day 7 after intracranial tumor implantation. Combined results from three independent experiments with log-rank test (**, P < 0.01; ***, P < 0.001). Median survival length and number of animals are indicated. d, days; F, female; M, male. C–F, Immunophenotyping was performed on tumor-infiltrating immune cells on day 18, 2 days after the last treatment. Data are combined from two independent experiments. n = 9–10 for the anti–PD-1 treatment group and n = 7–8 for the isotype antibody-treated group. C, Percentage of CD8⁺ T cells in CD45^hi cells. D, Proliferation marker Ki-67 expression in CD8⁺ T cells. Data are shown as mean ± SD of n = 5/group from one of two independently repeated experiments. E, Frequency of exhausted T-cell subsets in CD8⁺ T cells: TEX, PEX, and EFF. F, Percentages of intracellular CD8⁺ T cells expressing IFNγ, TNF, and granzyme B. Two-way ANOVA with the Tukey multiple comparisons test was performed (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4.

Immune cell–intrinsic and cell–extrinsic effect in GBM survival. A, A schematic of the generation of bone marrow chimera models. Immune profiling was performed from the tumor-bearing hemisphere on day 14 after tumor implantation (SB28, 10,000 cells/injection). i.c., intracranial. B, Frequency of T-cell subsets in CD45^hi cells. Data are combined from four independent experiments. n = 8–18 per group. One-way ANOVA with the Tukey multiple comparisons test (*, P < 0.05; **, P < 0.01). C, Percentage of exhausted T-cell subsets in CD8⁺ T cells. Data are combined from three independent experiments. n = 4–11 per group. One-way ANOVA with Tukey multiple comparisons test (*, P < 0.05; **, P < 0.01). D, Kaplan–Meier curves depicting survival of bone marrow chimeras after intracranial injection of SB28 cells. Data are combined from three independent experiments. n = 23–25 per group. Statistical significance was determined by the log-rank test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). d, days. E, Schematic showing the generation of mixed bone marrow chimera models. Immune profiling was performed from the tumor-bearing hemisphere on day 14 after tumor implantation (SB28, 10,000 cells/injection). F, Frequency of the PEX subset in male (CD45.1⁺) or female (CD90.1⁺) CD8⁺ T cells. Dotted line indicates cells from the same recipient. G, Ki-67 expression in male (CD45.1⁺) or female (CD90.1⁺) CD8⁺ T cells. Data are combined from two independent experiments. n = 6–7 per group. A paired t test was performed (*, P < 0.05; **, P < 0.01).

Figure 5.

Cell-intrinsic regulation of sex differences in T-cell function. A, Schematics depicting in vitro generation of exhausted T cells. B, Exhaustion markers and cytokine expression were measured on day 5 by flow cytometry after polyclonal stimulation with a stimulation cocktail for 4 hours. Data are shown as mean ± SD (n = 3) and are representative of three independent experiments. Two-way ANOVA with Tukey multiple comparisons test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). gMFI, geometric mean fluorescence intensity. C, qPCR analysis on exhausted T cells. Relative expression levels normalized to exhausted T cells from one male are shown (n = 4). An unpaired t test was performed. D, Male and female in vitro exhausted T cells were cultured with SB28-OVA cells for 24 hours, and viability of tumor cells was measured by flow cytometry. Multiple t test (*, P < 0.05; **, P < 0.01). E, A schematic depicting the adoptive transfer model. F, Kaplan–Meier curves depicting survival of male and female RAG1^−/− mice bearing SB28-OVA tumor cells after adoptive transfer of OT-I cells. Data shown are combined from three independent experiments and n = 10–14 each group. Statistical significance was determined by the log-rank test (*, P < 0.05; ***, P < 0.001). d, days.

Figure 6.

Sex differences in exhausted T cells in GBM patients. A, A gating strategy for exhausted T-cell subsets from GBM patient tumors. B and C, Frequency of PEX (CD8⁺KLRG1⁻PD-1⁺CXCR5⁻TCF1⁺TIM3⁻; B) and TOX expression (C) in CD8⁺ T cells from tumors of male (n = 18) and female (n = 14) patients with isocitrate dehydrogenase (IDH) wild-type GBM tumors. Unpaired t test (*, P < 0.05). gMFI, geometric mean fluorescence intensity. D,In vitro induction of exhaustion in human CD8⁺ T cells. E, Exhaustion marker expression in CD8⁺ T cells on day 12 after stimulation. Two-way ANOVA with Tukey multiple comparisons test (**, P < 0.01; ***, P < 0.001). gMFI, geometric mean fluorescence intensity. F, Intracellular expression of IFNγ⁺TNF⁺ in CD8⁺ T cells during repeated stimulation. Multiple unpaired t test was performed (*, P < 0.05; **, P < 0.01). Data are shown as mean ± SD (n = 3) and are representative of two independent experiments.

Figure 7.

Higher UTX (Kdm6a) expression in female exhausted T cells is associated with sex-biased T-cell exhaustion status. A, mRNA expression level of Kdm6a in murine and human in vitro exhausted T cells measured by qPCR analysis. Relative expression levels normalized to exhausted T cells from one male are shown. Unpaired t test (*, P < 0.05). B, Expression of UTX (encoded by Kdm6a) in murine and human in vitro exhausted T cells measured by flow cytometry. A representative histogram of UTX expression in murine cells is shown. Data are combined from two independent experiments. Unpaired t test (*, P < 0.05; **, P < 0.01). gMFI, geometric mean fluorescence intensity. C, UTX expression in CD8⁺ T cells isolated from SB28-bearing male and female B6 mice on day 14 after tumor implantation. Two-way ANOVA with the Tukey multiple comparisons test (*, P < 0.05). TIL, tumor-infiltrating lymphocyte. D, UTX expression in CD8⁺ T cells from SB28-bearing mixed bone marrow chimera mice on day 14 after tumor implantation. Paired t test (*, P < 0.05; ***, P < 0.001). E, Male and female OT-I cells were treated with GSK-J4 at varying doses during in vitro induction of exhaustion (days 2–5), and expression level of IFNγ and exhaustion markers was measured by flow cytometry on day 5. Data are shown as mean ± SD (n = 6) and are represen­tative of two independent experiments. Two-way ANOVA with the Tukey multiple comparisons test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Veh, vehicle. F, Proposed model of sex-biased T-cell phenotype and functionality mediated by the XCI gene Kdm6a in patients with GBM.