GCN2 is essential for CD8+ T cell survival and function in murine models of malignant glioma

Abstract

Amino acid deprivation is a strategy that malignancies utilize to blunt anti-tumor T-cell immune responses. It has been proposed that amino acid insufficiency in T-cells is detected by GCN2 kinase, which through phosphorylation of EIF2α, shuts down global protein synthesis leading to T-cell arrest. The role of this amino acid stress sensor in the context of malignant brain tumors has not yet been studied, and may elucidate important insights into the mechanisms of T-cell survival in this harsh environment. Using animal models of glioblastoma and animals with deficiency in GCN2, we explored the importance of this pathway in T-cell function within brain tumors. Our results show that GCN2 deficiency limited CD8⁺ T-cell activation and expression of cytotoxic markers in two separate murine models of glioblastoma in vivo. Importantly, adoptive transfer of antigen-specific T-cells from GCN2 KO mice did not control tumor burden as well as wild-type CD8⁺ T-cells. Our in vitro and in vivo data demonstrated that reduction in amino acid availability caused GCN2 deficient CD8⁺ T-cells to become rapidly necrotic. Mechanistically, reduced CD8⁺ T-cell activation and necrosis was due to a disruption in TCR signaling, as we observed reductions in PKCθ and phoshpo-PKCθ on CD8⁺ T-cells from GCN2 KO mice in the absence of tryptophan. Validating these observations, treatment of wild-type CD8⁺ T-cells with a downstream inhibitor of GCN2 activation also triggered necrosis of CD8⁺ T-cells in the absence of tryptophan. In conclusion, our data demonstrate the vital importance of intact GCN2 signaling on CD8⁺ T-cell function and survival in glioblastoma.

Keywords: CD8+ T-cell; GCN2; Glioma; ISRIB.

Fig. 1

Absence of GCN2 reduces CD8⁺ T-cell numbers in murine brain tumors. C57BL/6 (WT) and GCN2 KO mice were injected with 2 × 10⁵ GL-261 cells (glioma syngeneic cell line) intracranially, and after 14 days were euthanized for flow cytometric analysis. In a flow cytometric analysis of the total number and percentage of the different T-cell subsets infiltrating the brain tumor microenvironment. In b flow cytometric analysis of the total number of the different T-cell subsets from the spleen. Flow cytometry statistics in a and b were calculated, n = 5 per group, representative of two experiments. Unpaired T test analysis was used to calculate significance. p < 0.05*; p < 0.01**; p < 0.001***. Refer to Supplementary Fig. 7a for the gating strategy

Fig. 2

Glioma infiltrating GCN2 KO CD8⁺ T-cells are dysfunctional. GCN2 KO and WT mice were injected with 2 × 10⁵ GL-261 cells, and after 14 days were euthanized for flow cytometric analysis. In a GL-261 injected tumor-infiltrating lymphocytes were stimulated for 5 h with PMA/Ionomycin, and then stained and analyzed for CD44 mean fluorescence intensity (MFI) and cytokine secretion via flow cytometry. In b, 1 × 10⁶ heat shock killed GL-261 OVA cells were injected intraperitoneally into OT-1 and GCN2 KO OT-1 mice. After 14 days of engraftment, CD8⁺ T-cells from these mice were isolated and transferred i.v. into RAG1 KO mice previously inoculated with GL-261 OVA. Seven days post transfer, mice were euthanized and analyzed for CD44 MFI and cytokine secretion with flow cytometry. Flow cytometry statistics were calculated, n = 5 per group in a, n = 3 per group in b, representative of two experiments. Unpaired T test analysis was used to calculate significance. p < 0.05*; p < 0.01**; p < 0.001***. Refer to Supplementary Fig. 7b for the gating strategy

Fig. 3

GCN2 promotes antigen-specific CD8⁺ T-cell responses against glioma. In a, WT OT-1 and GCN2 KO OT-1 mice were challenged directly with 4 × 10⁵ GL-261 OVA and were analyzed for survival. In b, OVA activated CD8⁺ T-cells from WT OT-1 or GCN2 KO OT-1 mice were transferred i.c. into tumor-bearing Rag1 KO hosts and analyzed for survival. In c, OVA activated CD8⁺ T-cells from WT OT-1 and GCN2 KO OT-1 mice were isolated and labeled with cell proliferation dye eFluor 450 (GCN2 KO CD8⁺ T-cells) and cell proliferation dye eFluor 670 (WT OT-1 CD8⁺ T-cells), and were transferred at a 1:1 ratio into GL-261 OVA tumor-bearing mice either i.c. (left panel) or i.v. (right panel). After 3 days, mice were euthanized for flow cytometric analysis. In a, b, Kaplan–Meier curves were generated from n = 7 mice per group from two independent experiments, and statistical significance calculated using the Log-Rank analysis. In c, flow cytometry statistics calculated from n = 3 mice per condition. Unpaired T test analysis was used to calculate significance. p < 0.05*; p < 0.01**

Fig. 4

Inhibition of memory and function of in vivo activated CD8⁺ T-cells with GCN2 deletion. In a, b, 1 × 10⁶ heat shock killed GL-261 OVA cells were injected into OT-1 and GCN2 KO OT-1 mice. After 14 days, CD8⁺ T-cells from these mice were isolated and transferred i.v. into previously inoculated Rag1 KO mice (4 × 10⁵ GL-261 OVA). The first group of mice was analyzed for survival analysis (a) and the second group of mice was euthanized 7 days post-transfer for flow cytometric analysis (b). In a, the Kaplan–Meier curve was generated from n = 7 per group from two independent experiments, and statistical significance calculated using the Log-Rank analysis. In b, flow cytometry statistics calculated and shown as the total number and percentage positive population, n = 3 per group, representative of two experiments. Unpaired T test analysis was used to calculate significance. p < 0.05*; p < 0.01**. Refer to Supplementary Fig. 7c for the gating strategy

Fig. 5

Amino acid deficiency causes necrosis in the absence of GCN2 in CD8⁺ T-cells in murine glioma tumor models. In a, WT mice were injected with GL-261 (top panel) and CT2A (bottom panel) tumor cells, three mice per group. After 14 days of tumor growth, tumor (R. Hemi) and non-tumor (L. Hemi) hemisphere were isolated and measured for TRP/KYN ratios via LC–MS. In b, WT mice were injected with GL-261 (top panel) and CT2A (bottom panel) tumor cells, six mice per group. After 14 days, mice were euthanized and CD8β⁺ T-cells were isolated from the tumor (brain) and periphery (spleen), and their metabolites were measured using LC–MS. In c, total T-cells were cultured in serial dilutions of three amino acids, and at 24 and 48 h, cells were lifted and stained with annexin-V and fixable viability dye for flow cytometric analysis. Refer to Supplementary Fig. 7d for the gating strategy. In d, WT and GCN2 KO OT-1 CD8⁺ T-cells were transferred i.v. into previously inoculated with GL-261 OVA, Rag1 KO mice. Seven days post transfer, mice were euthanized, and tumor-infiltrating lymphocytes were stained with annexin-V and fixable viability dye for flow cytometric analysis. Refer to Supplementary Fig. 7e for the gating strategy. In a, the ratio of normalized peak area of TRP to KYN and in b is the normalized number of the peak area of tryptophan, arginine and lysine in the tumor versus non-tumor. In c, Two-way ANOVA followed by Sidak’s multiple comparisons test was used to calculate significance, n = 3 per group. In d, flow cytometry statistics calculated and shown as percentage positive population, n = 3 per group, representative of two experiments. Unpaired T test analysis was used to calculate significance. p < 0.05*; p < 0.01**; p < 0.001***; p < 0.0001****

Fig. 6

EIF2α inhibitor (ISRIB) increases CD8⁺ T-cell necrosis and deactivation in tryptophan starved conditions. In a, b, CD8⁺ T-cells were isolated from splenocytes of WT and GCN2 KO mice then cultured in − TRP, + TRP for 24 and 48 h in (a), and − TRP, + TRP or complete RPMI for 72 h in (b). After 24, 48, and 72 h cells were lifted and were lysed for western blot. In c, d, CD8⁺ T-cells from WT mice were cultured in RPMI, + TRP, − TRP, 200 nM ISRIB + TRP and 200 nM ISRIB − TRP, for 24, 48 and 72 h. At each time-point, cells were lifted and stained for flow cytometric analysis. In c, d, flow cytometry statistics calculated and shown as percentage positive population, n = 3 per group, representative of five experiments. One way ANOVA and Tukey’s post hoc was used to calculate significance. p < 0.05*; p < 0.01**; p < 0.001***. Refer to Supplementary Fig. 7d for the gating strategy