The stress kinase GCN2 does not mediate suppression of antitumor T cell responses by tryptophan catabolism in experimental melanomas

Abstract

Tryptophan metabolism is a key process that shapes the immunosuppressive tumor microenvironment. The two rate-limiting enzymes that mediate tryptophan depletion, indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), have moved into the focus of research and inhibitors targeting IDO and TDO have entered clinical trials. Local tryptophan depletion is generally viewed as the crucial immunosuppressive mechanism. In T cells, the kinase general control non-derepressible 2 (GCN2) has been identified as a molecular sensor of tryptophan deprivation. GCN2 activation by tryptophan depletion induces apoptosis and mitigates T cell proliferation. Here, we investigated whether GCN2 attenuates tumor rejection in experimental B16 melanoma using T cell-specific Gcn2 knockout mice. Our data demonstrate that GCN2 in T cells did not affect immunity to B16 tumors even when animals were treated with antibodies targeting cytotoxic T lymphocyte antigen-4 (CTLA4). GCN2-deficient gp100 TCR-transgenic T cells were equally effective as wild-type pmel T cells against gp100-expressing B16 melanomas after adoptive transfer and gp100 peptide vaccination. Even augmentation of tumoral tryptophan metabolism in B16 tumors by lentiviral overexpression of Tdo did not differentially affect GCN2-proficient vs. GCN2-deficient T cells in vivo. Importantly, GCN2 target genes were not upregulated in tumor-infiltrating T cells. MALDI-TOF MS imaging of B16 melanomas demonstrated maintenance of intratumoral tryptophan levels despite high tryptophan turnover, which prohibits a drop in tryptophan sufficient to activate GCN2 in tumor-infiltrating T cells. In conclusion, our results do not suggest that suppression of antitumor immune responses by tryptophan metabolism is driven by local tryptophan depletion and subsequent GCN2-mediated T cell anergy.

Keywords: AHR; CHOP; GCN2; IDO; T cells; TDO; tryptophan.

Figure 1.

T cell-specific Gcn2 knockout does not alter antitumor immune response to experimental melanoma. B16 melanoma cells were implanted into Gcn2^ΔLck mice and control littermates (n = 5). (A) Tumor growth was monitored for 15 d before tumors were excised and processed for flow cytometry. (B) Final tumor size prior to TIL isolation (day 15). Flow cytometric analysis of B16 TILs for (C) T cells, (D) CD4⁺ and CD8⁺ T cells, and (E) regulatory T cells, T_H1 cells, and IFNγ-secreting CD8⁺ T cells. All data are represented as mean ± SEM. For (A)–(D) one representative out of three experiments is shown, for (E) analysis was performed twice. Statistical significance was assessed using the two-tailed student's t test.

Figure 2.

T cell GCN2 is not critical for immune resistance to immune checkpoint blockade. Gcn2^ΔLck mice and control littermates were inoculated with B16 melanoma cells and treated with anti-CTLA4 or isotype control (n = 5). (A) Survival was assessed for 37 d post-inoculation. Mice were treated three times at indicated time points. Data from one out of two independent experiments are shown. Evaluation of survival patterns was performed by the Kaplan–Meier method and results were corrected for multiple testing according to Benjamini–Hochberg (*p < 0.05). Individual growth curves for the different groups are shown in (B).

Figure 3.

Immune checkpoint blockade does not reveal T cell-intrinsic differences as a result of Gcn2 deletion. TILs were isolated from B16 melanoma-bearing Gcn2^ΔLck mice and control littermates treated with anti-CTLA4 or isotype control at day 15 (n = 5). (A) T cell frequency and T cell count was assessed by flow cytometry. T cell infiltrates from anti-CTLA4-treated animals were further analyzed for (B) CD4⁺ and CD8⁺ T cell, (C) T_regs, T_H1 cells, and CTLs, (D) T_reg/T_H1 and T_reg/CTL ratios, as well as (E) Ki67 and (F) PD1 expression on T cell subsets. All data are represented as mean ± SEM. For (A) statistical significance was determined by one-way ANOVA in combination with Tukey's test, in (B)–(F) a two-tailed student's t test was used.

Figure 4.

GCN2 does not alter phenotype and function of gp100-specific CD8⁺ T cells. B16 melanoma cells were implanted into WT mice and either left untreated or received adoptively transferred gp100-specific CD8⁺ T cells isolated from pmel WT or pmel Gcn2^Δ mice in combination with gp100 peptide vaccination (n = 5). (A) Tumor growth was monitored for 15 d and (B) final tumor size was assessed prior to TIL isolation. One out of two independent experiments is shown. Frequency of (C) gp100-tetramer-specific cells out of CD8⁺ T cells and (D) PD1 expression was determined by flow cytometry. (E) Gp100-specific CD45⁺ CD8⁺ T cells were analyzed for their cytolytic and proliferative capacity after co-culture with B16 melanoma cells at indicated effector:target ratios by CD107a, granzyme B, and IFNγ expression and CFSE dilution (n = 1). For (A–(C), all data are represented as mean ± SEM. In vitro co-culture assays were performed as technical triplicates for CD107a, granzyme B, and IFNγ. For (B) and (C), statistical significance was determined by one-way ANOVA in combination with Tukey's test, and in (D), a two-tailed student's t test was used (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5.

T cell-specific Gcn2 knockout does not alter the growth of constitutively tryptophan-metabolizing B16 melanomas. Gcn2^ΔLck mice and control littermates were inoculated with B16 Tdo melanoma cells (n = 4). (A) Tumor growth was monitored for 16 d and tumors were excised for further analyses. (B) Tdo expression in B16 melanoma tissue was assessed by qRT-PCR in comparison to B16 control tumors. Data are represented as mean ± SEM. A two-tailed student's t test was used to assess significance (**p < 0.01).

Figure 6.

GCN2 is not activated in tumor-infiltrating T cells. (A) B16 melanoma cells were implanted into Gcn2^fl/fl mice and animals were treated with anti-CTLA4 or isotype control. T cells were purified from B16 tumor tissue when the tumor area exceed 150 mm² and GCN2 activation was assessed by Chop expression (n = 5). (B) Gcn2^ΔLck mice and control littermates were inoculated with B16 Tdo melanoma cells. Chop expression in purified tumor-infiltrating T cells was assessed by qRT-PCR 16 d post-inoculation (n = 3). For (A) and (B), a two-tailed student's t test was used to assess significance and data are represented as mean ± SEM. (C) Heatmap of the GCN2-ATF4-CHOP pathway in B16 melanoma TILs and spleen T cells generated from microarray gene expression data (n = 3). (D) Ido expression in B16 tumor tissue and Chop/Atf4 expression in TILs from matched samples were correlated and p-values were adjusted for multiple testing according to Benjamini–Hochberg (n = 5). (E) SKCM RNA sequencing data were retrieved from TCGA and correlation between Chop/Atf4 and Tdo and Chop/Atf4 and Ido expression was analyzed.

Figure 7.

Tryptophan is not depleted in B16 melanomas treated with immune checkpoint inhibitors or constitutively tryptophan-metabolizing B16 melanomas. (A) B16 melanoma cells were implanted into Gcn2^fl/fl mice and animals were treated with anti-CTLA4 or isotype control. Intratumoral tryptophan levels were determined by HPLC and MALDI-TOF MS imaging. Average tryptophan intensities measured by MALDI-TOF MS were extracted from DHB peak normalized images using mMass (n = 5). (B) Gcn2^fl/fl mice were inoculated with B16 Tdo and control melanoma cells. Tryptophan levels in B16 Tdo and control tumors were assessed by MALDI-TOF MS imaging and tryptophan intensities were obtained as described above (n = 4). All data are represented as mean ± SEM and a two-tailed student's t test was used to assess significance.