Depletion of CD4⁺ T cells induces a concomitant increase in the frequencies of exhausted CD8⁺ T cells and IL-17-poducing CD8⁺ T cells in the tumor

As a first step to investigate the regulatory mechanisms governing the exhaustion of CD8⁺ T subsets in tumor setting in vivo, we sought to determine the contribution of conventional CD4⁺ T cells, since depletion of CD4⁺ T cells is known to incapacitate the differentiation of cytotoxic CD8⁺ T cells in tumor and in infection.31 To this end, we employed a B16F10 lung metastasis model and analyzed TILs after treatment with anti-CD4 depleting antibody or control IgG. Of note, we observed a significant increase in the frequency of PD-1⁺ cells among CD8⁺ TILs in the lung of B16F10 melanoma-bearing mice treated with anti-CD4 (figure 1A,B). Further analyses of the PD-1⁺ cells revealed that anti-CD4 treatment substantially increased the proportion of Tim3⁺, TOX⁺, or TCF-1⁻ “terminally exhausted” CD8⁺ TILs (figure 1A,C‒E), indicating that depletion of CD4⁺ T cells promotes “terminal exhaustion” of CD8⁺ T cells in tumor.

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Figure 1

Depletion of CD4⁺ T cells concomitantly increases terminally exhausted CD8⁺ T cells and IL-17-producing Tc17 cells in tumor bearing mice. (A–E) CD8⁺ T cells in lung tumor were analyzed 15 days after B16F10 intravenous (i.v.) injection and CD4⁺ T-cell depletion by anti-CD4 Ab treatment (n=4). Control mice were treated with rat IgG2b isotype control Ab (n=4). (A) Representative contour plots of PD-1, Tim-3, Tox, and TCF-1 expression in CD8⁺ TILs. (B–E) Frequencies of PD-1⁺ (B), PD-1⁺Tim-3⁺ (C), PD-1⁺TOX⁺ (D), and PD-1⁺TCF-1⁻ (E) cells among CD8⁺ TILs are shown. (F) IL-17 and IFN-γ production by lung CD8⁺ TILs of B16F10 tumor-bearing control Ab-treated (n=5) or CD4-depleted mice (n=7) was compared. Representative contour plots and frequencies of IL-17⁺IFN-γ⁻ cells (Tc17) and IL-17⁻IFN-γ⁺ cells (Tc1) are shown. (G) Representative contour plots of IL-17 and IFN-γ production by lung CD8⁺ TILs of B16F10 tumor-bearing Foxp3 ^DTR mice after diphtheria toxin (DT) or PBS treatment (n=4 each). Frequencies of Tc17 and Tc1 cells among CD8⁺ TILs are shown. (H) Composition of IL-17⁺CD90⁺ lung TILs in B16F10 tumor-bearing control Ab-treated or CD4-depleted mice. (I–L) Frequencies of tumor-infiltrating Tc17 cells with or without anti-CD4 treatment in B16F10 subcutaneous (s.c.) injection (I), B16F10 intrasplenic (i.s.) injection (J), TC-1 i.v. injection (K), CCSP ^Cre /K-ras ^G12D spontaneous tumor models (L). Data are representatives of at least two independent experiments. Data are shown as mean±SD for (B-G), and mean+SD for (I-L). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DN17, CD4⁻CD8⁻IL-17⁺; Misc., miscellaneous.

To further investigate the contribution of CD4⁺ T cells to CD8⁺ T cell responses in the tumor, we comparatively examined the frequencies of IFN-γ⁺CD8⁺ T (Tc1) cells and IL-17⁺CD8⁺ T (Tc17) cells in the tumor sites. To our surprise, anti-CD4 treatment significantly increased the frequency of Tc17 cells, while minimally impacting that of Tc1 cells among CD8⁺ TILs in comparison with control IgG treatment (figure 1F). Unlike anti-CD4 treatment, depletion of Treg cells in B16F10-bearing Foxp3 ^DTR mice remarkably increased the frequency of Tc1 cells, while minimally impacting Tc17 cell population (figure 1G). Analysis of IL-17-producing lymphoid cells in the TILs revealed that anti-CD4 treatment specifically expanded the Tc17 cell population, while γδT17 cell and NKT17 cell populations were reduced (figure 1H). To determine whether the CD4⁺ T cell–mediated inhibition of Tc17 cell differentiation observed in the B16F10 lung metastasis model could also be true in other tumor models, we employed four additional well-established tumor models; a subcutaneous injection of B16F10 tumor model, a liver metastasis model induced by intrasplenic injection of B16F10, a TC-1 lung metastasis tumor model, and a spontaneous tumor model driven by Kras ^G12D mutant expression in lung epithelial cells. In all the tested murine tumor models, anti-CD4 treatment significantly increased the frequencies of Tc17 cells among CD8⁺ TILs (figure 1I‒L). Thus, conventional CD4⁺ T cells and Treg cells respectively control the differentiation of Tc17 and Tc1 cells in tumor-bearing mice in vivo in our experimental settings. Together, these results demonstrate that depletion of CD4⁺ T cells increases the frequency of both terminally exhausted CD8⁺ T cells and Tc17 cells in the tumor.

Tumor-infiltrating Tc17 cells are phenotypically and functionally distinct from Tc1 cells

Since the nature of tumor-infiltrating Tc17 cells remains incompletely defined, we comparatively analyzed the phenotypes of tumor-infiltrating Tc1 and Tc17 cells. Flow cytometric analysis revealed that, unlike Tc1 cells, the majority of Tc17 cells have CD103⁺KLRG1⁻ tissue-resident memory (Trm)-like phenotype (figure 2A). The Trm-like features of Tc17 cells were observed in all tested murine models of tumor (online supplemental figure S1A‒D). Intravascular labeling experiments showed that the majority of Tc17 cells in the tumor were protected from intravenous labeling, suggesting that they exhibit tissue-residency property (figure 2B). CD8⁺ Trm cells have recently emerged as critical players in tumor immunity.32 Of note, Tc17 cells were found to be CD127^hi (IL-7Rα^hi), while Tc1 cells were mixed populations of CD127⁺ and CD127⁻ (figure 2C). Moreover, compared with Tc1 cells, Tc17 cells in the tumor were Ki-67^hi, suggesting the highly proliferative potential of this unique CD8⁺ T cell subset (figure 2D). We also found that CD8α⁻ IL-17-producing TILs of CD4-depleted mice as well as Tc17 cells and Th17 cells from tumor-bearing mice treated with control IgG were CD103⁺KLRG1⁻ and highly expressed CD127, but only Tc17 cells were Ki-67^hi (online supplemental figure S1E‒M). Thus, CD103⁺KLRG1⁻CD127 ^hi Trm-like phenotype is likely a common feature of IL-17-producing cells in the tumor regardless of CD4 depletion. When we tested whether the blockade of IL-7R signaling pathway by anti-CD127 Ab treatment impacts Tc17 cell development, we did not see any change in Tc17 cell frequencies, but a significant decrease in Tc1 cell frequencies (online supplemental figure S1N). Cytotoxicity of CD8⁺ T cells is a hallmark of anti-tumor immunity. We found that the frequency of granzyme B⁺ cells was significantly lower in Tc17 cells than in Tc1 cells (figure 2E). Consistently, Tc17 cells were significantly less efficient than Tc1 cells in inducing antigen-specific lysis of target cells in in vitro cytotoxicity assay conditions (figure 2F), indicating that Tc17 cells are poorly cytolytic.

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Figure 2

Tumor-infiltrating Tc17 cells exhibit tissue resident memory-like phenotypes in mice and humans. (A–E) Lung CD8⁺ tumor-infiltrating lymphocytes (TILs) in CD4-depleted B16F10 tumor-bearing C57BL/6 mice were analyzed. (A) Representative contour plots of cell surface expression of CD103 and KLRG1 on Tc17 and Tc1 cells. (B) PerCP-Cy5.5-labeled anti-mouse CD8α Ab was intravenously (i.v.) injected into CD4-depleted B16F10 tumor-bearing mice 5 min before sacrifice for analysis. Representative contour plots of CD8 α-PerCP-Cy5.5⁺ cells among total CD8⁺ T cells in spleen and lung tumor are shown. (C) Representative histogram of CD127 expression and frequencies of CD127⁺ cells of Tc17 and Tc1 cells (n=4). (D) Representative histogram of Ki-67 expression and frequencies of Ki-67⁺ cells of Tc17 and Tc1 cells (n=7). (E) Frequencies of granzyme B(GzmB)⁺ cells of Tc17 and Tc1 cells (n=4). (F) Cytotoxicity of in vitro generated gp100-specific Tc1 cells or Tc17 cells (effector cells) against gp100-pulsed TC-1 tumor cells (target cells). Frequencies of Caspase-3(Casp3)⁺ among CTV-labeled target cells were analyzed 4 hours after co-culture with effector cells. (G–J) CD8⁺ TILs isolated from tumor tissues of patients with hepatocellular carcinoma (HCC) were analyzed by flow cytometry. (G) Representative contour plots of IL-17 and IFN-γ production by Tc17 and Tc1 cells in TILs of patients with HCC and frequencies of Tc17 and Tc1 cells among CD8⁺ TILs (n=11). (H) Cell surface expression of CD103 and KLRG1 on each CD8⁺ T cell subset. (I) Representative histogram of cell surface expression of CD127 on each CD8⁺ TIL subset and frequencies of CD127^hi cells (n=6). (J) Representative histogram of GzmB expression in each CD8⁺ TIL subset and frequencies of GzmB⁺ cells in each CD8⁺ TIL subset (n=6). Data are representatives of at least two independent experiments for (A–F) and (H–J) and combined results of two independent experiments for (G). Data are shown as mean±SD for (C-E, G, I, J) and mean±SEM for (F). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

To determine whether Tc17 cells found in human tumors also exhibit similar phenotypes as those observed in mice, we analyzed the phenotypes of CD8⁺ TILs isolated from patients with hepatocellular carcinoma (HCC) (online supplemental table S1). In addition to Tc1 cells, we observed a small, but distinct population of Tc17 cells (figure 2G). The Tc17 cells from patients with HCC uniformly exhibited CD103⁺KLRG1⁻ Trm-like phenotype, while Tc1 cells were found to be mixed populations of CD103⁺KLRG1⁻ and CD103⁻KLRG1⁺ cells (figure 2H). Moreover, more than 80% of Tc17 cells were CD127^hi, which was in sharp contrast to the CD127^lo/int-dominant phenotype of Tc1 cells, bearing less than 50% of CD127^hi cells on average (figure 2I). Unlike the high frequency of granzyme B⁺ cells in Tc1 (about 70%), tumor-infiltrating Tc17 cells were mostly granzyme B⁻ (figure 2J). Taken together, these results demonstrate that, unlike Tc1 cells, tumor-infiltrating Tc17 cells exhibit a Trm-like CD103⁺KLRG1⁻CD127^hi surface phenotype and poorly cytotoxic, which seems to be conserved between mouse and human.

IL-17-producing cells promote the exhaustion of tumor-infiltrating CD8⁺ T cells in vivo

Concomitant increase in the frequencies of Tc17 cells and PD-1^hiTim3⁺ cells by anti-CD4 treatment prompted us to hypothesize that IL-17-producing cells might exhibit “terminally exhausted” phenotypes. However, flow cytometric analysis of cell surface PD-1 and Tim-3 expression showed that Tc17 cells were PD-1^intTim3⁻, while PD-1^hiTim3⁺ “terminally exhausted” CD8⁺ TILs were confined to Tc1 population (figure 3A). Th17 cells are known to transdifferentiate into Th1 cells on repeated stimulation with IL-23 or IL-12.33 Moreover, in vitro–generated Tc17 cells have been shown to become Tc1 cells when transferred in vivo.34 To determine if in vivo–generated tumor-infiltrating Tc17 cells transdifferentiate into Tc1 cells in response to environmental stimuli in the TME, we employed Il17a ^Cre R26 ^YFP fate mapping (fm) mice in which IL-17-producing cells are permanently labeled with YFP. We injected anti-CD4 into B16F10 bearing Il17a ^Cre R26 ^YFP mice, and found that only a small fraction of CD8⁺YFP⁺ T cells (<10%) was IL-17⁻IFN-γ⁺ Tc1 cells, while the majority was still IL-17⁺ in the TME (figure 3B). These results suggest that in vivo–generated tumor-infiltrating Tc17 cells are stable in the TME in our experimental setting.

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Figure 3

Tc17 cells are phenotypically not exhausted but can promote the exhaustion of CD8⁺ T cells in the tumor. (A) Representative contour plots of PD-1, IL-17, and Tim-3 expression of lung tumor-infiltrating Tc17 and Tc1 cells in CD4-depleted B16F10 tumor-bearing mice. (B) Fate-mapping (fm) of Tc17 cells within the tumor microenvironment. Il17a ^Cre R26 ^YFP mice were intravenously inoculated with B16F10 tumor cells and were treated with anti-CD4 Ab. Representative contour plots of IFN-γ and IL-17 expression in IL-17fm-YFP⁺ cells and IL-17fm-YFP⁻ cells within the CD8⁺ tumor-infiltrating lymphocytes (TILs) and the frequencies of IL-17⁺IFN-γ⁻ and IL-17⁻IFN-γ⁺ cells among IL-17fm-YFP⁺ or IL-17fm-YFP⁻ CD8⁺ TILs. (C,D) C57BL/6 mice were adoptively transferred with in vitro differentiated CD45.1⁺ Pmel Tc17 cells 1 day before B16F10 intravenous (i.v.) injection. Control mice were injected with PBS and CD4⁺ T cells were depleted by anti-CD4. (C) Tumor foci count in the lung. (D) Representative contour plots of PD-1 and Tim-3 expression of CD45.1⁻ lung CD8⁺ TILs and frequencies of PD-1^hi and PD-1^hiTim-3⁺ cells among CD45.1⁻ lung CD8⁺ TILs are shown. Data are representatives of at least two independent experiments. Data are shown as mean±SD. *p<0.05 and ***p<0.001.

These results together raised a possibility that Tc17 cells contribute to the exhaustion of Tc1 cells in the TME. To test this possibility, we stimulated naïve CD8⁺ T cells isolated from CD45.1⁺ Pmel mice under Tc17-skewing conditions in vitro and adoptively transferred them into B16F10 tumor-bearing mice (CD45.1⁻) before analyzing host CD8⁺ TILs. The number of tumor foci was slightly increased in the recipients of Tc17 cells (figure 3C). Compared with those from the PBS-injected mice, host CD8⁺ TILs from the tumor-specific Tc17 cell-recipient mice showed a significant increase in Tim-3⁺ or TCF-1⁻TOX⁺ population. The increase in TCF-1⁻TOX⁺ population was associated with a concomitant decrease in TCF-1⁺TOX⁻ cell frequencies among the PD-1^hi population, although the frequency of PD-1^hi cells remained comparable (figure 3D and online supplemental figure S2A,B). Thus, Tc17 cells can accelerate the terminal exhaustion of tumor-infiltrating CD8⁺ T cells in vivo.

Based on the observed exhaustion-promoting function of Tc17 cells, we speculated that depletion of IL-17-producing cells could reverse the exhaustion of CD8⁺ TILs, thereby suppressing tumor growth in vivo. To prove this hypothesis, we established Il17a ^Cre R26 ^DTA mice in which all IL-17-producing cells can be depleted by expressing diphtheria toxin. We confirmed the depletion of IL-17-producing cells in the TILs by comparing YFP⁺CD90⁺ cell frequency between Il17a ^Cre R26 ^WT/YFP mice and Il17a ^Cre R26 ^DTA/YFP mice, both of which selectively express eYFP reporter protein in IL-17-producing cells (figure 4A). Indeed, compared with Il17a ^Cre R26 ^WT mice, Il17a ^Cre R26 ^DTA mice exhibited significantly lower tumor burdens as evidenced by tumor foci and lung weight in the B16F10 lung metastasis model (figure 4B). The frequency of PD-1^hiTim-3⁺ as well as PD-1^hiTCF1⁻ “terminally exhausted” population within CD8⁺ TILs was significantly lower in the latter group of mice (figure 4C,D). Similar reduction of PD-1^hiTim-3⁺ CD8⁺ TILs was observed in Il17a ^Cre R26 ^DTA mice bearing subcutaneous B16F10 without anti-CD4 treatment (figure 4E) as well as in a TC-1 lung metastasis tumor model (online supplemental figure 2C,D). These results collectively demonstrate that IL-17-producing cells promote the terminal exhaustion of CD8⁺ T cells in the tumor per se, regardless of tumor types, tumor injection routes, and CD4 depletion in our experimental settings in vivo.

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Figure 4

IL-17-producing cells promote CD8⁺ T cell exhaustion and tumor progression in vivo. (A) Il17a ^Cre R26 ^WT/YFP (n=8) or Il17a ^Cre R26 ^DTA/YFP mice (n=5) were intravenously (i.v.) injected with TC-1 tumor cells and were treated with anti-CD4 Ab. Representative contour plots of IL-17fm-YFP and CD90 expression in lymphocytes of lung tumor and frequencies of YFP⁺CD90⁺ cells among lymphocytes. (B) Il17a ^Cre R26 ^WT (n=7) or Il17a ^Cre R26 ^DTA mice (n=7) were i.v. injected with B16F10 tumor cells and were treated with anti-CD4. Representative photograph of lung tumor burdens and summary of lung weights at day 19 after tumor inoculation. (C,D) PD-1, Tim-3, and TCF-1 expression of lung CD8⁺ tumor-inflitrating lymphocytes (TILs) of CD4-depleted Il17a ^Cre R26 ^WT (n=6) or Il17a ^Cre R26 ^DTA mice (n=5) were analyzed at day 16 after B16F10 tumor injection. Frequencies of PD-1^hiTim-3⁺ cells (C) or PD-1^hiTCF-1⁻ cells (D) among CD8⁺ TILs. (E) Il17a ^Cre R26 ^WT mice (n=4) and Il17a ^Cre R26 ^DTA mice (n=5) were subcutaneously injected with B16F10 tumor cells. Representative contour plots of PD-1 and Tim-3 expression in CD8⁺ TILs of B16F10 tumor and the frequencies of PD-1^hi and PD-1^hiTim-3⁺ cells among CD8⁺ TILs at day 14 after tumor injection. Data are representatives of at least two independent experiments. Data are shown as mean±SD. *p<0.05, **p<0.01, ***p<0.001.

IL-17 deficiency delays the exhaustion of tumor-infiltrating CD8⁺ T cells

Since IL-17 plays pivotal roles in type 17 cell-mediated tissue inflammation and host defense,35 we wondered if blockade of IL-17 pathway could recapitulate the phenotypes observed in the tumor-bearing Il17a ^Cre R26 ^DTA mice. We compared the tumor growth and the exhaustion progression of CD8⁺ TILs between tumor-bearing wild-type and IL-17-deficient mice after anti-CD4 treatment. Indeed, a significantly lower tumor burden was observed in IL-17-deficient mice than wild-type mice, associated with a lower frequency of PD-1^hiTim3⁺ CD8⁺ TILs (figure 5A,B). Consistently, we also observed a significant reduction in PD-1^hiTim3⁺ CD8⁺ TIL frequencies of IL-17-deficient mice without CD4 depletion in the B16F10 subcutaneous model (figure 5C), indicating that the IL-17 deficiency attenuated the terminal exhaustion of tumor-infiltrating CD8⁺ T cells in vivo.

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Figure 5

IL-17 deficiency delays the exhaustion of tumor-infiltrating CD8⁺ T cells and tumor growth via suppressing the recruitment of CD11b⁺Gr-1^hi myeloid cells. (A,B) C57BL/6 (WT) or Il17a ^−/− (IL-17 KO) mice were intravenously (i.v.) injected with B16F10 tumor cells and were treated with anti-CD4 Ab. (A) Summary of lung weights of WT (n=6) and IL-17 KO (n=7) at day 17 after tumor inoculation. (B) Frequencies of PD-1^hiTim-3⁺ cells of lung CD8⁺ tumor-infiltrating lymphocytes (TILs) of WT (n=12) and IL-17 KO mice (n=13). (C) WT and IL-17 KO mice were subcutaneously injected with B16F10 tumor cells. Representative contour plots of PD-1 and Tim-3 expression in CD8⁺ TILs and frequencies of PD-1^hiTim-3⁺ cells in CD8⁺ TILs of WT (n=5) and IL-17 KO mice (n=5). (D) Frequencies of CD11b⁺Gr-1^hi cells among CD45⁺ cells in lung tumor of WT (n=12) and IL-17 KO mice (n=13) in (B). (E) Frequencies of CD11b⁺Gr-1^hi cells among CD45⁺ cells of lung tumor in CD4-depleted B16F10 tumor bearing Il17a ^Cre R26 ^WT mice (n=7) and Il17a ^Cre R26 ^DTA mice (n=7). (F) B16F10 tumor-bearing C57BL/6 mice were treated with anti-CD4 Ab alone (n=7) or anti-CD4 Ab plus anti-Ly6G Ab (n=7). PD-1^hiTim-3⁺ cells or PD-1^hiTOX⁺ cells among lung CD8⁺ TILs were analyzed. Data are representatives of at least two independent experiments for (A,C,E,F) and combined results of two independent experiments for (B,D). Data are shown as mean±SD. *p<0.05, **p<0.01.

In addition to the profound decrease of PD-1^hiTim3⁺ CD8⁺ T cells, we observed a remarkable reduction of CD11b⁺Gr-1^hi(Ly6G^hi) myeloid cells among the TILs of IL-17-deficient mice as well as in Il17a ^Cre R26 ^DTA mice in comparison with their respective controls (figure 5D,E). Conversely, anti-CD4 treatment increased the accumulation of CD11b⁺Gr-1^hi myeloid cells into the tumor, likely due to increase of Tc17 cells (online supplemental figure S3A,B). As we repeatedly observed a concomitant decrease in the frequencies of exhausted CD8⁺ TILs and CD11b⁺Gr-1^hi myeloid cells in Il17a ^Cre R26 ^DTA mice and IL-17-deficient mice, we sought to determine if CD11b⁺Gr-1^hi myeloid cells are involved in the IL-17-mediated exhaustion of CD8⁺ TILs. Depletion of the CD11b⁺Gr-1^hi myeloid cells by anti-Ly6G treatment partially reduced the frequency of PD-1^hiTim3⁺ or PD-1^hiTOX⁺ CD8⁺ TILs in anti-CD4-treated mice (figure 5F). Thus, IL-17-producing cells in the TME facilitate the terminal exhaustion of CD8⁺ TILs, at least in part, via the recruitment of CD11b⁺Gr-1^hi myeloid cells into the tumor.

Blockade of RORγt suppresses the exhaustion of CD8⁺ TILs induced by anti-CD4 or anti-PD-L1

Type 17 immunity exerts its effector functions via IL-17 as well as other pro-inflammatory cytokines such as IL-17F, IL-22, and GM-CSF, whose production largely depends on the activity of a transcription factor RORγt.36–38 To determine if targeting RORγt could reinvigorate the exhaustion of tumor-infiltrating CD8⁺ T cells in vivo, we examined the expression of PD-1, Tim-3, and TOX in CD8⁺ TILs in mice bearing B16F10 treated with vehicle or ursolic acid (UA), a small molecule inhibitor of RORγt.39 Treatment with UA remarkably reduced the tumor burden as well as the frequency of Tc17 cells among CD8⁺ TILs, while that of Tc1 cells was minimally affected (figure 6A‒C). Similar to the results observed in Il17a ^Cre R26 ^DTA mice and IL-17-deficient mice, UA treatment significantly decreased the frequency of PD-1^hiTim-3⁺ or PD-1^hiTOX⁺ terminally exhausted CD8⁺ TILs (figure 6D‒F).

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Figure 6

Inhibition of RORγt represses the exhaustion of CD8⁺ T cells induced by anti-CD4 or anti-PD-L1 in vivo. (A–F) C57BL/6 mice were intravenously (i.v.) injected with B16F10 tumor cells and were treated with anti-CD4 Ab every 4 days starting from day 6 after tumor injection. The CD4-depleted tumor-bearing mice were treated with ursolic acid (UA) or vehicle every other day starting from day 6 (n=8 each). Lung weight (A) and the frequencies of Tc17 (B), Tc1 (C), PD-1^hi (D), PD-1^hiTim-3⁺ (E), and PD-1^hiTOX⁺ cells (F) among lung CD8⁺ TILs were analyzed at day 15 after tumor injection. (G–I) CD4-depleted B16F10 tumor-bearing mice were either untreated or treated with UA or anti-PD-L1 or both. (G) Representative contour plots of PD-1 and Tim-3 expression in lung CD8⁺ TILs. (H,I) Frequencies of PD-1^hi (H) or PD-1^hiTim-3⁺ cells (I) among lung CD8⁺ TILs. Data are representatives of at least two independent experiments. Data are shown as mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (unpaired t-test for (A–F) and one-way ANOVA for (H,I)). ^#p<0.05 (unpaired t-test). n.s. not significant.

Blockade of PD-1/PD-L1 pathway is known to induce the expansion of PD-1^intTCF-1⁺ progenitor exhausted CD8⁺ TILs and also the terminal exhaustion of the progenitor exhausted CD8⁺ TILs.19 20 Indeed, we observed that blockade of PD-1/PD-L1 pathway by anti-PD-L1 treatment further increased the frequency of PD-1^hi population, particularly PD-1^hiTim-3⁺ terminally exhausted population among CD8⁺ TILs in anti-CD4-treated mice bearing B16F10 tumor (figure 6G‒I). UA treatment, however, significantly reduced the frequency of PD-1^hi or PD-1^hiTim-3⁺ CD8⁺ TILs induced by anti-PD-L1 in anti-CD4-treated mice (figure 6G‒I). These findings together demonstrate that blockade of RORγt efficiently represses the exhaustion of CD8⁺ TILs induced by anti-CD4 or anti-PD-L1 in tumor-bearing mice in vivo.

Type 17 gene set positively correlates with terminal exhaustion gene set in multiple human cancers

To investigate the clinical relevance of the type 17 immunity-mediated exhaustion of CD8⁺ TILs observed in animal models, we examined the expression of type 17 and exhaustion genes in human tumors by analyzing data from The Cancer Genome Atlas (TCGA). Significant heterogeneity in IL17A transcript expression was observed between individuals of the same cancer. In addition, we also noticed wide variability across various cancer types (figure 7A). We selected four cancers—colorectal cancer, melanoma, lung adenocarcinoma and liver cancer—for further analysis based on IL17A expression and whether immune checkpoint blockers were being used in the clinic for a given indication. We conducted correlation tests between type 17 signature and exhaustion signature gene sets, with each signature being defined by previously reported signature gene sets.28–30 Interrogation of the TCGA dataset revealed that type 17 gene expression was closely associated with exhaustion gene expression in three out of four tumor types examined: colorectal adenocarcinoma, liver hepatocellular carcinoma, and malignant melanoma (online supplemental figure S4A‒D). The highest correlation coefficient was scored by malignant melanoma (rho=0.4617, p<0.0001), while the lowest was observed in lung adenocarcinoma (rho=0.1026, p=0.0861).

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Figure 7

Expression of type 17 signature genes positively correlates with exhaustion signature genes in multiple human cancers. (A) IL-17A mRNA expression in tumor was compared across 31 cancer types. Red bars indicate mean of log₂(value+1). All data were obtained from TCGA using cbioportal website (www.cbioportal.org). (B–E) Correlation between type 17 signature genes expression and either progenitor exhausted T cell (Progenitor Exh.) signature genes expression (left) or terminally exhausted T cell (Terminally Exh.) signature genes expression (right) in colorectal adenocarcinoma (B), liver hepatocellular carcinoma (C), malignant melanoma (D), and lung adenocarcinoma (E). All data were obtained from TCGA. Statistical significance is shown with a Pearson correlation coefficient (r) or a Spearman correlation coefficient (rho).

By interrogating the correlation of type 17 gene set with either the progenitor exhausted (Progenitor Exh.) T cell gene set or the terminally exhausted (Terminally Exh.) T cell gene set,19 28 we found that type 17 gene expression level was tightly associated with expression level of Progenitor Exh. gene set and the correlation coefficient was higher than that of exhaustion signature gene set in all four tumor types (figure 7B‒E and online supplemental figure S4A‒D). Of note, the correlation of type 17 gene set with Terminally Exh. gene set was much stronger than that with Progenitor Exh. gene set, in which the correlation coefficient was over 0.6 in all four cancer types. While the highest correlation coefficient was scored by malignant melanoma (rho=0.8247, p<0.0001), the correlation coefficients for other three cancer types were also comparably high (rho=0.6712~0.6934, p<0.0001) (figure 7B‒E). Thus, type 17 gene expression positively correlates with exhaustion gene expression, particularly “terminal exhaustion” genes in multiple human cancers, which is consistent with the role of IL-17-producing cells in CD8⁺ TIL exhaustion in animal models of tumor in the present study. Together, these results strongly suggest a role for type 17 immunity in promoting terminal exhaustion of CD8⁺ T cells within the tumor in humans.