TIGIT predominantly regulates the immune response via regulatory T cells

Abstract

Coinhibitory receptors are critical for the maintenance of immune homeostasis. Upregulation of these receptors on effector T cells terminates T cell responses, while their expression on Tregs promotes their suppressor function. Understanding the function of coinhibitory receptors in effector T cells and Tregs is crucial, as therapies that target coinhibitory receptors are currently at the forefront of treatment strategies for cancer and other chronic diseases. T cell Ig and ITIM domain (TIGIT) is a recently identified coinhibitory receptor that is found on the surface of a variety of lymphoid cells, and its role in immune regulation is just beginning to be elucidated. We examined TIGIT-mediated immune regulation in different murine cancer models and determined that TIGIT marks the most dysfunctional subset of CD8+ T cells in tumor tissue as well as tumor-tissue Tregs with a highly active and suppressive phenotype. We demonstrated that TIGIT signaling in Tregs directs their phenotype and that TIGIT primarily suppresses antitumor immunity via Tregs and not CD8+ T cells. Moreover, TIGIT+ Tregs upregulated expression of the coinhibitory receptor TIM-3 in tumor tissue, and TIM-3 and TIGIT synergized to suppress antitumor immune responses. Our findings provide mechanistic insight into how TIGIT regulates immune responses in chronic disease settings.

Figure 7. TIM-3 and TIGIT synergize to suppress antitumor immunity.

(A) WT and Tigit^–/– mice (n = 5) were implanted s.c. with B16F10 melanoma cells and treated with 250 μg isotype or anti–TIM-3 (RMT3-23) Ab on days 3, 6, 9, and 12. Statistical comparisons are between WT plus Ig and Tigit^–/– plus Ig and between Tigit^–/– plus Ig and Tigit^–/– plus anti–TIM-3. ****P < 0.0001 by repeated-measures ANOVA with a Sidak test. Data are representative of 2 experiments. (B and C) B16F10 melanoma cells or RM-1 cells were administered i.v. to WT mice (n = 5 per group) that were treated i.p. with 250 μg isotype (cIg) and/or anti–TIM-3 (RMT3-23) Ab on days 0 and 3. (B) Graphs show the number of foci ± SEM in the lungs after B16F10 (left) or RM-1 (right) cell injections on day 14. Statistical comparisons are between Tigit^–/– plus cIg and Tigit^–/– plus anti–TIM-3. **P < 0.01 by Mann-Whitney U test. Data are representative of 2 experiments. (C) Survival of mice (n = 10 per group) after i.v. administration of B16F10 melanoma cells. Statistical comparisons are between WT plus cIg and Tigit^–/– plus cIg and between Tigit^–/– plus cIg and Tigit^–/– plus anti–TIM-3. ***P < 0.001 by log-rank test. Data were pooled from 2 independent experiments.

Figure 6. TIGIT signaling in Tregs.

CD4⁺ FOXP3⁺ Tregs from WT Foxp3-GFP–KI mice were sorted and stimulated with plate-bound isotype or agonistic anti-TIGIT Ab, together with anti-CD3 and anti-CD28, for 48 or 96 hours. Gene expression was then analyzed using a NanoString CodeSet. Bar graphs show the fold expression ± SEM of genes in Ab-treated versus isotype-treated cells at 48 and 96 hours. Data were pooled from 2 different experiments.

Figure 5. TIGIT expression on Tregs has a dominant role compared with CD8⁺ T cells in regulating antitumor responses.

(A) CD8⁺ T cells from WT or Tigit^–/– mice were mixed with WT CD4⁺ (FOXP3⁺ and FOXP3^–) T cells and injected i.v. into Rag^–/– mice (n = 5). Two days later, mice were implanted with B16F10 tumor cells. Graph shows tumor growth over time. Data are representative of 3 different experiments. (B) Tregs from WT or Tigit^–/– Foxp3-GFP–KI mice were mixed with WT CD8⁺ and CD4⁺ FOXP3^– cells and transferred into Rag^–/– mice (n = 5). Graph shows tumor growth over time. Data are representative of 5 different experiments. ***P < 0.001 by repeated-measures ANOVA with a Sidak test. (C–E) Seventeen days after implantation, TILs and DLNs were isolated from mice that received WT or Tigit^–/– Tregs. (C) Frequency ± SEM of TIGIT⁺ CD8⁺ TILs from each group (n = 9–10). Data were pooled from 2 independent experiments. (D) TILs were stimulated with anti-CD3 and anti-CD28 and analyzed for cytokine production. Graphs show IL-2⁺, TNF-α⁺, and IFN-γ⁺ percentages ± SEM within CD8⁺ TILs in each group (n = 4–5). *P < 0.05 by Mann-Whitney U test (C and D). (E) Representative plots show gp33 (control dextramer) or gp100 dextramer staining in CD8⁺ T cells from pooled DLNs of Rag^–/– mice. (D and E) Data are representative of 2 independent experiments.

Figure 4. TIGIT restrains antitumor immune responses.

(A) Growth of B16F10 melanoma or MC38 colon carcinoma in WT or Tigit^–/– mice (n = 5–6). Data are representative of 3 independent experiments. ***P < 0.001 and ****P < 0.0001 by repeated-measures ANOVA with a Sidak test. (B) WT or Tigit^–/– mice (n = 5) were treated i.p. with anti-asialoGM1 or anti-CD8β Abs or with their isotype controls (cIg) on days –1, 0, 7, and 14 after B16F10 tumor implantation. Tumor growth after anti-asialoGM1 (left) or anti-CD8β (right) treatment is shown. Data are representative of 2 independent experiments. Comparisons are between WT plus cIg versus Tigit^–/–, irrespective of cIg or anti-asialoGM1 treatment (left), and between cIg versus anti-CD8β in Tigit^–/– mice (right). ***P < 0.001 and ****P < 0.0001 by repeated-measures ANOVA with a Sidak test. (C and D) Total TILs were isolated from B16F10 tumor–bearing WT or Tigit^–/– mice (n = 4–5). (C) TILs were stimulated with 10 μg/ml gp100 peptide. Contour plots and bar graphs show the percentage ± SEM of granzyme B⁺ cells and the normalized MFI of granzyme B expression within CD8⁺ TILs. MFI was normalized to the mean of WT CD8⁺ TILs in each independent experiment. Data were pooled from 2 to 3 experiments. *P < 0.05 and **P < 0.01 by Mann-Whitney U test. (D) CT Violet–labeled TILs were stimulated with anti-CD3 for 4 days and then analyzed by flow cytometry. Representative histogram of CT Violet dilution in CD8⁺ TILs. Data are representative of 3 individual measurements.

Figure 3. TIGIT⁺ Tregs in the tumor tissue exhibit a more suppressive and activated phenotype.

TIGIT⁺ and TIGIT^– Tregs were isolated from B16F10 tumors implanted into Foxp3-GFP–KI mice (A, C, and D) or IL-10-Thy1.1 Foxp3-GFP–KI mice (B). (A) Gene expression was analyzed using a custom NanoString CodeSet. Bar graphs show the fold expression ± SEM of selected differentially expressed surface receptors (left), effector molecules, and transcription factors (TFs) (right) in TIGIT⁺ and TIGIT^– Tregs. Data are pooled from 2 different experiments. Selected genes with a fold change of greater than 2 are depicted. (B) Left panels: representative flow cytometric data showing IL-10-Thy1.1 staining in TIGIT^– and TIGIT⁺ Treg TILs. Right panel: frequency ± SEM of IL-10⁺ cells within TIGIT^– and TIGIT⁺ Treg TILs (n = 5). Data are representative of 2 independent experiments. **P < 0.01 by Mann-Whitney U test. (C) Tregs isolated from tumor tissue were cocultured with splenic CT Violet–labeled CD4⁺ T cells (1:8). CT Violet dilution was analyzed by flow cytometry 4 days later. Gates indicate the undivided CD4⁺ subsets. Data are representative of 2 experiments. (D) Representative contour plots of TIM-3 expression on TIGIT⁺ TIL Tregs or TIGIT⁺ naive Tregs. Data are representative of 5 individual mice.

Figure 2. TIGIT marks a dysfunctional CD8⁺ T cell subset.

TILs were harvested from WT mice (n = 5–20) or IL-10 Thy1.1 mice (n = 5) bearing B16F10 melanoma 12–15 days after tumor implantation when tumor sizes measured between 140 and 180 mm². (A) Frequency ± SEM of CD8⁺ TIGIT⁺ and TIGIT^– TILs producing IL-2, TNF-α, IFN-γ, and IL-10 (Thy1.1) or expressing surface CD107a after stimulation with anti-CD3 and anti-CD28. Data were pooled from 3 different experiments. (B) Representative flow cytometric data showing TIGIT coexpression with PD-1, TIM-3, and LAG-3 on CD8⁺ TILs. Data are representative of 5 individual mice. (C) Left panel: representative contour plot showing TIGIT coexpression with eomes in CD8⁺ TILs. Middle panel: representative histogram showing eomes expression in TIGIT⁺ and TIGIT^– CD8⁺ TILs. Right panel: mean fluorescence intensity (MFI) ± SEM for eomes in TIGIT⁺ and TIGIT^– CD8⁺ TILs (n = 8 mice). Data were pooled from 2 different experiments. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05 by Mann-Whitney U test.

Figure 1. TIGIT is enriched on TILs.

Spleen, tumor DLNs, and TILs were harvested from WT Foxp3-GFP–KI mice (n = 5) bearing B16F10 melanoma tumors and stained with Abs against CD4, CD8, and TIGIT. (A) Left panels: representative flow cytometric data showing TIGIT expression on CD4⁺ and CD8⁺ T cells in spleen, DLNs, and TILs. Right panel: frequency ± SEM of TIGIT⁺ cells. ****P < 0.0001 by 1-way ANOVA and Tukey’s multiple comparisons test. (B) Left panels: representative flow cytometric data showing TIGIT expression in CD4⁺ FOXP3^– (GFP^–) and CD4⁺ FOXP3⁺ (GFP⁺) in TILs. Right panel: frequency ± SEM of TIGIT⁺ FOXP3^– and FOXP3⁺ cells. CD4⁺ FOXP3⁺ percentages within total CD4⁺ T cells were 32.46% ± 4.27%. Data are representative of 2 to 3 experiments. ****P < 0.0001 by 1-way ANOVA and Tukey’s multiple comparisons test comparing TIGIT⁺ FOXP3⁺ cells across the indicated tissues.