TIGIT and PD-1 dual checkpoint blockade enhances antitumor immunity and survival in GBM

Abstract

The use of inhibitory checkpoint blockade in the management of glioblastoma has been studied in both preclinical and clinical settings. TIGIT is a novel checkpoint inhibitor recently discovered to play a role in cancer immunity. In this study, we sought to determine the effect of anti-PD-1 and anti-TIGIT combination therapy on survival in a murine glioblastoma (GBM) model, and to elucidate the underlying immune mechanisms. Using mice with intracranial GL261-luc⁺ tumors, we found that TIGIT expression was upregulated on CD8⁺ and regulatory T cells (Tregs) in the brain compared to draining cervical lymph nodes (CLN) and spleen. We then demonstrated that treatment using anti-PD-1 and anti-TIGIT dual therapy significantly improved survival compared to control and monotherapy groups. The therapeutic effect was correlated with both increased effector T cell function and downregulation of suppressive Tregs and tumor-infiltrating dendritic cells (TIDCs). Clinically, TIGIT expression on tumor-infiltrating lymphocytes was shown to be elevated in patient GBM samples, suggesting that the TIGIT pathway may be a valuable therapeutic target. Expression of the TIGIT ligand, PVR, further portended a poor survival outcome in patients with low-grade glioma. We conclude that anti-TIGIT is an effective treatment strategy against murine GBM when used in combination with anti-PD-1, improving overall survival via modifications of both the T cell and myeloid compartments. Given evidence of PVR expression on human GBM cells, TIGIT presents as a promising immune therapeutic target in the management of these patients.

Keywords: CD155; GBM; PD-1; PVR; TIGIT; checkpoint inhibitor; dendritic cell; glioma; immunotherapy.

Figure 1.

High PD-1 and TIGIT Expression in Patient Brain TILs. A. Representative histograms of PD-1 and TIGIT expression on tumor-infiltrating CD3⁺CD8⁺ and CD3⁺CD4⁺ T cells. Gray histograms represent isotype control, and red histograms represent the staining of PD-1 and TIGIT surface markers. B. PD-1 (40.19% ± 4.01) and TIGIT (54.48% ± 4.30) expressions on CD8⁺ T cells were elevated in patients with glioma. C. High expressions of PD-1 (43.92% ± 3.27) and TIGIT (41.78% ± 2.91) were found in tumor-infiltrating CD4⁺ T cells.

Figure 2.

TIGIT and PD-1 expression is upregulated in brain tumor infiltrating lymphocytes. A. Brain CD8⁺ cells had significantly higher expression of TIGIT (p = 0.0015) and B. PD-1 (p = 0.0001) compared to CD8⁺ cells in the CLN and spleen. C. CD4⁺FoxP3⁺ cells in the brain had similarly elevated expression of TIGIT (p < 0.0001) and D. PD-1 (p < 0.0001) compared to those in the CLN and spleen.

Figure 3.

TIGIT expression is upregulated at later time-points in a tissue specific pattern. Flow analysis was performed on days 13 and 20 for brain TILs and spleens. A. TIGIT expression on CD3⁺ cells in the brain was significantly higher on day 20 than 13 (p = 0.0490). There was no significance between the two time-points for PD-1 or CD226 expression on brain CD3⁺ cells (p = 0.7432 and 0.6690 respectively). B. Expression of TIGIT, PD-1, and CD226 on spleen CD3⁺ cells remained the same across the two time-points (p = 0.1846, 0.2879, and 0.7560 respectively).

Figure 4.

Anti-PD-1 and anti-TIGIT combination therapy improves long-term survival following both early and late treatment courses. A. Diagram depicting experiment set up including treatment schedules. Day 0, 130,000 GL261-luc⁺ cells were injected stereotactically into the right stratum of female C57 BL/6 J mice (N = 70). IVIS was used to confirm tumor presence on day 7, and the animals were randomized into 10 groups. Anti-PD-1 treatment was administered on days 10, 12, and 14 via i.p. injection at a dose of 200μg/animal. Anti-TIGIT treatment was also given via i.p. injections, 200μg/animal, every other day for a total of 5 doses starting on either day 8 (Group A), 10 (Group B), 12 (Group C), or 14 (Group D). Survival was monitored. B. Kaplan meier survival curve depicts the primary endpoint for each treatment group. Anti-PD-1 treatment alone resulted in 14.3% long-term survivors, compared to 0% in the control group (p = 0.0086). Anti-TIGIT monotherapy led to 0%, 57.1%, 14.3%, and 0% long-term survival using treatment schedules A, B, C, and D respectively (p = 0.1960, 0.0006, 0.0101, and 0.1032 respectively). All combination regimens led to significant improvements in survival compared to control, including 57.1%, 85.7%, 42.9% and 57.1% long-term survivors for Groups A-D respectively (p = 0.002, 0.0002, 0.0006, and 0.0032). Combination B was also significant compared to anti-PD-1 monotherapy (p = 0.0082), while combinations A and D also trended towards significance (p = 0.0982 and 0.0929 respectively). C. Survival curve demonstrating therapeutic effect in each of the four arms following repeat survival experiment using Group D anti-TIGIT antibody treatment course (N = 54). Anti-PD-1 monotherapy resulted in 16.7% long-term survival and a median survival of 28.5 days, compared to 0% long-term survival and 24.5 days median survival in the control group (p = 0.0175). Anti-TIGIT monotherapy was not significantly different from control, with 0% long-term survivors and 25.5 days median survival (p = 0.9062). Combination treatment conferred a median survival of 44 days, including 48.0% long-term survivors (p < 0.0001). Dual checkpoint blockade was led to a significant improvement in survival compared to both anti-PD-1 and anti-TIGIT monotherapy groups (p = 0.0366 and <0.0001 respectively).

Figure 5.

Anti-PD-1 monotherapy and combination treatment confers immunologic memory in long-term survivors. A. Representative IVIS images showing tumor presence in the control group with naïve wildtype mice, compared to absence of tumor in long-term survivors following anti-PD-1 or combination treatment on day 7 after rechallenge with GL261-luc⁺ cells. B. Kaplan meier survival curve demonstrating 100% long-term survival after tumor rechallenge of responding mice previously treated with anti-PD-1 or anti-TIGIT and anti-PD-1 dual therapy, versus 0% long-term survival of naïve mice without prior treatment (p = 0.0005).

Figure 6.

Combination treatment restores T cell effector function and anti-TIGIT therapy downregulates Treg suppressor phenotype. A. Frequency of brain infiltrating CD8⁺ cells and CD4⁺ cells were significantly elevated in the combination group relative to control, anti-PD-1 monotherapy, and anti-TIGIT monotherapy groups (p = 0.0059 and 0.0230 respectively). B. Combination treatment was correlated with significantly greater IFNγ expressing CD8⁺ and CD4⁺ cells (p = 0.0066 and 0.0014 respectively). IFNγ and TNFα dual-expressing CD8⁺ and CD4⁺ cells were also significantly higher in the combinatorial group (p = 0.0002 and 0.0008 respectively). C. Sample flow contour plots demonstrating shift in IFNγ expression in CD4⁺ and CD8⁺ cells across treatment arms. D. An overall significant difference was noted in the frequency of brain infiltrating CD4⁺FoxP3⁺ regulatory T cells (Tregs) in the four treatment arms (p = 0.0080). The percentage of Tregs was significantly reduced by anti-PD-1 and anti-TIGIT monotherapies (p = 0.0496 and 0.0209 respectively), but not significantly changed with combination treatment when compared to untreated, PD-1 monotherapy, and TIGIT monotherapy groups (p > 0.9999, 0.4824, and 0.2645 respectively). E. The expressions of TIGIT and PD-1 on Tregs were significantly different in the overall comparisons of all four treatment arms (p = 0.0042 and 0.0098 respectively). TIGIT expression on Tregs was significantly reduced in the anti-TIGIT monotherapy group only relative to control arm (p = 0.0082). PD-1 expression on Tregs demonstrated a significant increase in the anti-PD-1 group compared to control (p = 0.0145).

Figure 7.

Tumor infiltrating dendritic cells are reduced following anti-TIGIT and anti-PD-1 combination treatment. A. A significant overall difference in CD11b⁺CD11 c⁺ tumor infiltrating dendritic cells (TIDCs) was found in ANOVA analysis of the four treatment arms (p = 0.0069). Addition of anti-TIGIT to anti-PD-1 therapy significantly decreases the frequency of CD11b⁺CD11 c⁺ TIDCs relative to untreated group (p = 0.0169), while neither monotherapy groups were significantly different from control (p > 0.05). B. The expression of MHCII was significantly different in the overall comparison of all groups (p = 0.0018). Frequency of CD11b⁺CD11 c⁺ TIDCs with high MHCII expression (CD11b⁺CD11 c⁺MHCII^hi) is significantly lower in all treated groups relative to control (p = 0.0018). C. Representative flow contour plots showing relative MHCII expression on CD11b⁺CD11 c⁺ cells in the four treatment arms. D. Sample flow plots depicting populations of Ly6G⁺ and Ly6 C⁺ myeloid derived suppressor cells (MDSCs) across treatment groups.

Figure 8.

PVR expression confers poor overall survival in patients with low grade glioma. A Expression of PVR did not significantly affect survival in patients with glioblastoma (N = 584; p = 0.315). B. PVR expression was correlated with a significantly poorer survival for patients with low grade glioma (N = 513; p = 0.0121).