Combination checkpoint therapy with anti-PD-1 and anti-BTLA results in a synergistic therapeutic effect against murine glioblastoma

Abstract

Clinical trials involving anti-programmed cell death protein-1 (anti-PD-1) failed to demonstrate improved overall survival in glioblastoma (GBM) patients. This may be due to the expression of alternative checkpoints such as B- and T- lymphocyte attenuator (BTLA) on several immune cell types including regulatory T cells. Murine GBM models indicate that there is significant upregulation of BTLA in the tumor microenvironment (TME) with associated T cell exhaustion. We investigate the use of antibodies against BTLA and PD-1 on reversing immunosuppression and increasing long-term survival in a murine GBM model. C57BL/6 J mice were implanted with the murine glioma cell line GL261 and randomized into 4 arms: (i) control, (ii) anti-PD-1, (iii) anti-BTLA, and (iv) anti-PD-1 + anti-BTLA. Kaplan-Meier curves were generated for all arms. Flow cytometric analysis of blood and brains were done on days 11 and 16 post-tumor implantation. Tumor-bearing mice treated with a combination of anti-PD-1 and anti-BTLA therapy experienced improved overall long-term survival (60%) compared to anti-PD-1 (20%) or anti-BTLA (0%) alone (P = .003). Compared to monotherapy with anti-PD-1, mice treated with combination therapy also demonstrated increased expression of CD4+ IFN-γ (P < .0001) and CD8+ IFN-γ (P = .0365), as well as decreased levels of CD4+ FoxP3+ regulatory T cells on day 16 in the brain (P = .0136). This is the first preclinical investigation into the effects of combination checkpoint blockade with anti-PD-1 and anti-BTLA treatment in GBM. We also show a direct effect on activated immune cell populations such as CD4+ and CD8 + T cells and immunosuppressive regulatory T cells through this combination therapy.

Keywords: B and T lymphocyte attenuator; anti-BTLA; anti-PD-1; glioblastoma; glioblastoma immunotherapy; immune checkpoint inhibitor therapy.

Figure 1.

Timing of BTLA expression in tumor-bearing mice on days 7, 16, and 21. (a) Representative flow cytometry plots of TILs demonstrating BTLA expression on day 7, 16, and 21 on CD4+ FoxP3 + T cells, CD4+ FoxP3- T cells, and CD8 + T cells (b)Aggregate (n = 5) flow cytometry data of BTLA expression in PBMCs from untreated tumor-bearing mice on days 7, 16, and 21. (c) Aggregate (n = 5) flow cytometry data of BTLA expression in TILs of untreated tumor-bearing mice on days 7, 16, and 21. All plots represent mean with standard deviation. Comparison between two groups is made via the Student’s t test. (* p < .05)

Figure 2.

Flow cytometric analysis of T cell populations in mouse brain at day 16 post-tumor implantation. (a) Flow plots demonstrating CD4 and CD8 IFN-γ expression, 4–1BB expression, and CD4 Foxp3 presence in the brain. (b) Aggregate (n = 5 per group) data of control (no treatment), anti-PD-1, anti-BTLA, and combination therapy groups for CD4 and CD8 IFN-γ and CD4 Foxp3 presence in the brain. Differences between two treatment arms were analyzed via Student’s t test. (**** P < .0001, *** P < .001, ** P < .01, * P < .05)

Figure 3.

In vitro co-culture assay for IFN-γ secretion with ELISA. (a) CD8 + T cells were isolated via FACS microfluidic sorting from untreated mice and mice treated in vivo with anti-PD-1 monotherapy, anti-BTLA monotherapy, and combination anti-PD-1 and anti-BTLA. Cells were co-cultured with GL261 tumor cell lysate and CD11c+ dendritic cells. (b) Plot of concentration of IFN-γ in supernatant of co-culture wells. All plots represent mean with standard error. Differences between two treatment arms were analyzed via Student’s t test. (**** P < .0001, *** P < .001, ** P < .01, * P < .05)

Figure 4.

Survival schema and Kaplan-Meier curve. (a) Mice were implanted with 1.3e5 GL261-Luc2 cells with a stereotactic frame. On post-implantation day 7, mice were assessed for presence of tumor by injecting them with 200 μl of 1 mg/ml D-luciferin and imaged with IVIS (In Vivo Imaging Systems), and subsequently randomly assorted into four groups: control (no treatment), anti-BTLA monotherapy, anti-PD-1 monotherapy, and combination anti-BTLA and anti-PD-1 therapy. 5 mice in each arm were saved for blood and brain harvest to undergo flow cytometric analysis. Remaining 8 mice in each arm underwent a survival study and were rechallenged on day 60. (b) Treatment with anti-BTLA was on days 7, 10, and 14. Treatment with anti-PD-1 was on days 10, 12, and 14. (c) Kaplan-Meier curve demonstrating differences in overall long-term survival with combination therapy. (**** P < .0001, *** P < .001, ** P < .01, * P < .05)

Figure 5.

CD4+ and CD8 + T cell depletion Kaplan-Meier curve. Mice in the depletion arms underwent depletion of either CD4 + T cells or CD8 + T cells prior to treatment with anti-BTLA and anti-PD-1 therapy. There was no significant difference in survival between the control arm, CD4 + T cell depletion arm, and CD8 + T cell depletion arm. Combination therapy without any T cell depletion had significantly greater median overall survival compared to control (P = .004), CD4 + T cell depletion plus combination therapy (P = .036), and CD8 + T cell depletion plus combination therapy (P = .046)