Response to anti-PD-1 and anti-LAG-3 immune checkpoint blockade (ICB) is associated with induction of pro-inflammatory Tregs

Abstract

Abstract T cells upregulate the immune checkpoint, LAG-3, which is associated with reduced T cell effector function following repeated antigen exposure. Further, high LAG-3 expression on Tregs has been reported in patients with varying cancer types; thus, we hypothesized that the combination of aLAG-3 with another common ICB, aPD-1, would synergize to potently reinvigorate T cells to promote tumor regression and increase survival. Combined aPD-1/aLAG-3 immunotherapy significantly improved the survival of CT26 (BALB/c; colon carcinoma)and MCA-205 (C57BL/6; sarcoma) tumor-bearing mice compared to monotherapy. In-depth analysis of tumor-infiltrating lymphocytes (TIL) revealed that aPD-1/aLAG-3 therapy significantly increased the overall percentage, cytotoxicity (granzyme A), and cytokine production (TNF-a and IFN-g) of CD8+ TIL, and increased CD4+ Teff/Treg ratios compared to aPD-1 or aLAG-3 alone. These effects were enriched in responders to aPD-1/aLAG-3 therapy, which were designated as those exhibiting decreased tumor size on the day of harvest compared to maximum tumor growth post-implant. Surprisingly, Tregs isolated from tumors responding to aPD-1/aLAG-3 displayed increased proliferation, secretion of pro-inflammatory cytokines (TNF-a and IFN-g), and increased LAG-3 expression. In summary, these data suggest that aPD-1/aLAG-3 immunotherapy increased CD8+ TIL exhibiting enhanced effector function, increased CD4+ Teff/Treg ratios, and induced Treg pro-inflammatory responses. Together, these positive immunological changes led to a more immune stimulatory tumor microenvironment capable of supporting tumor regression and significantly improved tumor-free survival.