PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors

TLDR

Vaccination with irradiated B16 melanoma cells expressing GM‑CSF or Flt3‑ligand together with CTLA‑4 blockade promotes tumor rejection, yet T‑cell activity can still be suppressed by PD‑1/PD‑L1 or PD‑L1/B7‑1 interactions. Combined CTLA‑4 and PD‑1 blockade more than doubles B16 melanoma rejection rates, increases effector T‑cell infiltration and Teff‑to‑Treg and Teff‑to‑MDSC ratios, boosts IFN‑γ production, and shifts the tumor microenvironment from suppressive to inflammatory.

Abstract

Vaccination with irradiated B16 melanoma cells expressing either GM-CSF (Gvax) or Flt3-ligand (Fvax) combined with antibody blockade of the negative T-cell costimulatory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) promotes rejection of preimplanted tumors. Despite CTLA-4 blockade, T-cell proliferation and cytokine production can be inhibited by the interaction of programmed death-1 (PD-1) with its ligands PD-L1 and PD-L2 or by the interaction of PD-L1 with B7-1. Here, we show that the combination of CTLA-4 and PD-1 blockade is more than twice as effective as either alone in promoting the rejection of B16 melanomas in conjunction with Fvax. Adding αPD-L1 to this regimen results in rejection of 65% of preimplanted tumors vs. 10% with CTLA-4 blockade alone. Combination PD-1 and CTLA-4 blockade increases effector T-cell (Teff) infiltration, resulting in highly advantageous Teff-to-regulatory T-cell ratios with the tumor. The fraction of tumor-infiltrating Teffs expressing CTLA-4 and PD-1 increases, reflecting the proliferation and accumulation of cells that would otherwise be anergized. Combination blockade also synergistically increases Teff-to-myeloid-derived suppressor cell ratios within B16 melanomas. IFN-γ production increases in both the tumor and vaccine draining lymph nodes, as does the frequency of IFN-γ/TNF-α double-producing CD8 + T cells within the tumor. These results suggest that combination blockade of the PD-1/PD-L1- and CTLA-4-negative costimulatory pathways allows tumor-specific T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby shifting the tumor microenvironment from suppressive to inflammatory.