Combination anti–CTLA-4 plus anti–PD-1 checkpoint blockade utilizes cellular mechanisms partially distinct from monotherapies

Significance Immune checkpoint blockade therapy has become a critical pillar of cancer therapy. Here, we characterize the cellular mechanisms of monotherapy and combination anti–cytotoxic T lymphocyte antigen-4 plus anti–programmed cell death-1 therapy. Using high-dimensional single-cell profiling, we determine that combination therapy elicits cellular responses that are partially distinct from those induced by either monotherapy. In particular, combination therapy mediates a switch from expansion of phenotypically exhausted cluster of differentiation 8 (CD8) T cells to expansion of activated effector CD8 T cells. In addition, we systematically compare T cell subsets present in matched peripheral blood and tumor tissues to define what aspects of antitumor responses can be observed peripherally. These findings have significant implications for both the cellular mechanisms of action and biomarkers of response to monotherapies and combination therapy. Immune checkpoint blockade therapy targets T cell-negative costimulatory molecules such as cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1). Combination anti–CTLA-4 and anti–PD-1 blockade therapy has enhanced efficacy, but it remains unclear through what mechanisms such effects are mediated. A critical question is whether combination therapy targets and modulates the same T cell populations as monotherapies. Using a mass cytometry-based systems approach, we comprehensively profiled the response of T cell populations to monotherapy and combination anti–CTLA-4 plus anti–PD-1 therapy in syngeneic murine tumors and clinical samples. Most effects of monotherapies were additive in the context of combination therapy; however, multiple combination therapy-specific effects were observed. Highly phenotypically exhausted cluster of differentiation 8 (CD8) T cells expand in frequency following anti–PD-1 monotherapy but not combination therapy, while activated terminally differentiated effector CD8 T cells expand only following combination therapy. Combination therapy also led to further increased frequency of T helper type 1 (Th1)-like CD4 effector T cells even though anti–PD-1 monotherapy is not sufficient to do so. Mass cytometry analyses of peripheral blood from melanoma patients treated with immune checkpoint blockade therapies similarly revealed mostly additive effects on the frequencies of T cell subsets along with unique modulation of terminally differentiated effector CD8 T cells by combination ipilimumab plus nivolumab therapy. Together, these findings indicate that dual blockade of CTLA-4 and PD-1 therapy is sufficient to induce unique cellular responses compared with either monotherapy.