While PD-1 and CTLA-4 immune checkpoint antibodies have led to durable clinical activity in certain cancers, only a fraction of patients exhibit responses. In these responsive tumors, PD-1 and CTLA-4 antibodies are thought to interfere with tumor immunosuppression of T-cells; however, the exact mechanisms of action and potential synergism between these therapies remains unclear. As triple-negative breast cancers (TNBCs) are characterized by elevated expression of inflammatory and immunosuppressive molecules, as well as high levels of immune infiltrating T cells (TILs), we hypothesized that they would be susceptible to treatment with PD-1 and CTLA-4 antibodies. To test this hypothesis and further define the mechanisms of action of checkpoint blockade, we generated a model of murine TNBC (E0771) utilizing ovalbumin (OVA) as a defined antigen that is tumor-specific and recognizable by transgenic T cells (OT-I cells). Consistent with human TNBCs, E0771 tumors exhibit robust T cell infiltration, with >60% of CD4+ T cells being T-regulatory cells (Tregs), and tumor cells express high levels of PDL1. We found that despite the generation of systemic anti-tumor responses and the addition of OT-I cells, TNBC immunosuppression shielded tumors from immune mediated regression. We then tested the efficacy of anti-PD1 and anti-CTLA4 targeting antibodies to inhibit this tumor immunosuppression and demonstrate that they had an anti-tumor effect by blocking PD-1 signaling in the tumor microenvironment and reducing intratumoral Tregs, respectively. When combined, these distinct mechanisms of action led to regression of ~80% of tumors and were significantly associated with anti-tumor adaptive responses. T cell receptor (TCR) sequencing of TILS in treated mice demonstrated a hyperexpansion of several clones, while also a broadening of the total number of unique clones present. Surprisingly, we found that despite using a homogenous tumor model and adoptively transferring OT-1 cells, TCR sequencing revealed clonal populations that were almost entirely unique for each tumor, with the OTI TCR not representing an expanded clone. As such, our study demonstrates that dual CTLA-4 and PD-1 checkpoint blockade inhibits immunosuppression of T cells in the tumor microenvironment through different and complementary mechanisms to expand and broaden unique intrinsic T cell repertoires in the tumor.
Citation Format: Erika J. Crosby, Junping Wei, Xiao Yi Yang, Gangjun Lei, Tao Wang, Cong-Xiao Liu, Pankaj Agarwal, H. Kim Lyerly, Zachary C. Hartman. Checkpoint blockade elicits unique T cell expansion to promote tumor regression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A38.