PD-1 checkpoint therapy promotes pro-survival phenotype in regulatory T cells in a model of claudin-low breast cancer

Abstract

Abstract Results We have previously shown in our claudin-low genetically engeneired mouse model (GEMM) that Tregs are increased in the tumor microenvironment (TME), and express high levels of PD-1. We hypothesized that one cause for the lack of activity of α-PD-1 therapy in GEMMs and patients with claudin-low tumors was the activation of PD-1 expressing Tregs in the TME. To evaluate this hypothesis, Tregs were isolated from claudin-low tumors and functionally evaluated ex vivo. In vitro, PD-1 blockade decreased the suppressive activity of Tregs at high Treg:Tnaive cell ratios, but no difference was found in suppression at physiologically relevant ratios. We compared the transcriptional profiles of Tregs isolated from tumor bearing mice with or without α-PD-1 therapy using RNA-Seq. There were 27 signifcantly differentially regulated genes in Tregs from mice treated with α-PD-1 compared to untreated. These genes were associated with survival pathways using IPA with JUN, FOS (P < 0.001) and BCL2 (P = 0.02) upregulated in Tregs exposed to α-PD-1 treatment. Based on these data, we hypothesized that α-PD-1 treatment on Tregs results in a pro-survival phenotype. We found in vitro that Tregs in the presence of α-PD-1 proliferated significantly more than untreated Tregs (P < 0.0001). Importantly, PD-1 blockade did not significantly alter the expression of immunosuppressive proteins such as CTLA-4, CD25, TGFβ, or Il-10 as measured by flow cytometry. Conclusions We found that PD-1 blockade increases proliferation of Tregs in the T11 model, while not significantly altering suppressive capabilities thus leading to enhanced immunosuppression in the TME. Activation of Tregs by α-PD-1 therapy may be responsible for hyperacute tumor flares post therapy.