In-vitro effect of pembrolizumab on different T regulatory cell subsets

Abstract

Programmed death-1 (PD-1) and interactions with PD-L1 play critical roles in the tumor evasion of immune responses through different mechanisms including inhibition of effector T cell proliferation, reducing cytotoxic activity, induction of apoptosis in tumor-infiltrating T cells, and regulatory T cells (Treg) expansion. Effective blockade of immune checkpoints can therefore potentially eliminate these detrimental effects. The aim of this study was to investigate the effect of anti-PD-1 antibody, pembrolizumab, on various Treg subpopulations. Peripheral blood mononuclear cells (PBMC) from healthy donors (HD) and primary breast cancer patients (PBC) were treated in vitro with pembrolizumab, which effectively reduced PD-1 expression in both cohorts. We found that PD-1 was expressed mainly on CD4⁺CD25⁺ T cells, and pembrolizumab had a greater effect on PD-1 expression in CD4⁺CD25^- T cells, compared to CD4⁺CD25⁺ cells. In addition, pembrolizumab did not affect the expression levels of Treg-related markers including CTLA-4, CD15s, LAP and Ki-67. Moreover, we report that CD15s is mainly expressed on FoxP3^-Helios⁺ Treg in HD, but it is expressed on FoxP3⁺Helios^- Treg subset in addition to FoxP3^-Helios⁺ Treg in PBC. Pembrolizumab did not affect the levels of FoxP3^+/-Helios^+/- Treg subsets in both cohorts. Taken together, our study suggests that pembrolizumab does not affect Treg or change their phenotype or function but rather blocks signaling via PD-1/PD-L1 axis in activated T cells.