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

Programmed death-1 (PD-1) and interactions with PD-ligand 1 (PD-L1) play critical roles in the tumour evasion of immune responses through different mechanisms, including inhibition of effector T cell proliferation, reducing cytotoxic activity, induction of apoptosis in tumour-infiltrating T cells and regulatory T cell (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 cytotoxic T lymphocyte antigen-4 (CTLA-4), CD15s, latency-associated peptide (LAP) and Ki-67. Moreover, we report that CD15s is expressed mainly on forkhead box P3 (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 signalling via the PD-1/PD-L1 axis in activated T cells.