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 (T ) 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 T 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 T -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 T in HD, but it is expressed on FoxP3 Helios T subset in addition to FoxP3 Helios T in PBC. Pembrolizumab did not affect the levels of FoxP3 Helios T subsets in both cohorts. Taken together, our study suggests that pembrolizumab does not affect T or change their phenotype or function but rather blocks signalling via the PD-1/PD-L1 axis in activated T cells.