Effect of pembrolizumab on CD4+ CD25+ , CD4+ LAP+ and CD4+ TIM-3+ T cell subsets.

Tumor immune evasion involves the expansion of avidly proliferating immunosuppressive cells and inhibition of effector T cell proliferation. Immune checkpoints (IC) block the activation pathways of tumor-reactive T cells. IC pathways are often exploited by tumor cells to evade immune destruction, and blocking these pathways through IC inhibitors (ICI) has shown promising results in multiple malignancies. In this study, we investigated the effects of an ICI, pembrolizumab, on various T cell subsets in vitro. We compared the suppressive activity of CD4+ CD25+ regulatory T cells (conventional Treg ) with T cells expressing T cell immunoglobulin-3+ (TIM-3+ ) and latency-associated peptide (LAP)+ T cells. We found that LAP-expressing T cells were more suppressive than conventional Treg , but TIM-3-expressing T cells were not suppressive. Our results show that pembrolizumab does not modulate functions of Treg and mediates its immunostimulatory effects via the release of effector T cells from suppression. These findings may assist in the development of agents designed to intervene in IC pathways to overcome Treg resistance to ICI.

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.