Background Antibodies against programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have become established part of anti-cancer therapy. However, the mechanisms contributing to the therapeutic success have not been entirely uncovered by now. Here we focus on the impact of PD-1/CTLA-4-blocking antibodies on regulatory T cells (Tregs), which are known to be involved in tumor immune evasion in many cancer types.

Methods To evaluate how Tregs are affected by anti-PD-1/CTLA-4 therapy, we used a MYC-transgenic mouse model of spontaneously arising B-cell lymphoma, which can be effectively treated by immune checkpoint inhibition. Data were acquired by flow cytometry.

Results As earlier shown, Tregs were involved in immune escape of MYC tumors. The Treg to effector T cell (Teff) ratio was elevated within the CD4-positive cell compartment. Tumor-infiltrating Tregs were predominantly thymic Tregs, which recognized overexpressed tumor-derived self-peptides in an MHC class II-restricted manner and showed upregulated expression of activation markers, Foxp3, CD25 and IL-10. To examine whether these phenotypic alterations correlated with the immunosuppressive capability of Tregs, an in vitro suppression assay was established. In this setting, MYC Tregs turned out to suppress proliferation and IFN-γ release of Teff cells more effectively than wildtype Tregs. The suppression observed in vitro was mediated by cell contacts and IL-10. Further suppressive mechanisms are likely to play a role, such as competition for MHC-II ligands and a consumption of IL-2. To investigate if immune checkpoint blockade interferes with these Treg-dependent immunosuppressive pathways, MYC mice were treated with a combination of anti-PD-1 and anti-CTLA-4 antibodies. Tregs from treated MYC mice showed decreased expression of CD69, CD137, Foxp3, CD25 and IL-10 as compared to Tregs from untreated MYC mice. This correlated with a lower suppressive capacity of Tregs from treated animals in the in vitro suppression assay.

Conclusions Taken together, the data show that immune checkpoint blockade impairs the development of the suppressive phenotype of intratumoral Tregs. Thus, apart from the initially described Teff reactivation, other mechanisms are also relevant for unfolding the therapeutic effect of immune checkpoint inhibitors.

Ethics Approval All animal experiments were approved by Regierung von Oberbayern, approval number 55.2-1-54.