Tumor-associated Tregs obstruct antitumor immunity by promoting T cell dysfunction and restricting clonal diversity in tumor-infiltrating CD8+ T cells

Accumulation of regulatory T cells (Treg) has been described to often correlate with poor prognosis in many solid tumors. How Treg presence impinges on limited functionality and clonal composition of tumor-associated CD8 +T cells has important implications for their therapeutic targeting in the tumor microenvironment. In the present study, we investigated how accumulation of Tregs contributes to T cell dysfunction and clonal constriction of tumor-infiltrating CD8 +T cells.Resected melanoma and lung adenocarcinoma tissues from tumor-bearing mice or patients were analyzed. The proportions and phenotype as well as clonal diversity of tumor-associated CD8 +T cells were evaluated by flow cytometry and single-cell T-cell receptor (TCR) sequencing, respectively, at early or advanced tumor stages or under Treg depletion conditions. Furthermore, antigen-specific T cells were evaluated on adoptive transfer into tumor-bearing mice in the presence or absence of anti-CTLA-4 antibody or CTLA-4 Ig. Lastly, tumor-bearing mice were treated with anti-KLRG1 antibody and/or bromodomain inhibitor JQ1 with interleukin (IL)-2 immune complexes to determine therapeutic efficacy.We demonstrate that the emergence of exhaustion-like phenotype and impaired effector functionality in tumor-associated CD8 +T cells is positively correlated with Treg accumulation in the tumor bed and this dysfunctional phenotype becomes reversed on Treg reduction in murine melanoma and lung cancer models. Heightened tumor-associated Treg-expressed CTLA-4 is key to emergence and sustenance of this phenotype. Furthermore, TCR sequencing revealed a clonal shrinkage of tumor-infiltrating CD8 +T cells as tumor progressed, which was associated with reduced survival profile concomitant to increasing Treg proportions. Limited IL-2 availability was a key mechanism contributing to this peripheral repertoire reshaping as Treg depletion improved IL-2 levels, rescued CD8 +T cell viability, and improved their clonal diversity. Finally, targeted reduction of tumor but not peripheral Tregs through JQ1 and/or anti-KLRG1 antibody significantly improved antitumor response in melanoma-bearing mice when supplemented with IL-2 immune complexes.Collectively, our study reveals a bimodal program enacted by Tregs to support T cell dysfunction in the tumor bed and highlights a promising therapeutic regimen for localized reprogramming of the tumor microenvironment to curb Treg impairment of antitumor CD8 +T cell response in favor of improved antitumor immunity.