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.
Combined inhibition of BRAF/MEK is an established therapy for melanoma.,In addition to its canonical mode of action, effects of BRAF/MEK inhibitors on antitumor immune responses are emerging.,Thus, we investigated the effect of these on adaptive immune responses.,Sequential tumor biopsies obtained before and during BRAF/MEK inhibitor treatment of four (n = 4) melanoma patients were analyzed.,Multiplexed immunofluorescence staining of tumor tissue revealed an increased infiltration of CD4+ and CD8+ T cells upon therapy.,Determination of the T-cell receptor repertoire usage demonstrated a therapy induced increase in T-cell clonotype richness and diversity.,Application of the Grouping of Lymphocyte Interactions by Paratope Hotspots algorithm revealed a pre-existing immune response against melanoma differentiation and cancer testis antigens that expanded preferentially upon therapy.,Indeed, most of the T-cell clonotypes found under BRAF/MEK inhibition were already present in lower numbers before therapy.,This expansion appears to be facilitated by induction of T-bet and TCF7 in T cells, two transcription factors required for self-renewal and persistence of CD8+ memory T cells.,Our results suggest that BRAF/MEK inhibition in melanoma patients allows an increased expansion of pre-existing melanoma-specific T cells by induction of T-bet and TCF7 in these.,The online version of this article (10.1007/s00262-020-02804-4) contains supplementary material, which is available to authorized users.