biostudies-literatureDykema AGNIBIB NIH HHSNHGRI NIH HHSNCI NIH HHSNIGMS NIH HHSeadg1487https://www.ebi.ac.uk/biostudies/studies/S-EPMC10629528biostudies-literatureUnknown8(87)Regulatory T cells (T_reg) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating T_reg (TIL-T_reg) from anti-PD-1-treated and treatment-naive non-small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)-specific T_reg derived from a murine tumor model. We identified 10 subsets of human TIL-T_reg, most of which have high concordance with murine TIL-T_reg subsets. Only one subset selectively expresses high levels of <i>TNFRSF4</i> (OX40) and <i>TNFRSF18</i> (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in T_reg suppression, including <i>LAG3</i>. Functionally, the OX40^hiGITR^hi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-T_reg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-T_reg-expressing T cell receptors that are specific for TAA fully develop a distinct T_H1-like signature over a 2-week period after entry into the tumor, down-regulating <i>FoxP3</i> and up-regulating expression of <i>TBX21 (</i>Tbet)<i>, IFNG</i>, and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific T_H1-like T_reg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1-responding tumors. These findings demonstrate that TIL-T_reg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-T_reg may positively contribute to antitumor responses.Science immunologyLung tumor-infiltrating T&lt;sub&gt;reg&lt;/sub&gt; have divergent transcriptional profiles and function linked to checkpoint blockade response.PMC10629528P30 CA006973R37 CA251447R01 EB029455R01 HG010889R01 CA121113R01 HG009518T32 CA009110K99 HG011468T32 GM136577R01 CA142779Nishimoto MZhang JBom STaube JMitchell-Flack MSmith KNZhan WAye THKCherry CMHou WYegnasubramanian SAnagnostou VIonta NVanDyke DConnor SWang YZhang BDykema AGBrahmer JRCheung LSMunoz AJJi HSpicer JZeng ZCaushi JXTam ASingh DForde PMTomasovic LMZhang TLi TRashid RRayes RJi ZTirado LESpangler JBSawosik CAYang SPardoll DMfalseLung tumor-infiltrating T&lt;sub&gt;reg&lt;/sub&gt; have divergent transcriptional profiles and function linked to checkpoint blockade response.Regulatory T cells (T_reg) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating T_reg (TIL-T_reg) from anti-PD-1-treated and treatment-naive non-small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)-specific T_reg derived from a murine tumor model. We identified 10 subsets of human TIL-T_reg, most of which have high concordance with murine TIL-T_reg subsets. Only one subset selectively expresses high levels of <i>TNFRSF4</i> (OX40) and <i>TNFRSF18</i> (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in T_reg suppression, including <i>LAG3</i>. Functionally, the OX40^hiGITR^hi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-T_reg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-T_reg-expressing T cell receptors that are specific for TAA fully develop a distinct T_H1-like signature over a 2-week period after entry into the tumor, down-regulating <i>FoxP3</i> and up-regulating expression of <i>TBX21 (</i>Tbet)<i>, IFNG</i>, and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific T_H1-like T_reg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1-responding tumors. These findings demonstrate that TIL-T_reg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-T_reg may positively contribute to antitumor responses.2023-01-01T00:00:00Z2023 Sep2026-05-28T22:28:57.347Z2025-04-06T08:04:15.126ZS-EPMC106295283771350710.1126/sciimmunol.adg1487