Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response

Regulatory T cells (Treg) are conventionally viewed to suppress endogenous and therapy-induced anti-tumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNAseq/TCRseq of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment naive non-small cell lung cancers (NSCLC) with single cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Notably, one subset selectively expresses high levels of OX40 and GITR, whose engangement by cognate ligand mediated proliferative programs and NF-kB activation, as well as multiple genes involved in Treg suppression, in particular LAG3. Functionally, the OX40hiGITRhi subset in the most highly suppressive ex vivo and Treg expression of OX40, GITR and LAG3, correlated with resistance to PD-1 blockade. Surprisingly, in the murine tumor model, we found that virtually all TIL-Treg expressing T cell receptors that are specific for TAA fully develop a distinct Th1-like signature over a two-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFNy and certain pro-inflammatory granzymes. Application of a gene score from the murine TAA-specific Th1-like Treg 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-Treg partition into multiple distinct transcriptionally-defined subsets with potentially opposing effects on ICB-induced anti-tumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to anti-tumor responses. ### Competing Interest Statement V.A. receives research funding from Bristol-Myers Squibb and Astra Zeneca. J.M.T. receives research funding from Bristol-Myers Squibb and serves a consulting/advisory role for Bristol-Myers Squibb, Merck, and Astra Zeneca. J.R.B. serves an advisory/consulting role for Amgen, AstraZeneca, Bristol-Myers Squibb, Genentech/Roche, Eli Lilly, GlaxoSmithKline, Merck, Sanofi, and Regeneron, receives research funding from AstraZeneca, Bristol-Myers Squibb, Genentech/Roche, Merck, RAPT Therapeutics, Inc., and Revolution Medicines, and is on the Data and Safety Monitoring Board of GlaxoSmithKline, Janssen, and Sanofi. P.M.F. receives research support from AstraZeneca, BioNtech, Bristol-Myers Squibb, Novartis, Rogeneron , and has been a consultant for AstraZeneca, Amgen, Bristol-Myers Squibb, Iteos, Novartis, Star, Surface, Genentech, G1, Sanofi, Daiichi, Regeneron, Tavotek, VBL Therapeutics, Sankyo, and Janssen and serves on a data safety and monitoring board for Polaris. S.Y. receives research funding from Bristol-Myers Squibb/Celgene, Janssen, and Cepheid, has served as a consultant for Cepheid, and owns founders equity in Brahm Astra Therapeutics and Digital Harmonic. K.N.S. and D.M.P. have filed for patent protection on the MANAFEST technology (serial No. 16/341,862). D.M.P. is a consultant for Compugen, Shattuck Labs, WindMIL, Tempest, Immunai, Bristol-Myers Squibb, Amgen, Janssen, Astellas, Rockspring Capital, Immunomic, Dracen and owns founders equity in ManaT Bio, Inc., WindMIL, Trex, Jounce, Enara, Tizona, Tieza, RAPT and receives research funding from Compugen, Bristol-Myers Squibb, and Enara. K.N.S. has received travel support/honoraria from Illumina, Inc., receives research funding from Bristol-Myers Squibb, Anara, and Astra Zeneca, and owns founders equity in ManaT Bio, Inc. J.T received research funding from Akoya Biosciences and BMS. J.T. is a consultant/advisory board member for BMS, Merck, Astra Zeneca, Genentech, Akoya Biosciences, Lunaphore, and Compugen. J.T. received equipment, reagents, and stock options from Akoya Biosciences. The terms of all these arrangements are being managed by Johns Hopkins University in accordance with its conflict-of-interest policies.