T-cell responses to tumors are typically neutralized through a variety of mechanisms. While cytotoxic CD8 cells have been the focus of most efforts to exploit T-cell responses against tumors, recent studies have demonstrated the clinical potential of the CD4 tumor-infiltrating lymphocytes (TILs). However, the functional diversity characteristic of these cells has limited our ability to harness CD4 TILs to fight tumors. To address this issue, we have used new experimental strategies to characterize the heterogeneity of the antitumor CD4 response in vivo, combining (i) tracking of CD4 T-cells specific for a defined recombinant tumor antigen, both in the tumor microenvironment and draining lymph node (dLN), (ii) genome-wide mRNA sequencing at the single-cell level (scRNAseq) and (iii) new computational approaches to increase the resolution of subpopulations discovery and uncover robust transcriptional changes across populations. We find novel subpopulations of regulatory (Treg) and effector (Th1) TILs expressing diverse co-inhibitory gene programs unique to the tumor microenvironment. In contrast to TILs composition, we find an unexpected Follicular helper (Tfh)-like cells and non-classical Th1 response in dLN. Comparison of TIL subpopulations to those defined in an immune response to viral infection (LCMV) reveals gene expression patterns unique to cancer. Our study uncovers previously unknown CD4 T-cell effector programs that provide ineffective responses and fail to control tumor progression. Targeting these programs should provide new options for the design of improved immunotherapy strategies.

Citation Format: Assaf Magen, Jia Nie, Thomas Ciucci, Yongmei Zhao, Monika Mehta, Bao Tran, Sridhar Hannenhalli, Rémy Bosselut. Single-cell resolution profiling of tumor-reactive CD4 T-cells reveals immune landscape alterations [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A201.