Re-evaluating the melanoma TIL compartment and its unexpected spectrum of exhausted and functional T cells

Significant heterogeneity exists within the tumor infiltrating CD8 T cell population, and exhausted T cells harbor a subpopulation that may be replicating and retain signatures of activation, with potential functional consequences in tumor progression. Dysfunctional immunity in the tumor microenvironment is associated with poor cancer outcomes, making exploration of these exhausted but activated (Tex/act) subpopulations critical to the improvement of therapeutic approaches. To investigate mechanisms associated with Tex/act cells, we sorted and performed transcriptional profiling of CD8+ tumor infiltrating lymphocytes (TIL) coexpressing the exhaustion markers PD-1 and TIM-3, from large volume melanoma tumors. We additionally performed immunologic phenotyping and functional validation, including at the single cell level, to identify potential mechanisms that underlie their dysfunctional phenotype. We identified novel dysregulated pathways in CD8+PD-1+TIM-3+ cells that have not been well studied in TIL; these include bile acid and peroxisome pathway-related metabolism, and mammalian target of rapamycin (mTOR) signaling pathways, which are highly correlated with immune checkpoint receptor expression. Through bioinformatic integration of immunophenotypic data and network analysis, we propose unexpected targets for therapies to rescue the immune response to tumors in melanoma. ### Competing Interest Statement B. Gastman is a consultant/advisory board member for Merck. ### Funding Statement This research is supported by Cleveland Clinic and Case Western Reserve University internal funding. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Institutional Review Board of the Cleveland Clinic gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes RNA-seq data supporting this study have been deposited in the Gene Expression Omnibus (GEO) public database with accession number pending. * Tex/act : exhausted but activated T cell subpopulation TIL : tumor infiltrating lymphocyte mTOR : mammalian target of rapamycin ICR : immune checkpoint receptor TME : tumor microenvironment PD-1 : programmed cell death-1 SCC : cutaneous squamous cell carcinoma pCD8 : peripheral CD8 T cells MEL : melanoma IO : immunotherapy RIPA : radioimmunoprecipitation assay buffer GEO : Gene Expression Omnibus PCA : principal components analysis UMAP : uniform manifold approximation and projection MFI : mean fluorescence intensity HCC : hepatocellular carcinoma; GSVA : Gene Set Variation Analysis DEG : differentially expressed genes PBMC : peripheral blood mononuclear cells scRNAseq : single cell RNA sequencing Tim-3 : T cell immunoglobulin and mucin protein 3