Rapid in vitro generation of bona fide exhausted CD8+ T cells is accompanied by Tcf7 promotor methylation.

Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL) observed in chronic infection and cancer. Currentin vivomodels of CTL exhaustion using chronic viral infections or cancer yield very few exhausted CTL, limiting the analysis that can be done on these cells. Establishing anin vitrosystem that rapidly induces CTL exhaustion would therefore greatly facilitate the study of this phenotype, identify the truly exhaustion-associated changes and allow the testing of novel approaches to reverse or prevent exhaustion. Here we show that repeat stimulation of purified TCR transgenic OT-I CTL with their specific peptide induces all the functional (reduced cytokine production and polyfunctionality, decreasedin vivoexpansion capacity) and phenotypic (increased inhibitory receptors expression and transcription factor changes) characteristics of exhaustion. Importantly,in vitroexhausted cells shared the transcriptomic characteristics of the gold standard of exhaustion, CTL from LCMV cl13 infections. Gene expression of bothin vitroandin vivoexhausted CTL was distinct from T cells anergy. Using this system, we show thatTcf7promoter DNA methylation contributes to TCF1 downregulation in exhausted CTL. Thus this novelin vitrosystem can be used to identify genes and signaling pathways involved in exhaustion and will facilitate the screening of reagents that prevent/reverse CTL exhaustion. Author summary In this manuscript, we describe anin vitromethod that rapidly establishes large numbers of exhausted CD8+ T cells. The exhaustion of CTL induced by this method has been fully validated by multiple approaches (cytokine production, polyfunctionality, cytotoxicity,in vivoproliferation, inhibitory receptors, transcription factors, RNAseq and DNA methylation). This method will facilitate not only the study of T cell exhaustion but also the screening of drugs. As proof of point, we use this method to show that TCF-1 downregulation in terminally exhausted T cells is accompanied byTcf7DNA promoter methylation and show that a transmethylase inhibitor can prevent TCF-1 downregulation. Our method presents a critical resource for the study of CTL exhaustion and the screening of drugs and interventions.