Interleukin-4:STAT6 signaling delays protective CD8 T cell bystander activation by antagonizing IL-18 sensing

Memory CD8 T cells (Tmem) are activated into innate-like killers by cytokines such as IL-12, IL-15 and IL-18; but mechanisms regulating this phenomenon (termed bystander activation) are unclear. We show that basal IL-4 signals antagonize IL-18 sensing and blunt IFNg production following bystander activation of Tmem in mice. Furthermore, IL-4 treatment can act directly on Tmem in a STAT6-dependent manner, to limit their control of a bystander bacterial infection. Nevertheless, we find that IL-4 does not simply block bystander activation but rather alters Tmem gene expression to tune effector molecule expression. Defective bystander activation of homeostatic Tmem in some strains relates, in part, to IL-4 exposure, but we show that these differences are erased in Tmem produced by TCR activation leading to uniform IL-18 receptor expression and capacity for bystander activation/cytotoxicity. Our data demonstrate that bystander activation by inflammatory cytokines is subject to regulation by both IL-4 and by prior antigen experience. These findings underscore the importance of the cytokine milieu in dictating the ability of bystander CD8 Tmem to mediate pathogen control. Overall design: We isolated CD8 T cells from OT-I memory mice (mice with adoptively-transferred OT-I memory T cells that were expanded by VSV-OVA at least 30d prior). Cells were pooled from secondary lymphoid organs across 2 animals per sample. We divided cells for in vitro culture with media, IL-12/15, IL-12/15/18, or SIINFEKL peptide (TCR agonist) ± IL-4 (all cytokines at 100ng/mL, SIINFEKL at 1µM). We used these samples to test how IL-4 alters gene transcription during steady state (mock), bystander activation (IL-12/15, IL-12/15/18), and TCR activation (SIINFEKL). We used FACS to isolate live OT-I T cells, which we sorted into RLT buffer + beta-mercaptoethanol. We isolated RNAs, which were prepared into libraries by the University of Minnesota Genomics Core (UMGC) using Watchmaker Stranded mRNA prep kits. The UMGC pooled libraries, which were sequenced on an Aviti Freestyle Medium 2x150bp run. Runs generated =500 million pass filter reads for the pool, with all expected barcode combinations detected and mean quality scores =Q35.