Genome-wide analysis highlights the involvement of calcineurin/NFAT pathway signaling in transcriptional elongation in restimulated memory CD8 T cells (P1166)

Abstract

Abstract Memory T cells respond to antigen encounter more robustly than naïve T cells, but the cell-intrinsic molecular mechanisms underlying this accentuated responsiveness are unknown. Here we show that in in vitro-generated memory-like CD8+ T cells, perforin (Prf1) gene transcription in response to secondary stimulation requires all NFAT transcription factors present in immune cells (NFAT1, NFAT2 and NFAT4), activation of the phosphatase calcineurin, and store-operated Ca2+ entry mediated by the ER Ca2+ sensors Stim1 and Stim2. The calcineurin inhibitor cyclosporine A (CsA) blocked transcriptional elongation through the Prf1 gene, with much less effect on recruitment of RNA polymerase II (Pol II) and transcriptional initiation. The generality of this effect was confirmed by genome-wide RNA-sequencing and chromatin immunoprecipitation for Pol II, NFAT1, H3K4me3 and H3K36me3 histone modifications, and cyclinT1, a component of positive transcription elongation factor b (P-TEFb). Of 1340 genes upregulated upon restimulation, 199 genes were CsA-sensitive, with the majority of these genes affected by transcription elongation rather than Pol II recruitment. CyclinT1 binding to the transcription start sites of restimulation-induced genes overlapped with NFAT1 occupancy and was CsA-sensitive. Thus, a substantial fraction of the rapid TCR-induced memory CD8 T cell responses is regulated by transcription elongation via calcineurin/NFAT signaling and recruitment of P-TEFb.