RELA tunes innate-like interferon I/III responses in human T cells

In innate immune cells, intracellular sensors such as cGAS-STING stimulate type I/III interferon (IFN) expression, which promotes antiviral defense and immune activation. However, how IFN-I/III expression is controlled in adaptive cells is poorly understood. Here, we identify a transcriptional rheostat orchestrated by RELA that confers human T cells with innate-like abilities to produce IFN-I/III. Despite intact cGAS-STING signaling, IFN-I/III responses are stunted in CD4+ T cells compared with dendritic cells or macrophages. We find that lysine residues in RELA tune the IFN-I/III response at baseline and in response to STING stimulation in CD4+ T cells. This response requires positive feedback driven by cGAS and IRF7 expression. By combining RELA with IRF3 and DNA demethylation, IFN-I/III production in CD4+ T cells reaches levels observed in dendritic cells. IFN-I/III production provides self-protection of CD4+ T cells against HIV infection and enhances the elimination of tumor cells by CAR T cells. Therefore, innate-like functions can be tuned and leveraged in human T cells. To study transcriptional response to cGAMP, samples were harvested five hours following cGAMP (6 μg/ml) stimulation of activated CD4+ T cells. For RELA and RELA K5R analysis,activated CD4+T cells transduced with either pTRIP-SFFV-GFP or pTRIP-SFFV-GFP-RELA or pTRIP-SFFV-GFP-RELA K5R were harvested 4 days post-transduction. Total RNA was extracted (Macherey-Nagel #740955.50) and RNA integrity was verified using Agilent Bioanalyzer (Agilent RNA 6000 Nano kit #5607-1511), all samples had a RIN >9. RNA sequencing libraries were prepared from 500 ng of total RNA using the Illumina TruSeq Stranded mRNA Library preparation kit. A first step of polyA selection using magnetic beads was performed to focus sequencing on polyadenylated transcripts. After fragmentation, cDNA synthesis was performed and resulting fragments were used for dA-tailing and ligated to the TruSeq indexed adapters. PCR amplification was performed to create the final cDNA library (with 13 cycles). After quantification of PCR products, sequencing was carried out using 2*100 cycles (paired-end reads, 100 nucleotides) on a Novaseq6000 instrument, targeting 25M clusters.