Molecular basis for rapid activation of resident memory T cells (TRM) after antigen encounter in skin

Summary: Abstract Body: We have previously reported on a single cell RNA sequencing (scRNAseq) analysis of a temporal of TRM development after a cutaneous vaccinia virus infection. A unique feature of this time course analysis was a steadily increasing expression of AP-1 factor mRNA as TRM matured; this was not seen in TCM generated from the same infection. Analysis of >day 30 TRM by Assay for Transposase Accessible Chromatin sequencing (ATACseq) motif analysis revealed abundant AP-1 binding sites. While this accumulation of AP-1 mRNA was seen in TRM from multiple tissues, we asked whether AP-1 proteins could be detected in the mature TRM nucleus. Using skin >100 days after VACV infection and imaging by Cyclic Immunofluorescence microscopy (CyCIF), we could detect TRM that co-expressed cFos and JunB protein in a nuclear location, suggesting DNA binding of AP-1 complexes in resting skin TRM. Importantly, TRM in FFPE skin samples were not subjected to tissue digestion. This co-expression of cFos and/or JunB was seen in 39% of CD8+ CD103+ TRM. To further determine the location of these occupied AP-1 binding sites in the genome, we performed “cleavage under targets and release using nuclease” (CUT&RUN) analysis using a JunB-specific antibody. Among the top ten contiguous TF binding sites were interferon regulatory factors (IRF3/IRF8) sites and nuclear factor of activated T cells (NFAT, NFATC2) sites. Both NFAT/AP-1 and IRF8/AP-1 proteins bound to contiguous DNA sites form complexes which serve as potent transcriptional amplifiers for T cell activation. We suggest that the constitutive presence of DNA-bound cFos/JunB allows T cell receptor ligation (signal 1) and subsequent cytoplasmic NFAT nuclear translocation to rapidly initiate TRM activation. This obviates the temporal delay required for gene transcription and translation of AP-1 factors after signal 2 activation of conventional T cells, providing an explanation for the uniquely rapid kinetics of TRM activation in situ. N P. Smith¹, Y P. Yan¹, Y P. Pan², K B. Manakontreecheep¹, S B. Pant², Peter B. Sorger², Jason B. Williams², Alexandra C. Villani¹, Thomas S. Kupper² 1. Medicine, Massachusetts General Hospital, Boston, MA, United States. 2. Dermatology, Brigham and Women's Hospital, Boston, MA, United States. Adaptive and Auto-Immunity