BATF represses BIM to sustain tolerant T cells in the periphery (RNA-Seq)

T cells that encounter self-antigens after exiting the thymus avert autoimmunity through peripheral tolerance. Pathways for this include an unresponsive state known as anergy, clonal deletion, and T regulatory (Treg) cell induction. The transcription factor cues and kinetics that guide distinct peripheral tolerance outcomes remain unclear. Here, we found that anergic T cells are epigenetically primed for regulation by the non-classical AP-1 family member BATF. Tolerized BATF-deficient CD4+ T cells were resistant to anergy induction and instead underwent clonal deletion due to pro-apoptotic BIM (Bcl2l11) upregulation. During prolonged antigen exposure, BIM de-repression resulted in fewer PD-1+ conventional T cells as well as loss of peripherally-induced FOXP3+ Treg cells. Simultaneous Batf and Bcl2l11 knockdown meanwhile restored anergic T cell survival and Treg cell maintenance. The data identify the AP-1 nuclear factor BATF as a dominant driver of sustained T cell anergy and illustrate a mechanism for divergent peripheral tolerance fates. A combination of ATAC-seq and RNA-seq was used to interrogate freshly isolated spleen and lymph node polyclonal CD4 T cell subsets (Anergic, CD44high FOXP3– CD73high FR4high NRP1+; Teff, CD44high FOXP3– CD73low FR4low; Treg, FOXP3+ CD25+; Naïve, CD44low). Chromatin accessibility in sorted CD4+ T cell subpopulations was determined using the ATAC-seq. Briefly, 5 x 10^4 captured cells from each of the indicated groups (n = 2 or 3) were pelleted, washed with PBS, resuspended in cold sterile-filtered lysis buffer (10mM Tris-Cl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% Igepal CA-630), and immediately centrifuged for 10 minutes (500 x g, 4oC). Pellets were then resuspended in the transposition reaction mix (Nextera TD buffer, Nextera TDE1 transposase, nuclease-free H2O) and incubated for 30 minutes at 37oC. After transposition, DNA was purified with the MinElute PCR Purification kit (QIAGEN) according to the manufacturer’s instructions. Library amplification (Nextera dual-indexed), quality control (Agilent sizing and PicoGreen quantification), and sequencing (Illumina NovaSeq SP flow cell 2x50-bp paired-end run) were carried out at the University of Minnesota Genomics Center. Trimmed ATAC-seq reads (Trimmomatic 0.38.1) were aligned to the mm10 mouse genome assembly using Bowtie2 2.3.3.1 with the ‘--very-sensitive’ preset, then sorted and converted in SAMtools 1.13. Peak-calling with integrated PCR duplicate removal and Tn5 transposase cut site interval adjustment was performed on individual samples with Genrich 0.5 (github.com/jsh58/Genrich) in ATAC-seq mode. Reads mapping to mitochondrial DNA and ENCODE blacklist regions were excluded from analysis. From the individual peak-called samples, a total of 38,509 consensus peak sites were determined by DiffBind 2.10.0 (bioconductor.org/packages/release/bioc/html/DiffBind.html). Peaks were assigned to the gene with the nearest transcription start site using the annotatePeaks program in HOMER v4.11.1 with reference to the mm10 genome assembly and RefSeq annotation coordinates. For the RNA-seq analysis, total RNA was extracted from physically sorted CD4+ T cell populations using a QIAGEN RNeasy Kit according to the manufacturer’s instructions. Purified RNA quality control (Agilent sizing and RiboGreen quantification), library creation (Takara SMARTer Stranded Total RNA-seq Kit v2 Pico Input Mammalian), and sequencing (Illumina NovaSeq SP flow cell 2x50-bp paired-end run) were carried out at the University of Minnesota Genomics Center. RNA-seq reads were processed using an established pipeline (https://github.com/msi-ris/CHURP/wiki/PURR-Manual-Page). This pipeline incorporates the following software components: Trimmomatic, HISAT2, SAMtools, featureCounts, and edgeR. For visualization at select gene loci, ATAC-seq and RNA-seq mapped reads were summed from sample replicates using the bigwigCompare tool for display using Integrated Genomics Viewer.