Gene expression in anergic and naïve CD4 T cells deficient for Nr4a1 and Nr4a3

The NR4A family of nuclear receptors are upregulated by acute and chronic antigen stimulation, and play redundant, tolerogenic roles in T and B cells. To bypass their obligate function in the Treg lineage, we generated competitive chimeras with Nr4a1-/-Nr4a3-/- (double knockout or DKO) bone marrow mixed with congenically marked WT bone marrow at a 1:5 ratio. As a result of a defect in Treg production by DKO cells, Treg in mixed chimeras are almost exclusively of WT origin. By contrast, we generate an approximately 1:1 DKO:WT ratio in the peripheral T cell compartment because DKO cells escape negative selection. After reconstitution, we observed pronounced accumulation of anergic DKO CD4 T cells, but a defect in functional tolerance resulting in autoantibody production. Based on these studies and published literature, we hypothesize that the NR4A family regulate TCR-induced transcripts in both naive and anergic CD4 T cells. To identify such targets, we sorted naive and anergic CD4 T cells of either WT or DKO genotype (CD45.1+ or CD45.1-) on the basis of CD73/FR4/CD44 expression from these chimeras directly into RLT buffer. In parallel, we stimulated sorted naive CD4 T cells of each genotype in vitro with plate-bound anti-CD3 and anti-CD28 stimulation for 3 hrs and then generated RLT lysates. We made biological triplicate samples of each condition (naive WT and DKO, naive stimulated WT and DKO, anergic WT and DKO) resulting in 6 different populations x 3 replicates = 18 samples. Samples were subjected to library prep and bulk RNA sequencing to identify DEG. As described above, DKO and WT CD25-CD4+ T cells were sorted from competitive chimeras on the basis of CD62L/CD44/FR4/CD73 surface expression to identify either naïve (CD62LhiCD44loFR4loCD73lo) or anergic (CD62LloCD44hiFR4hiCD73hi) populations. Naïve and anergic cell populations were sorted directly into RLT buffer. In parallel, sorted naive CD4 samples were stimulated ex vivo withplate-bound anti-CD3 and anti-CD28 for 3 hrs. Libraries were constructed by the Emory EIGC core. Libraries were sequenced in an individual lane of an S1 flowcell on the NovaSeq6000 with a PE100 configuration using a NovaSeq 6000 SP Reagent Kit. Fastq files were trimmed for adapters and low quality base pairs using Fastp (https://academic.oup.com/bioinformatics/article/34/17/i884/5093234) then aligned to mouse genome assembly mm10 using STAR (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530905/). FeatureCounts (https://pubmed.ncbi.nlm.nih.gov/24227677/) was used to obtain read count data and a paired differential expression analysis comparing samples from the same chimeras was performed with edgeR (https://pubmed.ncbi.nlm.nih.gov/19910308/).