Strong basal/tonic TCR signals are associated with gene expression changes in naive CD4+ T cells

Naïve T cells constantly experience TCR signals in response to self-peptides presented by MHC (pMHC) in vivo. The intensity of these basal or tonic TCR signals can be indirectly read out by expression of surrogate markers of tonic TCR signaling, including the Nur77-GFP transgene and Ly6c. The strength of tonic TCR signaling varies broadly across the naïve CD4+ T cell population and is correlated with functional heterogeneity.Cells that experience the most basal TCR signaling (Nur77-GFP hi, Ly6c lo) are hyporesponsive to peptide-MHC stimulation. Here we examine whether tonic TCR stimulation is associated with differences in mRNA expression. Naïve, CD4 T cells (FoxP3-RFP negative, CD44lo, CD4+) were sorted from mouse spleens plus lymph nodes into four populations based on the expression of Nur77-GFP and Ly6c. Cells were sorted directly into RLT buffer. Sorted cells were either Nur77-GFPlo Ly6c+ (population A), Nur77-GFP intermediate Ly6c+ (population B), Nur77-GFP intermediate Ly6c- (population C) or Nur77-GFP hi Ly6c- (population D). We made biological triplicate samples for a total of 12 samples (4 populations x 3 replicates).