Next Generation Sequencing Facilitates Quantitative Analysis of WT and Il2ra mut/mut memory OT-I cell transcriptomes

Cognate antigen signals control CD8+ T cell priming, expansion size and effector versus memory cell fates, however, it is not clear whether they can also modulate the functional features of memory CD8+ T cells. We observed that OT-I cells that were primed with weak cognate antigen signals incorporate more cytokine signals, leading to a hypothesis that CD8+ T cells that receive weak TCR signals require cytokine signals to form functional memory. Using a previously described mouse model in which IL-2 signaling via its high affinity receptor CD25 is selectively impaired, the “Il2ramut/mut” mouse, we conducted a comparative analysis of gene expression and epigenetic landscape of Il2ramut/mut and WT OT-I memory cells that were primed with strong (Lm-Ova N4) versus weak (Lm-Ova T4). RNA seq data showed that both TCR and IL-2 priming signals have minimal effect on gene expression in resting memory CD8 T cells, but they significantly modify the epigenetic landscape of the memory CD8 T cells. These findings have important contributions to the current understanding of how priming signals program memory CD8 T cells in vivo. Overall design: We adoptively transferred Il2ramut/mut and WT OT-I cells to sets of hosts and immunized them with Lm-Ova N4 (4 replicates) or Lm-Ova T4 (4 replicates). Spleens of the hosts were harvested 3-4 weeks later and memory OT-I cells were enriched by negative selection (dynabeads) and flow-sorted (Aria III) before proceeding to first strand cDNA synthesis and library preparation for sequencing (RNA-seq).