Transcriptome analysis of reactivated T H 1 cells reveal distinct differences between priming and reactivation processes

Abstract

As a key player of the adaptive immune system CD4+ T cells play a critical role in orchestrating an effective immune response during infections, autoimmunity or cancer. Few studies have addressed antigen-mediated reactivation of T effector cells in peripheral tissues, as opposed to the well-studied primary activation (priming) in lymph nodes. We hypothesize that T effector cell reactivation is distinct from naïve T cell priming beyond linage differentiation, focusing on TH1 effector cells. We employed OT-II/Nur77GFP mice that allowed direct identification of T cell receptor (TCR) activated cells as Nur77 expression is up-regulated by TCR stimulation and correlates with the strength of TCR stimulation. Isolated naïve CD4 T cells (naïve resting), were either stimulated (naïve stimulated) or differentiated into TH1 (effector resting) and consecutively reactivated in an in vitro restimulation assay (effector stimulated). We correlated Nur77-GFP expression to the expression of TH1 lead cytokine IFNγ via qRT-PCR and flow cytometry and found that the maximum of restimulation is reached after 6-8 hours. While Nur77-GFP expression declines slowly after 4 hours in effector cells, IFNγ expression increases for up to 8 hours. In naïve T cells we correlated Nur77-GFP expression to IL2 expression and found that after 4 hours of stimulation IL2 expression reaches a saturation and nearly 90% of cells are Nur77-GFP positive. Transcriptome analysis of sorted naïve (CD62L+, CD44-, CD69-, Nur77-GFP-), active naïve (CD62Lint, CD44-, CD69+, Nur77-GFP+), TH1 (CD62L+, CD44+, Nur77-GFP-) and reactivated TH1 cells (CD62L+, CD44+, Nur77-GFP+) revealed 11899 significantly differentially expressed genes of overall T cell differentiation and activation (naïve vs reactivated effector), 10840 of naïve T cell activation (naïve vs active naïve), 11583 of TH1 differentiation (naïve vs effector) and 10736 of TH1 reactivation (effector vs reactivated effector). Furthermore 3107 genes were predominantly specifically regulated in reactivated TH1 cells. Of these, 447 are solely regulated in reactivated TH1 cells and do not appear in the other comparisons. We here report a unique signature to T effector cell reactivation that goes beyond lineage alterations.