RNA expression of reactivated Antigen-specific memory CD4 T cells previously exposed to tolerogenic or immunogenic stimulation

Activated and memory CD4 T cells play important roles in many autoimmune diseases often acting as upstream co-ordinators of inflammation and tissue destruction. A major therapeutic strategy is to turn off or tolerize these CD4 T cells thereby providing a cure. While much is understood about inducing tolerance in naïve CD4 T cells, little is known about whether or how to induce tolerance in previously activated or memory CD4 T cells. Here, we use RNA-sequencing to investigate the consequences of activating mouse CD4 memory T cells with antigen delivered in the absence of adjuvant, a classic tolerance-inducing strategy for naïve T cells. To examine the long term consequences of exposing memory CD4 T cells to tolerizing signals, we first reactivated them with antigen delivered with (control) or without (experimental) adjuvant, rested the cells and then reactivated them with antigen and adjuvant 5 days prior to isolating RNA for gene expression analysis. Overall design: We used a triple transgenic reporter mouse, TRACE, to identify antigen specific CD4 T cells. In TRACE mice, T cell activation drives expression of rtTA via the interleukin 2 promoter. Reporter mice are supplied with a diet containing doxycycline for a total of 7 days starting 2 days prior to immunisation. Binding of rtTA to doxycycline activates the tet-ON promoter that drives expression of Cre recombinase which removes a stop codon blocking EYFP expression from the Rosa locus. Thus, CD4 T cells activated in the presence of doxycycline become permanently EYFP+. TRACE mice were immunized subcutaneously in the scuff with OVA protein, conjugated to 200nm polyethylene beads delivered with anti-CD40 and polyIC. EYFP+ CD4 T cells were isolated from TRACE mice 8 days post-immunisation using a CD4 T cell enrichment kit (EasySep, StemCell Technologies) before further purification via FACS-sorting. EYFP+ CD4 T cells were transferred intravenously to sex-matched, naïve C57BL/6 recipients and administered with OVA peptide alone, or OVA peptide with LPS 14 days later. A further 30 days later, all mice were immunized with OVA protein with alum intraperitoneally. EYFP+ CD4 T cells from lymphoid organs were FACS-sorted 5 days later. Each sorted sample was then processed to extract total RNA using the RNeasy Micro kit (Qiagen).