MRTF-SRF signalling in the T cell IL2 response

The transcription factor SRF is a master regulator of growth factor inducible and cytoskeletal gene expression. Two transcription cofactor families, the TCFs & the MRTFs, are responsible for SRF's activity to direct transcriptional programme in response to extracellular signalling through the Ras-ERK and Rho-actin pathways respectively. We are investigating the role of the SRF network in the response to infection, using the Listeria monocytogenes model. We find that inactivation of the network impairs the ability of CD8 T cells to proliferate in response to listeria and to generate memory cells. Surprisingly, the defect reflects impaired MRTF-SRF signalling rather than the TCF-SRF signalling pathway usually associated with proliferation. Moreover, the defec doesn'tt lie downstream of the initial activation of the TCR, appearing instead to arise from an inability of CD8 T cells to present IL-2 to each other in paracrine fashion. Consistent with these observations, we find that while cultured CD8 MRTF-null T cells can be effectively activated by TCR crosslinking invitro, their ability to form clusters, which is IL-2-dependent, is impaired. Moreover, as expected, pilot experiments indicate that these cells exhibit deficits both in the basal levels of MRTF-SRF target gene expression, and in their ability to induce these genes in response to IL-2 stimulation: Overall design: T cells are activated by TCR crosslinking, then maintained in IL-12 before stimulation with IL-2 for various times. CD8+ T cells are sorted from pooled LN from 5 mice, each reconstituted with WT or MRTFf/f bone marrow (the latter rendered MRTF-null by a 15-day tamoxifen treatment). Cells are rested in full media for 90 min in incubator at 2x10^6 cells/ml before stimulation with plate-bound antiCD3+antiCD28 (5ug/ml) in a 6-well plate for 24h (6x10^6 cells per well). Cells were washed and transferred to a new plate containing 20ng/ml IL-12 (1x10^6 cells per well) for 12h before stimulation with 20ng/ml IL-2 for various times.IL-2R expression was analyzed by FACS and RNA was prepared for analysis from the following samples in triplicate: 1 Primary cells t = 0 2 TCR-activated cells t = 24h 3 Rested cells + IL-12 t = 36h 4 IL-2-stimulated cells +30min 5 IL-2-stimulated cells +60min 6 IL-2-stimulated cells +120min sequence total RNA (rRNA-depleted) in order to capture intronic sequences - see below. TOTAL samples 36