CD4+ T cell culture:
250ml of peripheral blood from a normal human female was separated through a Ficoll-histopaque (Sigma) gradient to obtain peripheral blood mononuclear cells (PBMCs). CD4+ T cells were isolated from the PBMCs using Human CD4 Naïve Separation Kit (Stem Cell Technologies). The purified cells were transferred to RPMI 1640 culture medium (+ 10% FBS, 100U/ml Penicilin, Mediatech) at 37°C and 5% CO2, activated with Dynabead Human T-Activator CD3/CD28 (Invitrogen) for 48h, and cultured in the presence of 30 U/ml of IL2. The cells were expanded for 7 days at a density between 0.5x106 to 2x106 cells/ml. Aliquots of 40x106 cells were collected in freezing media (90% FBS/10% DMSO) for storage at -80°C. Frozen cell aliquots were thawed for further expansion by culturing in RPMI supplemented with IL2 for an additional 7 days, then split in two groups for a 24h re-stimulation with T-activator beads or no stimulation (“resting”).
Transfection and siRNA-mediated knockdowns:
Cells were transfected by Amaxa Nucleofection (Lonza) following the manufacturer’s instructions. Briefly, 4E+6 cells were pelleted and resuspended in 100 ul of supplemented SE cell line solution. siRNA was added to a final concentration of 300 nM. The cell suspension was then transferred to cuvettes and nucleofected using program CL-120. Cells were cultured for 48h prior to activation with Dynabead Human T-Activator CD3/CD28 (Invitrogen). After and additional 24h in culture, the cells were collected and flash frozen in liquid nitrogen. Cell pellets were subsequently used for RIP.
Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2.We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as “central” interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, “peripheral” interactomemembers (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.