Authors:Jana Niemz, Stefanie Kliche, Marina C. Pils, Eliot Morrison, Annika Manns, Christian Freund, Jill R. Crittenden, Ann M. Graybiel, Melanie Galla, Lothar Jänsch and Jochen Huehn
Using quantitative phosphopeptide sequencing of unstimulated versus stimulated primary murine Foxp3+ regulatory and Foxp3− conventional T cells (Tregs and Tconv, respectively), we detected a novel and differentially regulated tyrosine phosphorylation site within the C1 domain of the guanine-nucleotide exchange factor CalDAG GEFI. We hypothesized that the Treg-specific and activation-dependent reduced phosphorylation at Y523 allows binding of CalDAG GEFI to diacylglycerol, thereby impacting the formation of a Treg-specific immunological synapse. However, diacylglycerol binding assays of phosphomutant C1 domains of CalDAG GEFI could not confirm this hypothesis. Moreover, CalDAG GEFI−/− mice displayed normal Treg numbers in thymus and secondary lymphoid organs, and CalDAG GEFI−/− Tregs showed unaltered in vitro suppressive capacity when compared to CalDAG GEFI+/+ Tregs. Interestingly, when tested in vivo, CalDAG GEFI−/− Tregs displayed a slightly reduced suppressive ability in the transfer colitis model when compared to CalDAG GEFI+/+ Tregs. Additionally, CRISPR-Cas9-generated CalDAG GEFI−/− Jurkat T cell clones showed reduced adhesion to ICAM-1 and fibronectin when compared to CalDAG GEFI-competent Jurkat T cells. Therefore, we speculate that deficiency in CalDAG GEFI impairs adherence of Tregs to antigen-presenting cells, thereby impeding formation of a fully functional immunological synapse, which finally results in a reduced suppressive potential.