The guanine-nucleotide exchange factor CalDAG GEFI fine-tunes functional properties of regulatory T cells

Abstract

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.