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  • 1 Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany
  • 2 Otto-von-Guericke-University, 39120 Magdeburg, Germany
  • 3 Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany
  • 4 Free University Berlin, 14195 Berlin, Germany
  • 5 Massachusetts Institute of Technology, Cambridge, MA, United States
  • 6 Hannover Medical School, 30625 Hannover, Germany
  • 7 Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany
Open access

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.

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