Extra-hematopoietic immunomodulatory role of the guanine exchange factor DOCK-2 [MethylCap-Seq]

The molecular basis of stromal immunomodulation is still unresolved. Here, we show a novel function for dedicator of cytokinesis 2 (DOCK2) in regulating extra-hematopoietic immune function of three independent stromal cell sources: induced pluripotent stem cells (iPSC)-derived mesodermal stromal cell (iPS-MSC), severe combined immunodeficiency (SCID) patient-derived fibroblasts and CRISPR/Cas9 knockout cells (iPS-MSCDOCK2KO). We first reprogrammed human mesenchymal stromal cells (MSC) into iPSC before differentiating the iPSCs back into MSC. Immature iPS-MSCs lacked immunosuppressive potential. Successive phenotypic maturation facilitated immunomodulatory function, while maintaining clonogenicity, comparable to parental MSCs. Sequential RNA-seq displayed time-dependent immune-related gene expression eventually resembling parental MSCs. SCID patient-derived fibroblasts harboring bi-allelic DOCK2 mutations also showed significantly reduced immunomodulatory capacity compared to non-mutated fibroblasts. CRISPR/Cas9-mediated DOCK2 knockout in healthy iPSCs resulted in significantly reduced immunomodulatory capacity, reduced F-actin stress-fibers, and a disturbed subcellular localization of CDC42 activation. We provide first evidence for extra-hematopoietic immunomodulation by the guanin exchange factor DOCK2. This suggests that persisting immune disease after successful blood stem cell transplantation in SCID patients could in part be due to loss-of-function DOCK2 mutations. MethylCap-seq analysis of the maturation of induced pluripotent stem cells (iPSCs) derived from Mesenchymal stromal cells (bone marrow and unbilical cord derived MSCs) into MSCs compared to their parental MSCs