DOCK2 Is Critical for FC to Induce Regulatory T Cells

Abstract

Abstract 1885CD8+/TCR− graft facilitating cells (FC) significantly enhance engraftment of hematopoietic stem cells (HSC) in both allogeneic and syngeneic recipients. We have shown that DOCK2−/− FC are compromised in cell migration in vitro and facilitating HSC engraftment in vivo. DOCK2 activates the small GTPase Rac and is indispensable for migration of neutrophils, lymphocytes and plasmacytoid dendritic cells (DC). A recent study demonstrated that DOCK2−/− DC were defective in antigen uptake and presentation. FC are a heterogeneous cell population, with a predominant subpopulation resembling plasmacytoid precursor DC. We previously found that FC induced antigen-specific regulatory T cells (Treg) in vitro and in vivo. In the present study, we evaluated whether DOCK2−/− FC could induce Treg. 5×105 wild-type (WT) sorted CD4+CD25− splenocyte T cells were incubated with 1×105 WT FC or DOCK2−/− FC at a 5:1 ratio in U bottom 96-well plates with long-term culture medium (IMEM with 20% horse serum, 10−6M hydrocortisone and 50μM β-mercaptoethanol) for up to 6 days. The co-cultures were harvested and CD4+CD25+ T cells were analyzed for FoxP3 by flow cytometry intracellular staining. WT CD4+CD25− splenocytes were cultured as negative control. Phagocytosis of APC-labeled polystyrene beads by WT and DOCK2−/− FC was assessed after incubating FC with the beads for 3 hours. We found that FC induced the generation of CD4+CD25+FoxP3+ Tregin vitro as expected, while DOCK2 deficiency completely abrogated the ability of FC to induce Treg generation. Moreover, the uptake of beads by DOCK2−/− FC was significantly impaired compared to their uptake by WT FC. In summary, DOCK2−/− FC were unable to induce the generation of CD4+CD25+FoxP3+ Tregin vitro and were compromised in phagocytosis. [Display omitted] Disclosures:Ildstad:Regenerex, a biotech start-up company: Equity Ownership.