FcRγ-Dependent Facilitating Cells Are Direct Inducers of Regulatory T Cells.

Facilitating cells (FC) are CD8⁺/αβγδTCR⁻ bone marrow-derived cells that promote allogeneic stem cell (SC) engraftment without graft vs. host disease (GVHD) and induce donor-specific transplantation tolerance. However, the mechanism of FC-mediated allogeneic SC engraftment is not known. We have previously demonstrated that allogeneic SC engraftment promoted by FC result in no clinical evidence of GVHD and is associated with increased number of CD4⁺25⁺ regulatory T cells post transplant compared to recipients of bone marrow T cells that develop severe GVHD. This is consistent with the hypothesis that FC facilitate allogeneic SC engraftment and induce tolerance through the induction of a regulatory T cell network. Here we report that CpG activation of toll-like receptor 9 (TLR9) on FC induce CD4⁺25⁻ naïve T cell differentiation into CD4⁺25⁺ regulatory T cells. CpG stimulated and unstimulated CD8⁺αβγδTCR⁻ FC isolated from bone marrow of C57/BL6 mice by flow cytometric cell sorting FC were co-cultured with splenic CD4⁺25⁻ T cells for 5 days. CpG stimulated FC co-culture gave rise to CD4⁺25⁺ T cells as determined by mRNA expression of the CD4⁺25⁺ regulatory T cell marker, FoxP3. In contrast, unstimulated FC in co-culture did not induce regulatory T cells. Because FcRγ is the dominant ITAM receptor (immunoreceptor tyrosine-based activating motif), on FC and given that in vivo studies have demonstrated a requirement for FcRγ expression on FC, we hypothesized that FcRγ gene expression is increased within activated FC. FcRγ gene expression in CpG stimulated and unstimulated FC were compared by real-time PCR analysis. CpG-mediated TLR signaling within FC result in increased gene expression of FcRγ. Taken together, these studies demonstrate that CpG stimulated FC induce the generation of CD4⁺25⁺ regulatory T cells in vitro and CpG activation results in increased gene expression of FcRγ, suggesting a requirement for FcRγ signaling in FC-mediated induction of regulatory T cells. These findings provide the first mechanistic evidence that FC are direct inducers of regulatory T cells. Further characterization of cooperative TLR and FcRγ signaling pathways in FC will be critical to defining the mechanism of FC-mediated SC engraftment and the identification of potential therapeutic targets for the clinical induction of tolerance in the future.