We recently reported induction of high levels of chimerism and donor-specific tolerance without GVHD in mismatched related and unrelated kidney transplant recipients through administration of CD8⁺TCR^– bone marrow facilitating cells (FC) with donor HSC. The predominant subpopulation of FC resembles plasmacytoid dendritic cell (preDC) precursors. Activated preDC induce generation of regulatory T cells (Treg) and IL-10-producing type 1 regulatory T (Tr1) cells from naÏve CD4 T cells in vitro. FC induce antigen-specific Treg in vitro and in vivo. After stimulation by CpG ODN, FC produce IL-6 and IL-10, both of which are the major inducers of Tr1 cells. We therefore evaluated whether FC also induce differentiation of naÏve CD4 T cells into Tr1 cells. Sorted CD4⁺CD25^– T cells were incubated with syngeneic or allogeneic FC at a 5:1 ratio in long-term culture medium (LTCM) with CpG ODN. In some co-cultures, naÏve CD4 T cells were separated from FC by Transwells, or co-cultured with FC sorted from dedicator of cytokinesis 2 (DOCK2) knockout mice, which are deficient in cell migration. After 6 days, the cells were stimulated for 4 hours with PMA and ionomycin, and analyzed for FoxP3, IL-17 and IL-10 expression. Functional phenotype of DOCK2^-/- FC was determined by co-transplantation of HSC with FC in vivo. NaÏve CD4 T cells cultured in LTCM with or without CpG ODN showed no positivity for IL-10 and FoxP3. In contrast, co-culturing naÏve CD4 T cells with allogeneic or syngeneic FC induced CD4⁺CD25^– T cells to differentiate into CD4⁺CD25^–FoxP3^–IL-10⁺ Tr1 cells and also into Treg without affecting Th17 cells. Separating naÏve CD4 T cells from FC in co-culture by Transwells completely abrogated the capability of FC to induce Tr1 cells and Treg. Similarly, co-culture of DOCK2^-/- FC with naive CD4 T cells failed to induce Tr1 and Treg. Moreover, deficiency of DOCK2 in FC abolished FC function in transplantation models. In summary, our results indicate that FC induce de novo differentiation of Tr1 cells from naÏve CD4 T cells in a contact-dependent manner. This conversion is critically dependent upon DOCK2 signaling. Cell migration might be crucial for FC to induce generation of Tr1 cells, which may contribute to prevention of GVHD and promotion of HSC engraftment.

Ildstad, S.: Other, Regenerex LLC, CEO.

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