Thymocyte/splenocyte-derived CD4+CD25+treg stimulated by anti-CD200R2 derived dendritic cells suppress mixed leukocyte cultures and skin graft rejection

Background. CD200 delivers immunoregulatory signals following engagement of its receptor, CD200R. A family of CD200Rs (CD200R1-4) has been described. Spleen expresses cell surface CD200R1, while bone marrow shows predominantly expression of cell surface CD200R2/R3. We showed that dendritic cell precursors (DCp) cultured with anti-CD200R2/3 develop the capacity to induce CD4(+)CD25(+)regulatory T cells (Treg) from peripheral lymphocytes. We now characterize DCs involved in induction of antigen-specific Treg from thymocytes or peripheral T cells, and the properties of Treg cells maintained in long-term culture. Methods. Bone marrow DCp (C3H or BL/6 origin) were cultured for 8 days with GMCSF, ILA and anti-CD200R2, or with CD200Fc and a previously described peptide inhibitor of CD200R1 to allow preferential engagement of nonCD200R1 receptors by CD200. Mixed leukocyte cultures (MLCs) were initiated with allogeneic responder lymphocytes/thymocytes (BL/6 or C3H) and mitomycin-c treated DCs to induce Treg. Treg cells were maintained by reculture with DCs derived in the same manner and IL-2, cloned at limiting dilution, and tested for their ability to suppress MLCs and skin graft rejection in vivo. Results. Foxp3(+) CD4(+)CD25(+) Treg were derived from 60-hr thymocyte and splenocyte T cell cultures using both DC populations. Cloned C3H Treg (Foxp3(+)) suppressed both C3H anti-BL/6 reactivity in a fresh MLC and rejection of BL/6 skin allografts in C3H recipients; the converse was true for BL/6 Treg. Conclusions. We conclude that CD200 triggering of bone-marrow DCs in the absence of CD200R1 engagement induces CD4(+)CD25(+) Treg, and these cloned antigen-specific Treg may have clinical utility.