Thymocyte/Splenocyte-Derived CD4+CD25+Treg Stimulated by Anti-CD200R2 Derived Dendritic Cells Suppress Mixed Leukocyte Cultures and Skin Graft Rejection

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Abstract

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, IL-4 and anti-CD200R2, or with CD200Fc and a previously described peptide inhibitor of CD200R1 to allow preferential engagement of non-CD200R1 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.

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