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, 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.
Gorczynski
Reginald M
RM
Transplant Research Division, Toronto Hospital, University Health Network, Ontario, Canada. reg.gorczynski@utoronto.ca
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002999:Clone Cells; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000276:immunology*; D006084:Graft Rejection / Q000517:prevention & control; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D008809:Mice, Inbred C3H; D064446:Orexin Receptors; D010641:Phenotype; D011956:Receptors, Cell Surface / Q000276:immunology*; D015375:Receptors, Interleukin-2 / Q000032:analysis; D016038:Skin Transplantation / Q000276:immunology*; D013154:Spleen / Q000166:cytology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D013950:Thymus Gland / Q000166:cytology*
D000906:Antibodies; D000954:Antigens, Surface; C501103:Cd200r1 protein, mouse; D064446:Orexin Receptors; D011956:Receptors, Cell Surface; D015375:Receptors, Interleukin-2