Potent Immunosuppression by a Bivalent Molecule Binding to CD200R and TGF-βR

Background. The novel immunosuppressive molecule, CD200, has been reported to induce immunoregulation after interaction with its receptor(s), CD200R(s), in part at least through augmented induction of regulatory T-cell populations. Independent studies have also described increased expression of indoleamine-2,3-dioxygenase after CD200R triggering, whereas others have provided evidence that TGF-beta is important for the induction or function of many populations of regulatory T cells. We have asked whether a hybrid molecule in which a soluble fusion protein containing CD200, CD200Fc, was linked to TGF-beta through a glycine linker (Gly(6)) functions as a superior immunosuppressant molecule when compared with CD200Fc or TGF-beta alone, or in combination. Methods. The hybrid molecule CD200FcGly(6) TGF-beta was expressed by transient transfection in CHO cells and purified over a protein A column. Functional activity of this and recombinant CD200Fc or TGF-beta alone were assessed in mixed leukocyte cultures (MLCs) and in skin graft rejection in vivo. Results. Immunosuppression mediated by CD200FcGly(6) TGF-beta is dependent on both functional CD200 and TGF-beta moieties, as indicated by inhibition of suppression using anti-CD200 or anti-TGF-beta antibodies. Using as responder cells, using antigen-presenting cell from mice with a deletion of the CD200R gene and responder T cells from mice with siRNA-mediated suppression of expression of the TGF-beta II receptor, we show that suppression follows binding to TGF-beta RII on T cells, and CD200R1 on antigen-presenting cells. Indoleamine-2,3-dioxygenase inhibitors did not attenuate suppression by CD200FcGly(6) TGF-beta. Conclusion. CD200FcGly(6) TGF-beta is a potent immunosuppressant in vivo and in vitro.