Blocking CD226 Promotes Allogeneic Transplant Immune Tolerance and Improves Skin Graft Survival by Increasing the Frequency of Regulatory T Cells in a Murine Model.

Regulatory T cells (Tregs) play key roles in maintaining peripheral tolerance and preventing autoimmune disease. Treg modulation could be helpful in treating malignancies, autoimmune disease, and allergies, as well as to facilitate organ transplantation. Signals transduced by co-stimulatory molecules are essential for Treg differentiation, homeostasis, and function. One well-known active receptor, CD226, also known as DNAM-1 or PTA1, is an adhesion molecule that interacts primarily with CD155 and is involved in Treg differentiation and immune tolerance to transplanted tissue. Anti-CD226 monoclonal antibody (mAb) and truncated recombinant CD226 proteins were employed to manipulate the CD226 signal. Various T cell markers on freshly isolated splenocytes and T lymphocytes were characterized by flow cytometry Cell proliferation was measured by carboxyfluorescein succinimidyl ester dye, mRNA transcripts by q-RT PCR, and protein expression by western blotting. A BALB/c-to-C57BL/6 skin allograft model was used to determine the effects of CD226 blocking treatment. We observed that both intact extracellular domains of CD226 were necessary for functional interaction of the receptor with its ligand CD155, even though one domain was shown to bind CD155 with lower affinity in a solid binding assay. Importantly, CD226 mAb promoted Treg expansion in a mixed lymphocyte culture and inhibited the cytotoxicity of effector cells. In allogeneic skin transplant mice, administering CD226 mAb reduced inflammation and prolonged allogeneic graft survival, with an increase in the frequency of Tregs. Our results reveal the mechanism underlying CD226-CD155 interactions and indicate that CD226 signals can be manipulated to promote Treg expansion. Moreover, we provide new evidence that suggests the therapeutic potential of anti-CD226 with allogeneic transplantation.