In animals with transplant tolerance without lympho-hemopoietic chimerism peripheral T cells reactive to donor are not deleted, but their capacity to effect rejection is inhibited by alloantigen specific CD4⁺CD25⁺T regulatory cells. Detection of tolerance by in vitro assays has not been established.

Here we compared the proliferation of CD4⁺, CD4⁺CD25⁺ and CD4⁺CD25^– T cells from animals tolerant to a fully allogeneic allograft to these subsets from naive animals. Paradoxically tolerant CD4⁺CD25⁺T cells did not proliferate to specific donor, but did to third party, whereas naive CD4⁺CD25⁺T cells proliferated to all alloantigens. Proliferation of tolerant and naive CD4⁺T cells to specific donor and self was similar, and removal of CD25⁺ cells enhanced proliferation except that of tolerant cells to specific donor. These studies paradoxically suggested tolerant CD4⁺CD25⁺ T cells were not inhibiting proliferation of CD4⁺CD25^–T cells to specific donor.

It is known that tolerant CD4⁺CD25⁺T cells are short lived, and require cytokines other than IL-2 to survive. We showed that tolerant CD4⁺CD25⁺T cells response to specific donor but not to third party or self could be enhanced by adding either IFNγ or IL-5 to culture, but not by other cytokines including IL-4, IL-10, IL-13, and TGF-Β. Receptors for IL-5 and IFN-γ are expressed by tolerant but not naive CD4⁺CD25⁺T cells. Responses of tolerant CD4⁺T cells are inhibited by addition of either IFNγ or IL-5.

These findings were consistent with transplant tolerance being mediated by CD4⁺CD25⁺T cells that is short lived ex vivo. The tolerance mediating CD4⁺CD25⁺FOXP3⁺ T regulatory cells only survive is stimulated by specific donor alloantigen and either IFNγ or IL-5 produced by alloactivated Th1 and Th2 cells. One or more of these differences in reactivity of CD4⁺T cell subsets may provide a test for transplant tolerance.

CITATION INFORMATION: Hall B, Robinson C, Plain K, Verma N, Tran G, Carter N, Nomura M, Boyd R, Hodgkinson S. In Vitro Reactivity of CD4⁺ T Cells Subsets from Hosts with Antigen Specific Transplant Tolerance Mediated by CD4⁺CD25⁺T Cells. Am J Transplant. 2016;16 (suppl 3).

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