Blockade of CD86 and CD40 induces alloantigen-specific immunoregulatory T cells that remain anergic even after reversal of hyporesponsiveness.

The generation of immunoregulatory T cells that block the B7(CD86/CD80)-CD28 and/or CD40-CD154 costimulatory pathways has great potential for the induction of long-term transplantation tolerance. In a human polyclonal in vitro model, combined monoclonal antibody (mAb) blocking of the costimulatory ligands CD40 and CD86 lead to allospecific T-cell anergy that cannot be reversed by antigenic rechallenge in the presence of IL-2. Although antigenic restimulation with IL-2 restored the proliferative response, subsequent antigenic restimulation of the restored anergic cells in a tertiary mixed lymphocyte culture still resulted in nonresponsiveness. Importantly, these anergic T cells suppress the response of naive alloreactive T cells in an antigen-specific way via linked recognition. Suppression may partially depend on local IL-10 production, while transforming growth factor-beta (TGF-beta) did not play a role. Irrespective of the monoclonal antibody combination used, blast formation occurred in a subset of CD4(+) cells. These cells were characterized by a sustained CD45RA expression, an increased T-cell receptor density, and a lower level of CD4 expression. A reduced number of CD45RO(+)/CD8(+) T cells was observed whenever anti-CD86 was combined with anti-CD40, which was reflected by an even more attenuated cytotoxic T-cell function. This indicates the importance of CD40-CD154 in the generation of cytotoxic T cells in this transplantation model. We hypothesize that in our model, anergy is induced in the CD4(+) T-cell subset, whereby CD8(+) cytotoxic effector function is impaired by the lack of both CD40-CD154 signaling and cytokine-mediated help. This costimulatory ligand-directed mAb approach might well be used for the ex vivo generation of antigen-specific immunoregulatory T cells applicable in adoptive immunotherapy.