COSTIMULATORY PATHWAYS ARE ACTIVE IN XENOGENEIC IMMUNE RESPONSES.

Abstract

36 Costimulatory blockade has been shown in this and other laboratories to prevent allograft rejection. Recent advances in the control of the innate humoral response to discordant xenografts have focused attention on potential means of preventing subsequent cellular and acquired humoral reactivity. A variety of costimulatory pathway blockers have been tested in mixed lymphocyte reactions (MLR) for their ability to prevent human peripheral blood mononuclear cell (PBMC) activation when stimulated by irradiated porcine splenocytes. The results indicate that human CTLA4-Ig is capable of attenuating the xenoreactive PBMC proliferation by 60%, anti-human CD40L (5C8) by 52%, anti-human CD80 (1F1) by 13%, and anti-human CD86 (3D1) by 28%. Furthermore, combinations of these reagents proves to have synergistic effects. The combination of hCTLA4-Ig/5C8 attenuates the response by 76% and the 1F1/3D1/5C8 combination 72%. Consistent with this observation, LPS stimulated porcine splenocytes have been shown by flow cytometry to specifically bind hCTLA4-Ig, 1F1, and 3D1. A panel of 12 anti-human CD80 and CD86 antibodies have been screened with only 1F1 and 3D1 exhibiting cross reactivity to pig. In addition, using irradiated human PBMCs as stimulators and porcine PBMC's as the responding cells revealed that the combination of 5C8 and hCTLA4-Ig is also capable of blocking pig cell proliferation. It would thus appear that costimulatory ligands are sufficiently homologous, such that they support cross species ligand/receptor interaction which is important in the xenoreactive response between human and porcine cells. Preliminary evidence also suggests that blockade of CD86 on the porcine microvascular endothelial cell (PMVEC) inhibits their killing by human NK cells. This is consistent with the constitutive expression of CD86 on PMVEC's that we have observed. Furthermore, we have demonstrated that CD86 is upregulated by exposure to human T and NK cell and human T cell conditioned media. We conclude that costimulatory molecules play an important role in cellular xenoreactivity and that the same reagents used to prevent allograft rejection may function in the prevention of xenograft rejection. These results and the known effects of 5C8 on isotype switching and maturation of acquired humoral responses suggests efficacy of these reagents in modulating both the humoral and cellular immune response. We are now in the process of initiating these studies in a pig to primate model.