ANTIGEN PAINTING TO A XENOGRAFTED HEART'S CONTENT: TH2 CYTOKINE-INDUCED CHANGES IN PROTEOGLYCAN EXPRESSION RESULT IN ENDOTHELIAL COATING BY HOST MHC CLASS I AND REGULATORS OF COMPLEMENT ACTIVATION.

Abstract

33 Historically, definitions of xenograft accommodation were so vague that therapeutic approaches and mechanisms responsible for long-term survival, despite the presence of normal host antibody and complement (C) responses, were unknown. Recently, we have shown that brief decomplementing with cobra venom factor (CVF) plus daily cyclosporin (CsA) result in permanent survival of >75% of hamster->rat cardiac xenografts. Evaluation of accommodated xenografts showed antibody and C deposition and a Th2 cytokine response, in conjunction with infiltration by macrophages, NK and T cells; we now provide further insights into the mechanisms of accommodation in this model. Analysis with monoclonal antibodies (mAbs) of cardiac xenografts in which accommodation was induced by 1-2 doses of CVF plus daily CsA showed high levels of endothelial expression of hamster-specific heparan sulphate and CD44(hyaluronic acid receptor). Accordingly, baby hamster kidney cells (BHK) cells were cultured in the presence of rat Th1 (IL-2, IFN-γ) or Th2 (IL-4, IL-10) cytokines and evaluated (flow cytometry, ELISA) for changes in proteoglycan expression. Th2 but not Th1 cytokines induced dramatic increases in proteoglycan and CD44 production. Increased proteoglycan expression in vitro led to markedly enhanced binding of rat MHC class I and Crry (a rat complement regulatory protein with properties shared by both CD46 and CD55), as assessed using rat-specific mAbs. Moreover, the latter binding was associated with increased resistance to complement- or NK cell-mediated lysis in vitro. Consistent with these data, immunohistologic assessment of accommodated hamster->rat cardiac xenografts vs. rejecting or normal hamster hearts showed dense vascular labeling for hamster proteoglycans and CD44, plus endothelial staining with mAbs to rat MHC class I and Crry molecules throughout follow-up (day 4 - day 100). Together, these studies show that rat Th2 cytokines can induce increased expression of proteogylcans by hamster cells in vitro and in vivo, and such expression of highly charged molecules facilitates the coating or“antigen painting” of vascular surfaces by host molecules. i.e. Local binding of Crry appears to make hamster cells more resistant to attack by rat C, the concentration of MHC class I dampens rat NK cell-mediated lysis, and CD44 expression may contribute to selective T cell recirculation through the xenograft. These findings indicate mechanisms for xenograft (and potentially allograft) accommodation which are testable in large animal models, and suggest means to promote long-term survival with minimal host toxicity.