Galectin 9 - a Novel Regulator of Humoral Rejection

Abstract

Introduction: The negative TIM-3:Galectin-9 costimulatory pathway has recently been established as a central master switch that modulates alloreactive Th1/Th17/Treg CD4+ effector T cell differentiation through regulation of the IL-6 production by CD4+ T cells. As the role of this pathway in the induction of tolerance by CTLA4-Ig is not known, we tested the effects of Galectin-9 blockade or deficiency in a murine model of cardiac transplantation. Methods: B6 mice were treated with a single dose of CTLA4-Ig (0.5 mg) and used as recipients of BALB/c hearts. Galectin-9 was targeted by either use of RG9-1, a blocking antibody against Gal9, or by using Gal9 gene-deficient (Gal9−/−) instead of wild-type recipients. Graft survival was assessed by daily palpation. Intragraft and systemic immune responses were assessed by graft histopathology/immunohistochemistry, ELISPOT and Flow Cytometry. Results: Both strategies of targeting Gal9 abrogated the tolerogenic effects of a single dose of CTLA4-Ig (1A). Abrogated graft survival was accompanied by increased frequencies of IFN-γ-, IL-17, and IL-6-producing alloreactive splenocytes (1B). Similarly, Gal9−/− recipients treated with multiple doses of CTLA4-Ig could not be tolerized (MST 78.5 vs. >150 days, n=6/6, p=0.002). After Gal9 blockade, grafts showed increased mononuclear cell infiltrates, extended areas of severe necrosis, and strong positivity for C4d, when compared to controls. We also found expansion of B cells, with no change in the overall number of splenic T cells (1C), and increased formation of DSA in the sera of graft recipients (1D), which correlated with the extent of myocardial necrosis. To test whether the effects of Gal-9 are dependent on the presence of B cells, we used B cell-deficient μMT mice as recipients. In the absence of B cells, Gal9 did not abrogate the tolerogenic effects of CTLA4-Ig (1E). Given the increased production of IL-6 after Gal9 blockade, and the importance of IL-6 for the induction of Tfh cells, we also tested the effects of Gal9 blockade in IL-6−/− recipients. In the absence of IL-6, Gal9 blockade had no effect on the tolerogenic effects of CTLA4-Ig (1F). Moreover, we showed in vitro that Gal9−/− CD4+ T cells stimulates naive B cells more powerfully than their WT counterparts in vitro (as assessed by B cell blast formation), and also induce enhanced production of IgG by naive B cells. Conclusions: These data suggest that absence of Gal-9 abrogates the tolerogenic effects of CTLA4-Ig and that graft rejection is switched to a humoral mechanism, in which B cells and IL-6 produced by CD4+ T cells are key players. This model provides a unique in vivo model of antibody-mediated rejection.[Figure 1]