IFN-γ Production Is Specifically Regulated by IL-10 in Mice Made Tolerant with Anti-CD40 Ligand Antibody and Intact Active Bone

Abstract

We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-γ production that is donor specific, and a reduction in the frequency of IFN-γ-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-γ responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-β, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-γ response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-γ production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-γ levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-γ. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-γ production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.