B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy

Abstract

Background: In the pig-to-baboon artery patch model with no immunosuppressive therapy, a graft from an α1,3-galactosyltransferase gene-knockout (GTKO) pig elicits a significant anti-nonGal IgG response, indicating sensitization to the graft. A costimulation blockade-based regimen, e.g., anti-CD154mAb or anti-CD40mAb, prevents sensitization. However, neither of these agents is currently FDA-approved. The aim of the present study was to determine the efficacy of FDA-approved agents on the T and B cell responses.

Methods: Artery patch xenotransplantation in baboons was carried out using GTKO/CD46 pigs with (n = 2) or without (n = 1) the mutant transgene for CIITA-knockdown. Immunosuppressive therapy consisted of induction with ATG and anti-CD20mAb, and maintenance with different combinations of CTLA4-Ig, tacrolimus, and rapamycin. In addition, all 3 baboons received daily corticosteroids, the IL-6R blocker, tocilizumab, at regular intervals, and the TNF-α blocker, etanercept, for the first 2 weeks. Recipient blood was monitored for anti-nonGal antibody levels by flow cytometry (using GTKO/CD46 pig aortic endothelial cells), and mixed lymphocyte reaction (MLR). CD22⁺B cell profiles (naïve [IgD⁺/CD27^-], non-switched memory [IgD⁺/CD27⁺], and switched memory [IgD^-/CD27⁺] B cell subsets) were measured by flow cytometry. At 6 months, the baboons were euthanized and the grafts were examined histologically.

Results: No elicited anti-pig antibodies developed in any baboon. The frequency of naïve memory B cells increased significantly (from 34% to 90%, p = 0.0015), but there was a significant decrease in switched memory B cells (from 17% to 0.5%, p = 0.015). MLR showed no increase in the proliferative T cell response in those baboons that had received CTLA4-Ig (n = 2). Histological examination showed few or no features of rejection in any graft.

Conclusions: The data suggest that immunosuppressive therapy with only FDA-approved agents may be adequate to prevent an adaptive immune response to a genetically-engineered pig graft, particularly if CTLA4-Ig is included in the regimen, in part because the development of donor-specific memory B cells is inhibited.

Keywords: Artery patch; CTLA4-Ig; FDA-approved agents; Pig; Rapamycin; Tacrolimus; Xenotransplantation.