In vitro recognition and impairment of pig islet cells by baboon immune cells: similarity to human cellular reactions.

Abstract

Grafting pig islets into patients with type 1 diabetes requires control of the strong cellular xenogeneic rejection. This in vitro study compared the cellular reaction of baboons and humans to pig islet cells (PICs) to confirm the validity of using these animals for further in vivo preclinical trials. Baboon or human peripheral blood mononuclear cells (PBMCs) or subsets were co-incubated with PICs from specific pathogen-free adult pigs for 7 days to determine the mechanisms and intensity of PBMC proliferation. Interleukin (IL) 10 and interferon (IFN) gamma secretion were assessed by enzyme-linked immunosorbent assay. Because proliferation was not indicative of aggression, a test based on perifusion analysis of the alteration of basal and stimulated insulin releases from PIC incubated with different baboon and human cells was developed. Baboon PBMCs strongly proliferated in response to PICs (stimulation index [SI]=24.8+/-6.9 [n=8] vs. 23.9+/-3.4 [n=34] for human PBMCs), showing considerable variation in intensity among animals (2.3<SI<63) and humans (1.8<SI<97). PBMC proliferation was inhibited in baboons and humans by anti-CD4 (% inhibition of SI: 71+/-10% and 75+/-7%, respectively) and anti-DR (75+/-35% and 80+/-6%) monoclonal antibodies (MoAbs) or by depletion of MHC class II+ cells (99+/-1% and 90+/-6%). Blocking by anti-CD8 or anti-CD16 MoAbs was weaker and variable among both animals and humans. IL-10 production by baboon and human PBMCs in response to PICs increased more than IFN-gamma production after 2 days of coculture, but the IL-10/IFN-gamma ratio was inverted after 5 days of coculture. After 7 days (and even after only 2 days) of coculture with baboon (n=8) or human (n=18) PBMCs, basal and glucose-stimulated insulin secretions from PICs were almost completely abolished (P<0.0001). The drop in insulin release could have mainly resulted from lysis of PICs, because the number of PICs decreased by 78% after 7 days of co-incubation with PBMCs. A decrease of insulin release by PBMCs was reproduced with plastic-adherent cells and was abolished by depletion of MHC class II+ cells or by addition of 100 microg/ml gadolinium (which inhibits macrophages), but not by cyclosporine. In baboons, as in humans, insulin release was also decreased after coculture of PICs with enriched T lymphocytes remixed with antigen-presenting cells (APCs). This study provides the first data on in vitro comparison of baboon and human cell-mediated recognition and impairment of PICs. Proliferation of PBMCs against PICs involves mainly CD4 T cells, with indirect recognition mediated by baboon or human MHC class II+ APCs. The Th2/Th1 profile of cytokines secreted in response to PICs was similar in baboon and human PBMCs. The model based on alteration of insulin secretion indicates that PIC impairment by whole mononuclear cells was strong and rapid and that a crucial role was played by MHC class II+ and plastic-adherent cells. Two mechanisms appear to be responsible for the role of these cells: (1) early and strong direct effect, which is potentially involved in vivo in primary nonfunction of islets aggressed by monocytes and macrophages; and (2) presentation of PIC xenoantigens, which leads to impairment by T lymphocytes possibly involved in in vivo-specific cellular rejection. The mechanisms and intensity of baboon cellular reactions to PICs in vitro were similar to those observed in humans, which suggests that the baboon is a suitable model for the study of cellular mechanisms during preclinical trials of pig islet xenografts.