Immunological characterisation of human anti-pig responses in vitro

Abstract

Xenotransplantation of organs or cells might solve the current problems of organ shortage. However, several problems need to be solved before xenotransplantation can become a successful clinical practice. Ethical, physiological, microbiological and immunological barriers need to be overcome. Presently, efforts are concentrated to providing porcine organs or cells for transplantation. This is mainly due to ethical and microbiological barriers that prevent the use of primates as donors for clinical transplantation. But pig to human transplantation is discordant, since human beings have natural antibodies against pig cells, which mediate hyperacute rejection of vascularised grafts. Therefore initial xenotransplantation trials to use xenogeneic cells for transplantation has been performed, since such cells might be insensitive to the detrimental effects of preformed natural antibodies.

In this thesis, we have characterised human natural antibody reactivity against pig cells in vitro, with special reference to porcine islet-like cells. We have also characterised the cellular immune response activated directly by porcine xenogeneic stimulator cells or indirectly by porcine peptides presented by human antigen presenting cells. Xenogeneic natural human antibodies against porcine cells were mainly of IgM, IgG2 and IgA subclasses. Only 30% of all samples tested contained IgG3 antibodies, capable of mediating antibody dependent cellular cytotoxicity (ADCC) of pig Iymphoid cells. In vitro cultured fetal porcine islet cells were insensitive to the cytotoxic effects of natural antibodies as well as to ADCC effects. This finding was a prerequisite for the clinical trial.

A characterisation of the cellular human anti-pig response was carried out in vitro, evaluating both the direct pathway of activation by porcine stimulator cells as well as the indirect pathway mediated by porcine peptides presented by human responder antigen presenting cells. Direct xenoactivation of CD4+ T cells led to proliferation, expression of functional IL-2 receptors and cytokine production with de novo synthesis of IL-2 and IFN-y in magnitude and character comparable to that induced by allogeneic stimulator cells. CD8+ T cells were less efficiently stimulated directly. The frequency of xenoreactive proliferating as well as of cytotoxic precursor cells was approximately 10 x lower than in the allogeneic combination. Indirect xenoactivation resulted preferentially in proliferation and CD25 expression in the CD4+ T cell population.

Xenostimulation by porcine Iymphocytes in vitro led to the formation of both MHC-restricted cytotoxic T cells as well as to an increase in non-MHC restricted natural killer (NK) cells. In vitro activation of purified human NK cells was strictly dependent on human IL-2. Fetal porcine islet like cells were insensitive to resting human NK cells but sensitive to in vitro activated NK cells. Ten diabetic patients received fetal porcine xenogeneic islet cells. All patients promptly synthesised high levels of IgM and IgG antibodies reactive against pig cells, which reached a peak at 30-50 days. The humoral immune response included IgGI and IgG3 antibodies, indicative of a class switch. Xenoimmune antibodies mediate ADCC against pig blood mononuclear cells, fibroblasts as well as fetal porcine islet-like cells. Specificity determinations of xenoimmune antibodies have been performed with alpha-1,3-galactosyltransferase transfected Raji cells expressing Gal-alpha-1,3-Gal epitopes. The results indicated that xenoimmune antibodies were directed against Gal-alpha-1,3-Gal epitopes on target cells.