Expression of Gal alpha(1,3)gal on neonatal porcine islet beta-cells and susceptibility to human antibody/complement lysis.

Abstract

Neonatal porcine pancreases may be a potential source of islets for transplantation into patients with type 1 diabetes; however, whether these cellular grafts will be susceptible to damage by human natural antibody-mediated rejection remains controversial. Although we and others have demonstrated that porcine islets bind human IgG and IgM, it remains unknown if they express the xenoreactive antigen Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (Gal epitope). In this study, by using the Gal-specific lectin IB4 for immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis, we determined which cell types present in porcine neonatal islet cell (NIC) aggregates express the Gal epitope and which ones are susceptible to lysis by activation of the human complement. After FACS analysis, 30.0 +/- 3.0% of porcine NICs were shown to express Gal, whereas 70.0 +/- 2.0% did not. Histological assessment of Gal-expressing cells revealed that 54.9 +/- 8.8% stained positive for either insulin or glucagon. In contrast, 68.8 +/- 8.4% of the Gal-negative population stained positive for the pancreatic hormones insulin and glucagon. Incubation of either the Gal-positive or -negative cells with human AB serum plus complement for 1.5 h resulted in the lysis of >90% of the cells. These results demonstrate that porcine NIC aggregates are composed of Gal-expressing cells and that expression of Gal is not restricted to nonendocrine cells. Furthermore, both Gal-positive and Gal-negative cells are susceptible to human antibody/complement-mediated cytolysis, suggesting that this form of immunological destruction is an obstacle that will need to be overcome before porcine NIC aggregates can be used clinically.

In vitro and in vivo expression of Galalpha-(1,3)Gal on porcine islet cells is age dependent

“…The expression of Gal on specific porcine islet endocrine cells remains unclear. Some studies have shown that Gal is not expressed on adult or fetal porcine islet endocrine cells (Oriol et al 1993, McKenzie et al 1994, 1995a, while we have demonstrated that Gal is expressed in both insulin-and glucagon-producing neonatal porcine islet cells (Rayat et al 1998). A recent study has shown that fetal porcine islet cells consistently express high levels of Gal epitope, whereas most adult islet cells are void of this epitope .…”

“…However, some immature endocrine cells, scattered between the duct epithelial cells or inside the clusters, were weakly positive for Gal antigen. Based on our previous study (Rayat et al 1998), we hypothesize that immature endocrine cells and some ductal cells present in NPI are the cells that we previously detected to express Gal antigen. To test this hypothesis, we further examined the presence of Gal in NPI and correlated this expression with cytokeratin 7 (CK7)-positive ductal cells in various porcine islet cell preparations.…”

Porcine Islet Xenotransplantation for the Treatment of Type 1 Diabetes

“…During both in vitro and in vivo maturation models of NPI, the expression of Gal is shown to reduce significantly as the precursor cells evolve into mature cells . Regardless, it has been demonstrated that NPI expressing and not expressing Gal can be susceptible to hyperacute rejection in vitro, suggesting that Gal is not the only xenoantigen responsible for this phenomenon (Rayat et al, 1998). Islet xenografts are also mainly revascularized by recipient endothelial cells (Nyqyist et al, 2005).…”

“…Second, NPI express antigens on their surface that can predispose the tissue to rejection upon transplantation. Although the most studied of these antigens is galactose (1,3) galactose (Gal), it is not likely the only xeno-antigen responsible for rejection (Rayat et al, 1998). A concern regarding the use of porcine tissue in humans is the possible transmission of xenosis, in particular, the transmission of porcine endogenous retrovirus (PERV).…”

Beta Cell Replacement Therapy

“…One source that has been identified is porcine islets, which secrete physiologically functional insulin but can be highly immunogenic to humans and carry a risk of cross-species infection by pig endogenous retroviruses. A particular surface epitope that has led to increased xenoreactivity is galactose-1,3-galactose (Rayat et al, 1998;Korbutt et al, 1997). Through genetic engineering of pig herds, a knockout animal which lacks this epitope has been successfully bred (Puga Yung et al, 2009).…”