Limited specificity of xenoantibodies in diabetic patients transplanted with fetal porcine islet cell clusters. Main antibody reactivity against α‐linked galactose‐containing epitopes

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Abstract
The immunological specificity of antibodies formed as a result of xenotransplantation of fetal porcine islet‐like cell clusters to diabetic patients was characterized. High titer increases were recorded against porcine cells, solubilized membrane fractions of porcine cells, and purified MHC class I antigens. However, titer increases were also noted against ssDNA and dsDNA and against pig thyroglobulin but not against actin, myoglobin, or haptenated BSA. Antibody titers against tetanus toxoid were unaffected. The reactivity against porcine RBC could be completely blocked by absorption with pig thyroglobulin. Since pig thyroglobulin contains the galαl‐3gal antigen, the reactivity against RBC was most probably mainly due to antibodies against this oligosaccharide epitope. The reactivity against porcine mononuclear cells was only partially absorbed by pig thyroglobulin, indicating a heterogeneity of the clonal response. These conclusions were substantiated by data showing that the antigenic determinants on pig thyroglobulin were completely destroyed by treatment with α‐ but not with β‐galactosidase. Further studies showed that immune reactivity against pig RBCs, platelets, islet‐cells, endothelial cells and pig MHC class I molecules, caused by xenoimmunization, was almost completely blocked by either absorption of antibodies on a column of Sepharose beads coated with galα1‐3gal or by pretreatment of the antigen fractions with α‐galactosidase. Western blot experiments revealed that both the natural and xenoimmune antibodies reacted with a large number of different glycoproteins. There was no difference in heterogeneity of the response when comparisons were made between pre‐and posttransplantation sera, nor was there any difference of patterns caused by IgM or IgG antibodies. Absorption studies revealed that this epitope was present in a large number of different glycoproteins, a conclusion verified by staining with the eluate from a thyroglobulin‐immunosorbent column or with the eluate from the galα1‐3gal‐coupled Sepharose column. Our findings demonstrate that the xenoimmune response is mainly directed against oligosaccharide residues and that there is a limited clonal heterogeneity of antibodies in xenografted patients. There was no direct evidence of reactivity against any porcine proteins nor of specific immunization against porcine MHC peptides. The data form a basis for a future understanding of means to cope with and prevent the rejection of xenogeneic islet cells in man. We also suggest, based on the specificity found in xenoimmunized patients sera, that a major part of naturally occurring antibodies might be specifically reactive with carbohydrate antigens.