Immunoselection of human lymphocytes producing xenoreactive natural antibodies against Galocl,3Gal terminal residues

Abstract: In the pig-to-human xenograft combination, human xenoreactive natural antibodies (XNA) bind to carbohydrate moieties, especially those with Galα1,3Gal terminal residues, expressed on the surface of most cells. The aim of this study was to select the cells that produce XNA by functionally characterizing a subset of cells for future studies on immunosuppression of XNA formation. We especially addressed the question whether XNA could be produced by CD5+ B lymphocytes, a subset of cells producing antibodies of high connectivity and polyreactivity, possibly involved in autoimmune processes. For that purpose we used porcine thyroglobulin (PTg), which expresses several Galα,3Gal terminal residues, for the immunoselection of XNA-producing cells. On FACS analysis, biotinylated PTg appeared to react with 5% of the B lymphocytes but the proportion of CD5+ B lymphocytes was not enriched in the PTg-reactive population. Similarly, magnetic beads coated with PTg were used to sort lymphocytes with Ig receptors recognizing PTg. Two and one-half percent of cells reacted, mainly B lymphocytes (89%), but they were not enriched for CD5+ B lymphocytes. When PTg-reactive lymphocytes were cultivated in presence of irradiated T cells and stimulated with PWM, the synthesis of Galα1,3Gal reactive antibodies, mainly of the IgG class, was demonstrated. The results of this study suggest that a high percentage of B lymphocytes react with Galα1,3Gal terminal residues. Such XNA-producing cells are not particularly common in the CD5+ subset.