Genes coding for anti-Gal in knock-out mice for the α1,3Galactosyltransferase gene: analysis by hybridomas

Abstract

THE interaction between the natural anti-Gal antibody and the a-gal epitope (Gala1-3Galb1-4GlcNAc-R) on xenograft cells results in rejection of the xenograft. Moreover, activity of anti-Gal increases by manyfold in xenograft recipients as a result of immune response to a-gal epitopes on the graft. This elicited anti-Gal greatly exacerbates xenograft rejection. A potential method for prevention of anti-Gal production in xenograft recipients is the treatment of such patients with anti-idiotype antibodies for anti-Gal. Such antibodies may neutralize anti-Gal and destroy cells capable of producing this antibody. To determine the possible diversity of such anti-idiotypes, the anti-Gal Ig genes have to be analyzed. As a first step, we studied the diversity of the immunoglobulin genes coding for this antibody in a1,3galactosyltransferase (a1,3GT) knock-out (KO) mice. These mice lack a-gal epitopes and are capable of producing large amounts of anti-Gal, upon immunization with xenograft cell membranes that express a-gal epitopes. Anti-Gal hybridomas were produced by fusion of splenocytes from KO mice, immunized with rabbit red cell membranes, with the mouse myeloma fusion partner X63.Ag.653. Hybridomas from several mice were screened for secretion of anti-Gal in ELISA with synthetic a-gal epitopes linked to BSA, as solid-phase antigen, as previously described. The specificity of these monoclonal antibodies was confirmed by demonstrating their specific binding to glycoproteins that express the a-gal epitope, such as mouse laminin and bovine thyroglobulin. Three hybridomas producing anti-Gal IgM were isolated, each from a different mouse. The Ig genes in these hybridomas were cloned and sequenced. All three anti-Gal clones contained heavy chain (VH) genes derived from the germline Ig gene VH22.1 and light chain (VL) genes derived from the germline Ig gene K5.1 (Table 1). These anti-Gal coding genes contained various somatic mutations in CDR1 and CDR2 regions. Some mutations were also found in frame work (FR) 2 and 3 regions. In addition, the CDR 3 regions differed significantly from one clone to the other. Interestingly, the sequences of the VL genes were much more conserved than those of the VH genes. Analysis of replacement (R) versus silent (S) mutations demonstrated a much higher proportion of R mutations in the CDR regions than in the FR regions (Table 1). Overall, these findings indicated that the Ig gene repertoire for anti-Gal is highly restricted in KO mice, and it consists primarily of one light and one heavy chain Ig genes. This limited repertoire is likely to reflect the structural