Gal mismatch alone causes skin graft rejection in mice

Background. Elimination of galactose-alpha1,3-galactose (Gal), the major xenoantigen between pig and human, may extend pig-to-human xenograft survival beyond the current barrier of acute vascular rejection. However, it has been suggested that Gal is an essential molecule in the pig and that the generation of a Gal-deleted (Gal KO) pig will not be possible. Should this be the case, understanding the Gal-mediated immune response will be crucial in developing strategies to overcome pig xenograft rejection in humans. There are no existing models of xenograft rejection in which the sole difference between donor and recipient is Gal. We describe a model of exclusively Gal-mismatched skin graft rejection. Methods. The survival of Gal(+/+) skin grafts on Gal KO mice with the same genetic background was analyzed. To examine innate anti-Gal immunity, Gal KO recipients that were also deficient in T and B cells (RAG-1 KO) were used. To study the role of cognate immunity, recipients were sensitized with a primary Gal(+/+) allograft before receiving a second Gal(+/+) graft that was otherwise isogeneic. To test the role of anti-Gal antibodies in this model, recipients were passively immunized with a non-complement-fixing anti-Gal monoclonal antibody. Results. Gal KO mice chronically reject Gal(+/+) skin grafts by 100 days at a rate of 48% (n=25) on a BALB/c background and 25% (n=8) on a C57BL/6 background. The grafts had an infiltrate that consisted predominantly of CD4(+) T cells and macrophages, whereas recipients deficient in T and B cells were incapable of rejection and survived for more than 120 days (n=5). Sensitization with a primary Gal(+/+) allograft increased the incidence and the tempo of rejection of a second Gal-only mismatched skin graft with 99% rejection that ranged from 11 to 45 days (n=26). Passive transfer of mouse IgG, anti-Gal monoclonal-antibody-induced rejection in Gal KO and RAG-1/Gal double-KO recipients at a rate of 92% (n=13). Conclusions. We have established a model to study rejection based solely on a Gal mismatch. Our results indicate that non-complement-fixing anti-Gal antibody can cause rejection in the acute vascular rejection time frame and that T-cell-mediated chronic rejection will be a further barrier to overcome if Gal cannot be deleted from the pig.