Anti-Gal antibody-mediated skin graft rejection requires a threshold level of Gal expression.

Background: Despite overcoming xenograft hyperacute rejection (HAR), Gal (galactose-α1,3-galactose) expression may not be completely eliminated from the α1,3-galactosyltransferase gene knockout (Gal KO) pig because of alternative galactosyltransferases. Whether low levels of “residual” Gal are still susceptible to either complement fixing or non-complement fixing antibody beyond the HAR barrier remains unknown. Furthermore, it would be impossible to analyze the immune response specific to low-level Gal in a xenograft setting given the multitude of xenoantigens that could induce a recipient response. To investigate this question, we therefore used a skin graft model in BALB/c mice where the sole difference between donor and recipient was the expression of Gal, where rejection is caused by passively administered anti-Gal monoclonal antibody and where HAR does not occur. Methods: Gal expression over time was examined by immunohistochemistry in wildtype-to-Gal KO skin grafts. Graft rejection in response to passively administered anti-Gal monoclonal antibody at early and late time points was studied to determine changes in susceptibility to antibody. To independently test the effect of reduced Gal expression on antibody-mediated rejection, we used two separate lines of α1,2-fucosyltransferase transgenic mice as skin donors in the model. These mice have known reduced but different levels of Gal as determined by flow cytometry on peripheral blood leukocytes. Results: Gal expression on skin grafts diminished with time with a corresponding reduction in susceptibility to antibody-mediated rejection. Skin grafts at day 30 (n = 7) and 150 (n = 11) had a rejection rate of 100% and 45% respectively in response to non-complement fixing anti-Gal antibody administered to the recipient. Similar results were demonstrated with a complement fixing anti-Gal antibody. When α1,2-fucosyltransferase transgenic mice skin was used in the model, the line with lowest level of Gal expression was resistant to antibody-induced rejection with a rate 0% (n = 9) vs. 60% (n = 5) in the alternative line with relatively more Gal expressed but still much less than normal mice. Conclusions: Resistance to anti-Gal antibody-mediated damage in the model was observed in skin grafts 100 to 150 days post-grafting but not earlier and was associated with a reduction in Gal expression. It is possible that below a threshold level of Gal expression, the grafts were not susceptible to anti-Gal antibody. [ABSTRACT FROM AUTHOR]