Characterization of antibody binding to swine leukocyte antigen class II

Abstract

Though the elimination of carbohydrate xenoantigens has reduced the antibody barrier to clinical xenotransplantation, identification of additional targets of rejection could further increase the immunologic compatibility of pig tissues with humans. Many patients in need of organ transplantation have antibodies to proteins encoded by the human major histocompatibility complex (MHC) which have high similarity to their swine homologs. The goal of this thesis was to determine if the class II genes of the swine MHC can bind human antibodies. To characterize antibody binding effect to class II swine leukocyte antigens (SLA), a constitutively positive SLA class II cell was created through transfection with the human class II transactivator (CIITA). Cells expressing only SLA-DR or SLA-DQ were also created using the CRISPR/Cas9 gene knockout tools. These various lines were incubated with human sera and tested for binding to IgM and IgG in a flow cytometry crossmatch (FCXM). The results demonstrate reliable antibody binding to each of the SLA class II –DR and –DQ derivatives. A two-way paired t-test revealed statistical difference in total sera binding between to the DR(+)DQ(+) and DR(-)DQ(-) clones for IgG (p = 0.0059) but not IgM (p = 0.2460). Looking at the subset of individuals with and without anti-HLA class II sensitization, statistical difference was noted for IgG (p = 0.0229) but not IgM (p = 0.3045). Examining further the role of DR(+) vs DQ(+), statistical analysis revealed difference in the DR(+)DQ(-) vs. the DR(-)DQ(+) FCXM (p = 0.0099), the DR(+)DQ(-) vs. the DR(+)DQ(+) FCXM (p = 0.0192), and the DR(-)DQ(-) parent vs. DR(+)DQ(+) FCXM (p = 0.0329). No difference was found in the DR(-)DQ(+) vs. DR(+)DQ(+) FCXM (p = 0.1601). The results of this project suggest that SLA class II, specifically SLA-DQ, could be a target of antibody binding and cross-reactive anti-HLA class II antibodies may be capable of binding SLA class II.