A Novel Assay to Screen Porcine MHC Class II Antibodies in Potential Xenotransplant Recipients

Abstract

BackgroundWell‐elucidated in clinical allotransplantation is the role of recipient's donor specific antibodies and graft rejection, specifically the antibodies targeted at Human Leukocyte Antigen (HLA) class II. To determine whether cross‐reactive or preformed antibodies exist to Swine Leukocyte Antigen (SLA) class II, an assay was developed to screen the patients on the Indiana University (IU) transplant waiting list.Methods69 patients on the IU transplant waiting list were selected for study based on their levels of anti‐HLA class II antibodies. Wild‐type pig red blood cells were used to absorb the sera, removing anti‐carbohydrate antibodies and leaving anti‐MHC antibodies. This sera was screened on an immortalized fibroblast cell line transfected with a version of the human class II transactivator (CIITA), producing a constitutively activate SLA class II cell line. A cell that incorporated the antibiotic resistance gene but not the CIITA was chosen as a negative control. Following incubation, the sera was evaluated for presence of IgM and IgG antibody binding by flow cytometry.ResultsCells lacking expression of SLA class II demonstrated decreased IgM and IgG binding compared to cells with 100% SLA class II DR and DQ expression (Figure 1). Of note are several individuals whose sera preferentially binds the class II positive cell (the dots beneath the diagonal line). The difference in binding between a positive and negative cell is statistically different for IgG (p = 0.0048) but not IgM (p = 0.5102). Increased binding is found when evaluating those individuals with anti‐HLA class II antibodies (Figure 2). The statistical significance holds when comparing the change in binding on the SLA class II positive and negative cells for individuals with and without anti‐HLA class II antibodies for IgG (p = 0.0341) and IgM (p = 0.2602).ConclusionsExpression of SLA class II results in increased IgM and IgG human antibody binding, demonstrating the presence of human anti‐pig class II antibodies. The existence of this humoral barrier suggests that antibody‐mediated binding to SLA class II may be an issue faced in clinical xenotransplantation.Support or Funding InformationThis publication was made possible in part by Grant Number UL1 TR001108 (A. Shekhar, PI) from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.