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HLA-Class I Sensitized Renal Transplant Patients have Antibody Binding to SLA-Class I Epitopes.

BACKGROUND
Highly sensitized patients are difficult to match with suitable renal allograft donors and may benefit from xenotransplant trials. We evaluate antibody binding from sensitized patients to pig cells and engineered single allele cells to identify anti-HLA antibody cross-species reactivity with swine leukocyte antigen (SLA). These novel testing strategies assess HLA/SLA epitopes and antibody binding patterns and introduce genetic engineering of SLA epitopes.


METHODS
Sensitized patient sera was grouped by calculated panel reactive antibody (cPRA) and luminex single antigen reactivity profile and was tested with cloned GGTA1/CMAH/B4GalNT2 glycan knockout (KO) porcine cells. Pig reactivity was assessed by direct flow cytometric crossmatch (FCXM) and studied following elution from pig cells. To study antigenicity of individual Class I HLA and SLA alleles in cells, irrelevant sera binding to lymphoblastoid cells was minimized by CRISPR/Cas9 elimination of endogenous Class I and Class II HLA, b-cell receptor, and Fc-receptor genes. Native HLA, SLA, and mutants of these proteins after mutating 144K to Q were assessed for antibody binding.


RESULTS
Those with predominately anti-HLA-B&C antibodies, including Bw6 and Bw4 sensitization, frequently have low pig reactivity. Conversely, antibodies eluted from porcine cells are more commonly anti-HLA-A. Single HLA/SLA antigen expressing engineered cells show variable antigenicity and mutation of 144K-to-Q reduces antibody binding for some sensitized patients.


CONCLUSIONS
Anti-HLA antibodies cross-react with SLA Class I in predictable patterns which can be identified with histocompatibility strategies and SLA Class I is a possible target of genetic engineering.