HLA-sensitized human sera retain cross-reactivity to genetically modified porcine cells via SLA epitopes
Sang-Ik Cho1, Ji-Jing Yan2, Beom Seok Kim2,3, Nayoung Ko4, Joohyun Shim4, Hyunil Kim4, Eun-Jee Oh5.
1Department of Medical Sciences, Graduate School of the Catholic University of Korea, Seoul, Korea; 2The Research Institute for Transplantation, Yonsei University College of Medicine, Seoul, Korea; 3Division of Nephrology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea; 4Optipharm, Inc., Cheongju, Korea; 5Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
Introduction; Genetically modified pigs are promising organ sources for xenotransplantation, but the cross-reactivity of pre-existing anti-human leukocyte antigen (HLA) antibodies against pig antigens remains a key barrier. This study evaluated the humoral immune response of human sera-classified by HLA antibody reactivity-to genetically modified porcine peripheral blood mononuclear cells (PBMCs), aiming to characterize cross-reactive antibody reactivity patterns.
Methods; Porcine PBMCs were isolated from wild-type (WT), α1,3-galactosyltransferase knockout (GTKO), triple-knockout (TKO) and quadruple-knockout (QKO) pigs (Optipharm Inc.) using density gradient centrifugation. Human sera from 61 kidney transplant patients were classified into four HLA antibody reactivity groups: I+II+ (n=10), I+ (n=10), II+ (n=10), I–II– (n=31) using single-antigen bead (SAB) assays. Flow cytometry crossmatch (FCXM) measured IgG/IgM reactivity via median fluorescence intensity (MFI). Complement-dependent cytotoxicity (CDC-NIH/AHG) assays assessed functional antibody activity. Red blood cell (RBC) absorption removed major carbohydrate xenoantigens, while antibody elution from QKO PBMCs identified bound cross-reactive HLA antibodies via SAB. HLA eplet compatibility with swine leukocyte antigen was analyzed using structural analysis.
Results; Human sera showed strong IgG/IgM reactivity to WT PBMCs, while TKO and QKO showed markedly reduced antibody binding. For QKO pigs, sera from HLA class I+II+ patients demonstrated the highest IgG reactivity, with significantly higher median MFI ratios compared to HLA I–II– sera (p < 0.05). IgM responses to QKO were low across all groups. RBC absorption abolished reactivity to WT PBMCs but not QKO PBMCs, confirming elimination of major carbohydrate xenoantigens (α-Gal, Neu5Gc, Sda) in QKO. CDC assays revealed stronger cytotoxicity in HLA-reactive sera, correlating with FCXM results. Eluted antibodies from QKO PBMCs included HLA-A*01:01, B*44:02, and B*15:12, but not high-MFI alleles like A*02:01, indicating selective cross-reactivity. Structural analysis confirmed HLA eplets 144KR and 163LS/G as potential mediators of SLA cross-reactivity.
Conclusion; Histocompatibility assays, including optimized FCXM, effectively reflect immunological compatibility. HLA antibody profiles critically influence residual humoral responses to genetically modified pig cells, suggesting the need for comprehensive pre-transplant assessment in xenotransplantation.