Evaluation of primate anti-pig xenoreactive immune responses using immortalized aortic endothelial cells from genetically modified pigs.

Porcine vascular endothelial cells are primary targets of humoral and cellular immune responses in pig-to-primate organ xenotransplantation. Although porcine aortic endothelial cells (PAECs) are widely used to evaluate primate anti-pig immune responses, their limited proliferative capacity poses a significant obstacle to repeated and standardized experimentation. To overcome this challenge, we established immortalized PAEC lines (iPAECs) derived from WT, GTKO, and GTKO/CMAHKO/B4GALNT2KO (triple-knockout, TKO) donor pigs using stable transduction of the SV40 large T antigen. All iPAEC lines expanded stably beyond passage 20, maintained high CD31 expression, and preserved genotype-specific xenoantigen profiles. Anti-pig IgM/IgG binding and complement-dependent cytotoxicity (CDC) against these cells were measured with flow cytometry using pooled rhesus monkey sera (n=20) or human sera (n=20). Compared with WT iPAECs, GTKO iPAECs demonstrated significantly reduced IgM/IgG binding and CDC with human and rhesus sera. Additional deletion of Sda and Neu5Gc in TKO iPAECs markedly reduced human serum reactivity, whereas rhesus sera retained relatively high antibody binding and CDC against TKO iPAECs. Notably, the humoral immune responses remained consistent across primary cells and passages P5, P10, and P20, validating the long-term efficacy of iPAECs as an experimental tool. Preformed anti-pig antibodies in 30 rhesus monkeys were further evaluated using TKO iPAECs and TKO peripheral blood mononuclear cells (PBMCs). The results demonstrated a significant correlation between the two target cell types, supporting their interchangeability for humoral immune assessment. Furthermore, xenogeneic MLR assays confirmed that iPAECs could stimulate human CD4+ and CD8+ T-cell proliferation, especially following porcine IFN-γ-induced upregulation of SLA-I and SLA-II expression. Collectively, these findings establish donor-derived iPAECs as a reliable, reproducible, and genetically defined in vitro platform to comprehensively evaluate humoral and cellular xenogeneic immune responses to genetically modified porcine endothelium.