Background: The creation of genetically-modified pigs has brought us closer than ever to clinical xenotransplantation. However, the optimal immunosuppressive doses still need to be established to reduce T cell-mediated rejection, especially in renal xenograft. Here we assessed the effect of immunosuppressives on human T cell proliferation, induced by renal microvascular endothelial cells (RMEC) from GGTA1/CMAH knockout (KO) pigs, which lack expression of αGal and Neu5Gc antigens.

Methods: Renal cells from GGTA1/CMAH KO pigs were isolated using collagenase perfusion. RMEC were sorted using endothelial cell marker CD31. The phenotypic and functional features of RMEC were analyzed using flow cytometry, confocal microscopy, and qPCR. Flow cytometry-based mixed lymphocyte reaction (MLR) was performed using CFSE-labeled human CD4 T cells and irradiated effectors: GGTA1/CMAH KO renal cells, splenocytes, and human splenocytes.

Results: GGTA1/CMAH KO renal cells lacked both αGal and Neu5Gc expression and had markedly reduced bindings by human IgG and IgM compared to those of Domestic and GGTA1 KO pigs using flow cytometry analysis. GGTA1/CMAH KO RMEC, which expresses both endothelial marker CD31 and SLA class II, consisted of 15-20% of the renal cells in culture. The RMEC also expressed von Willebrand factor and SLA class I by flow cytometry and confocal imaging. Comparison of GGTA1/CMAH KO CD31- renal cells by qPCR showed that the RMEC had not only expressed significant higher levels of CD31, SLA class II, and von Willebrand factor, but also costimulatory molecules including CD86 and ICAM-1. Furthermore, time course analysis (1day to 14 days) revealed that the expansion of human CD4 T cells in MLR reached maximum levels by day 14. The expansion of T cells was significantly upregulated (3-fold) by RMEC compared to CD31- renal cells, but the response of human CD4 T cells to pig splenocytes and human splenocytes were increased (around 2 fold) compared to RMEC. Moreover, FK 506 (10ng/ml) significantly decreased human CD4 T cell proliferation induced by RMEC, pig splenocytes, and human splenocytes, respectively at 2-fold, 4.5-fold, and 5-fold.

Conclusion: RMEC are useful for in vitro assessment of the immunoreactivity of genetically modified pigs deficient in GGTA1/CMAH genes and the therapeutic levels of immnosuppressives.

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