Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses

Abstract

Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, β4galNT2, SLA-I α chain, and β2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.

Figure 1

Morphology of primary pLDEC (passage 12) and WT ipLDEC (passage 25 after immortalization).

Figure 2

Phenotype of GM ipLDEC. Flow cytometric analysis of the absence of αGal (A), Sda (C), and SLA-I (D), and reduction of Neu5Gc (B) on ipLDEC (due to uptake of Neu5Gc glycan from Neu5Gc-rich FBS in culture medium).

Figure 3

Genotype of GM ipLDEC. Each gRNA targeted region was amplified by PCR. Sanger sequencing revealed mutations in GGTA1, CMAH, β4galNT2, and B2M genes.

Figure 4

Characterization of primary pLDEC and immortalized porcine liver-derived endothelial cells (ipLDEC). Porcine cells were analyzed for endothelial cell markers, including cell surface expression of CD31, intracellular expression of von Willebrand factor (VWF), and E-selectin (rhTNF-α stimulation) by flow cytometry. SLA-I and SLA-II expression in porcine cells before and after porcine IFN-γ stimulation (pIFN-γ) were examined by flow cytometric analysis using specific antibodies. Primary pLDEC (passage 15–20) and ipLDEC (passage 20–30 after immortalization) were used for characterization.

Figure 5

Comparison of human serum antibody binding to TKO and 5GKO ipLDEC lines. Human sera from 20 patients on the kidney transplant wait-list were incubated with TKO (GGTA1/CMAH/β4galNT2) or 5GKO (GGTA1/CMAH/ β4galNT2/SLA-I α chain/B2M) ipLDEC. Subsequently, fluorescent conjugated anti-human IgG or IgM was used to evaluate human IgG or IgM binding to the cells, with mean fluorescence intensity (MFI). Mann–Whitney test was used to analyze the differences between TKO and 5GKO ipLDEC lines. Human serum IgG exhibited significantly decreased binding to 5GKO ipLDEC compared to TKO ipLDEC (p < 0.0001). Human serum IgM binding to 5GKO ipLDEC was also significantly reduced compared to TKO ipLDEC, (p = 0.0443). Unfilled dots and triangles represented low PRA (< 10%) serum samples (n = 2). All other serum samples (n = 18) shown in dots were from individuals with high PRA (> 90%).

Figure 6

Human NK cell degranulation following porcine cell stimulation. Human PBMCs from three donors were treated with human IL-2 (20 ng/mL) for 5 days to expand NK cells, then cocultured with WT, TKO, and 5GKO ipLDEC for 2 h. Cells were stained with antibodies to CD45, CD3, CD56, and CD107a. Flow cytometry analysis was gated on NK cells (CD3^-CD56⁺) and monitored for the percentage of CD107a positive cells (left) and surface expression of CD107a (MFI) (right). Statistical significance was calculated by one-way ANOVA, Tukey’s multiple comparisons test (ns not significant; ***p < 0.001; ****p < 0.0001).