Introduction: Pig-to-human xenotransplantation (XTx) offers a promising solution to address the persistent organ shortage for clinical transplantation. However, incompatibilities between pig and human species result in destructive human immune responses and ultimate failure of pig tissue/organ grafts. Genetic modification of pigs is an essential approach to overcoming these obstacles. Pig-to-nonhuman primate (NHP) XTx is the standard preclinical model, which monitors NHP recipient survival (as surrogates for humans) after receiving life-supporting pig tissue/organ transplants. Pig-to-NHP XTx is a long process involving generation of GM pigs, XTx surgeries, and post-transplant animal care. It usually takes more than a year to complete one study. In addition, antibody reactivity to CMAH deficient porcine cells differs between humans and NHPs, suggesting the potential limitation of NHP models for examining specific genetic modification(s). Here, we report the development of an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro.  

Method: A porcine liver derived endothelial cell (pLDEC) was characterized and immortalized (ipLDEC) for genetic modification. Five genes including GGTA1, CMAH, β4galNT2, SLA-I a chain, and b2-microglobulin,which are responsible for the production of major xenoantigens (aGal, Neu5Gc, Sda, and SLA-I), were sequentially disrupted in immortalized porcine endothelial cells using the CRISPR/Cas9 technique. Human antibody reactivity to various genetically modified (GM) ipLDEC was examined by a flow cytometry-based assay. Human natural killer (NK) cell activation to ipLDEC stimulation was monitored by CD107a expression on CD3-CD56+ human PBMCs.  

Results: The elimination of aGal, Neu5Gc, Sda, and SLA-I (5GKO, GGTA1/CMAH/β4galNT2/SLA-I a-chain/B2M) dramatically reduced the antigenicity of the porcine cells compared to TKO (GGTA1/CMAH/β4galNT2), despite the use of high panel reactive antibody (PRA) human sera (>90%) (Figure 1). Notably, these cells still retained their ability to provoke human natural killer cell activation (Figure 2). 

Conclusion: Establishment of an in vitro model system to evaluate human immune responses provides an efficient approach to test GM pig-to-human compatibility. Our study indicates that further genetic manipulation of the pig genome to mitigate human NK responses is required, which may contribute to the long-term survival of pig xenografts in humans.