J. M. Ladowski1, G. Martens1, L. Reyes1, M. Tector1, A. J. Tector1 1University Of Alabama at Birmingham,Birmingham, Alabama, USA
Introduction: Xenotransplantation is a solution to the growing need for life-saving transplantable organs. Recent advances in genetic engineering allow for rapid manipulation of the swine genome. We hypothesize that genetically engineered cells possessing recipient-matched class I major histocompatibility complex (MHC), on a swine MHC deficient background, would reduce both antibody- and cellular-mediated rejection.
Methods: Two CRISPR gRNA plasmids were designed to remove the entire coding sequence of the swine class I MHC and co-transfected with a third plasmid containing a promoterless Hygromycin resistance gene surrounded by PhiC31 recombinase sequences, followed with a promoterless human class I MHC cDNA. The response of MHC-matched individuals to the human MHC expressing swine cell was evaluated in a flow cytometry crossmatch (FCXM), 24-hour IFN-y ELISPOT assay, and a mixed lymphocyte reaction (MLR) to measure an immediate, recall, and delayed response.
Results: A cell line expressing human class I MHC was successfully generated using the described approach. Individuals with no preformed to the class I MHC chosen in this experimental model demonstrated significantly less IgG antibody binding to the human MHC positive swine cell compared to the MHC deficient parent line (one-way ANOVA, p < 0.0001). The ELISPOT revealed significantly more IFN-y release for both MHC-matched and non-MHC-matched individuals in response to the human MHC positive swine cell line (paired two-tailed Wilcoxon test, p = 0.0078 and 0.0156 respectively). A human MHC expressing swine cell elicited less, but not significant, proliferation in the MLR assay compared to the swine MHC expressing cell for MHC-matched but significantly less for non-MHC-matched individuals (Figure 1 unpaired, two-tailed Wilcoxon test p = 0.1250 and 0.0312 respectively).
Conclusion: The development of the CRISPR/Cas9 system allows for complex genetic engineering strategies to be achieved rapidly. This study demonstrates that expression of human MHC on a MHC devoid swine cell can reduce the humoral and cellular response for MHC-matched individuals, but may result in a higher recall response as measured by IFN-y production.
Figure 1: Results of the proliferation in the CFSE-based MLR of HLA-A2 positive (Figure 1A) and HLA-A2 negative (Figure 1B) PBMC responders to the HLA-A2+ AEC (Lane 1) and SLA class I positive AEC (Lane 2). An unpaired, two-tailed Wilcoxon test for the four HLA-A2 positive responders found no statistical significance between the HLA-A2 positive AEC vs the SLA class I positive AEC (p = 0.1250). For the six HLA-A2 negative responders, statistical difference was found between the HLA-A2 positive AEC vs the SLA class I positive AEC (*, p = 0.0312).
