Elimination of GGTA1, CMAH, β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

Abstract

Background: Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic, while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs. Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection (HAR) that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure, in which process the MHC II molecule plays critical roles.

Methods: Thus, we generate a 4-gene (GGTA1, CMAH, β4GalNT2, and CIITA) knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously.

Results: We successfully obtained 4KO piglets with deficiency in all alleles of genes, and at cellular and tissue levels. Additionally, the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping. Piglets have survived for more than one year in the barrier, and also survived for more than 3 months in the conventional environment, suggesting that the piglets without MHC II can be raised in the barrier and then gradually mated in the conventional environment.

Conclusions: 4KO piglets have lower immunogenicity, are safe in genomic level, and are easier to breed than the model with both MHC I and II deletion.

Keywords: CD4 T cell; genetically edited pig; immune rejection; major histocompatibility complex II; xenotransplantation.

FIGURE 1

Construction and evaluation of 4KO piglets in xenotransplantation. (A) Schematic diagram of method for generating 4KO piglets. (B) Schematic of the 10 modified alleles. We generated the 4KO edits using CRISPR–Cas9 with gRNAs targeting the 2 copies of GGTA1, 2 copies of CMAH, 4 copies of β4GalNT2 and 2 copies of the CIITA. (C) Deficiency of GGTA1, CMAH, β4GalNT2, and CIITA can impair the antigens of αGal, Neu5Gc, SDa, and SLA class II molecules, respectively. Flow cytometry was used to verify the deficiency of αGal, Neu5Gc, SDa, and SLA class II‐DR molecules at the cellular level in 4KO piglets. PBMCs of 4KO pigs had negative population cells (WT = 3, 4KO = 3). (D) Immunofluorescence results showed that αGal, Neu5Gc, SDa, and SLA class II molecules were not expressed in kidney tissue of 4KO piglets, and GGTA1, CMAH, β4GalNT2, and CIITA are genes were used to knockout the antigen of αGal, Neu5Gc, SDa and SLA class II molecules, respectively. Scale bar: 20 μm. (E) Verification by HE staining of the viability of 4KO tissue after gene editing; Scale bar: 100 μm.

FIGURE 2

Changes in the pig immune system induced by CIITA deficiency (WT = 3, 4KO = 3): (A) Percentage of the population of CD3 in WT and 4KO. (B) Percentage of the population of γδT in WT and 4KO. (C) 4KO CD3 T cells are not significantly different from WT. (D) 4KO γδT are not significantly different from WT. (E) Percentage of the population of CD4+ T and CD8+ T in WT and 4KO. (F) 4KO CD4+ T cells significantly decreased relative to WT; 4KO CD8+ T cells significantly increased relative to WT. (G) B cell population of WT and 4KO PBMCs.(n = 3). (H) No significant difference between WT and 4KO in the population of B cells. (I) Percentage of 4KO NK cell population significantly decreased relative to WT. (J) NK cell population of WT and 4KO PBMCs. **p < 0.01; *p < 0.05.

FIGURE 3

(A) Verifying the function of CIITA deficiency, T cell proliferation was analyzed based on dilution of the proliferation dye CSFE with each cell division, Proliferation of human CD4+ T cells is depicted as percent of divided cell (WT = 3; 4KO = 3). (B) 4KO has lower immunogenicity to lead human CD4+ T proliferation than WT (WT = 3; 4KO = 3). (C) Verifying the Serum‐mediated antibody binding reaction of WT and 4KO, compared with WT, 4KO show significantly reduced binding to human IgG (a) and IgM (WT = 8; 4KO = 8). (D) Verifying the Human complement‐dependent cytotoxicity of WT and 4KO, 4KO show significantly lower antibody‐dependent complement cytotoxicity compared with WT (WT = 5; 4KO = 5). Unpaired two‐tailed t‐test; **p < 0.01; *p < 0.05).