Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes.
BACKGROUND
Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes.


METHODS
The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons.


RESULTS
Cells from GGTA1/CMAH/β4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Non-human primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased non-human primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/β4GalNT2.


CONCLUSIONS
Inactivation of the β4GalNT2 gene reduces human and non-human primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of non-human primates toward GGTA1-/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/β4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the non-human primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/β4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
Jose L
JL
Estrada
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
Greg
G
Martens
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
Ping
P
Li
Yerkes National Primate Research Center, Atlanta, GA, USA.|Emory Transplant Center and Department of Surgery, Emory University, Atlanta, GA, USA.
Andrew
A
Adams
Emory Transplant Center and Department of Surgery, Emory University, Atlanta, GA, USA.
Kenneth A
KA
Newell
Emory Transplant Center and Department of Surgery, Emory University, Atlanta, GA, USA.
Mandy L
ML
Ford
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
James R
JR
Butler
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
Richard
R
Sidner
Department of Surgery, Indiana University Health, Indianapolis, IN, USA.
Matt
M
Tector
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.|IU Health Transplant Institute, Indianapolis, IN, USA.
Joseph
J
Tector
D000818:Animals; D030801:Animals, Genetically Modified; D015478:Antigens, Heterophile / Q000235:genetics / Q000276:immunology*; D005700:Galactosyltransferases / Q000235:genetics*; D006084:Graft Rejection / Q000235:genetics / Q000276:immunology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014183:Transplantation, Heterologous
D015478:Antigens, Heterophile; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine
CRISPR; Cas9; antibody; genetic engineering; primate; swine; xenoantigen; β4GalNT2