In vitro and in vivo immune assessments of TKO pig skin grafts in New World (squirrel) monkeys
Hidetaka Hara1,2, Jeremy B Foote3, Christophe Hansen-Estruch1, Mohamed H Bikhet1, Huy Q Nguyen1, Mariyam Javed1, Max Oscherwitz1, Dalis E Collins4, David Ayares5, Takayuki Yamamoto1,6,7, Timothy W King1,8, David KC Cooper1,6.
1Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, United States; 2College of Veterinary Medicine, Yunnan Agricultural University, Kunming, People's Republic of China; 3Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, United States; 4Animal Resources Program, University of Alabama at Birmingham, Birmingham, AL, United States; 5Revivicor Inc., Blacksburg, VA, United States; 6Center for Transplantation Sciences, Massachusetts General Hospital/Harvard Medical School, Boston, MA, United States; 7Department of Transplant Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, MA, United States; 8Department of Plastic and Reconstructive Surgery, Loyola University, Chicago, IL, United States
Introduction: Following extensive burns, providing sufficient autografts may not be possible. Gene-edited pigs may be alternative sources of skin for temporary coverage of burns in human patients. Many humans have minimal or no natural antibodies to triple-knockout (TKO) pig cells and so TKO pigs might be suitable sources of skin. However, all Old World nonhuman primates (e.g., baboons) have natural cytotoxic antibodies to TKO pig cells and, therefore, are not optimal recipients of TKO organs or skin. In contrast, in New World monkeys (e.g., squirrel monkeys), serum antibody binding to, and cytotoxicity of, TKO pig cells are similar to those of humans. Therefore, New World monkeys are preferred surrogates for humans as recipients of pig skin grafts.
Methods: We compared the survival of skin grafts from pigs with 9 genetic manipulations (TKO with 6 further gene edits) with autografts and allografts in squirrel monkeys. The additional effect of applying topical tacrolimus ointment to the pig grafts was assessed. Monitoring for rejection was by (i) macroscopic examination, (ii) histopathological examination of skin biopsies, and (iii) measurement of anti-monkey and anti-pig IgM and IgG antibodies.
Results: Autografts (n=5) survived throughout the 28 days of follow-up without histopathological features of rejection. The median survival of allografts (n=6) was 14 days, and of pig xenografts (n=12) was 21 days (not significantly different), irrespective of topical tacrolimus treatment. Allotransplantation was associated with an increase in anti-monkey IgM, but the anticipated subsequent rise in IgG had not yet occurred at the time of euthanasia. Pig grafts were associated with increases in anti-pig IgM and IgG, but these did not cross-react with monkey antigens. In all cases, histopathologic features of rejection were similar, with loss of epithelial viability leading to ulceration, fibrosis, and inflammation. The numbers of infiltrating CD3+T cells were similar in allografts and xenografts. Tacrolimus-untreated pig skin xenografts exhibited the highest numbers of IgG+B cells compared to untreated allografts and tacrolimus-treated xenografts.
Conclusions: Skin grafts from 9-gene pigs survive at least as long as monkey skin allografts (and trended to survive longer), suggesting that they are a realistic clinical option for the temporary treatment of burns. Although monkeys with pig skin grafts developed anti-pig IgM and IgG antibodies, these did not cross-react with monkey antigens, indicating that sensitization to pig antigens would not be detrimental to a subsequent skin allograft.
This study was supported by Department of Defense grant WB1XWH-20-1-0559 and in part by NIH NIAID U19 grant AI090959. The squirrel monkeys in our studies were from the Michale E. Keeling Center, MD Anderson Cancer Center, Bastrop, TX, which is supported by NIH grant P40 OD24628-01..
