Porcine SIRPA binds to human CD47 to inhibit phagocytosis: Implications for human hematopoietic stem cell transplantation into severe combined immunodeficient (SCID) pigs

Authors: BOETTCHER, ADELINE - Iowa State University; CUNNICK, JOAN - Iowa State University; POWELL, ELLIS - Orise Fellow; EGNER, TIMOTHY - Iowa State University; CHARLEY, SARA - Iowa State University; Loving, Crystal; TUGGLE, CHRISTOPHER - Iowa State University

Submitted to: Xenotransplantation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/18/2018
Publication Date: 10/12/2018
Citation: Boettcher, A.N., Cunnick, J.E., Powell, E., Egner, T.K., Charley, S.E., Loving, C.L., Tuggle, C.K. 2018. Porcine signal regulatory protein alpha binds to human CD47 to inhibit phagocytosis: Implications for human hematopoietic stem cell transplantation into severe combined immunodeficient pigs. Xenotransplantation. 26(2):e12466. https://doi.org/10.1111/xen.12466.
DOI: https://doi.org/10.1111/xen.12466

Interpretive Summary: Pigs are often used as an animal model for human research needs, including transplantation studies. Pigs without an immune system, due to a mutation in a gene involved in development of the immune system, serve as a valuable model for research. In order to put a human immune system into pigs, it is important to show that human cells transplanted into a pig will not be recognized by pigs cells and then destroyed. Cells from immune-deficient pigs were mixed with cells from mice, pigs, or people to determine if pig cells destroyed human cells. Research findings indicate that human cells look enough like pig cells, and thus, the pigs cells do not interpret the human cells as foreign and do not actively destroy the human cells. A specific interaction between a protein on human cells and a protein on pig cells was required to prevent the destruction of human cells, and is a well characterized interaction between immune cells and other cells of the body. The findings are important for researchers using immune-deficient pigs for studies relevant to pig disease, but also for research requiring the transfer of human immune cells into immune-deficient pigs.

Technical Abstract: Background Severe combined immunodeficient (SCID) pigs are an emerging animal model being developed for biomedical and regenerative medicine research. SCID pigs can successfully engraft human induced pluripotent stem cells and cancer cell lines. The development of a humanized SCID pig through xenotransplantation of human hematopoietic stem cells (HSCs) would be a further demonstration of the value of such a large animal SCID model. Xenotransplantation success with HSCs into non-obese diabetic (NOD)-derived SCID mice is dependent on the ability of NOD mouse signal regulatory protein alpha (SIRPA) to bind human CD47, inducing higher phagocytic tolerance than other mouse strains. Therefore, we investigated whether porcine SIRPA binds human CD47 in the context of developing a humanized SCID pig. Methods Peripheral blood mononuclear cells (PBMCs) were collected from SCID and non-SCID pigs. Flow cytometry was used to assess if porcine monocytes could bind to human CD47. Porcine monocytes were isolated from PBMCs and were subjected to phagocytosis assays with pig, human, and mouse red blood cell (RBC) targets. Blocking phagocytosis assays were performed by incubating human RBCs with anti-human CD47 blocking antibody B6H12 and non-blocking antibody 2D3 and exposing to human or porcine monocytes. Results We found that porcine SIRPA binds to human CD47 in vitro by flow cytometric assays. Additionally, phagocytosis assays were performed and we found that porcine monocytes phagocytose human and porcine RBCs at significantly lower levels than mouse RBCs. When human RBCs were pre-incubated with CD47 antibodies B6H12 or 2D3, phagocytosis was induced only after B6H12 incubation, indicating the lower phagocytic activity in porcine monocytes requires interaction between porcine SIRPA and human CD47. Conclusions We have shown the first evidence that porcine monocytes can bind to human CD47 and are phagocytically tolerant to human cells, suggesting that porcine SCID models have the potential to support engraftment of human HSCs.