HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses

Abstract

The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1-/HLAG1+ pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1+ pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger’s sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous β2-microglobulin (β2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4+ and CD8+ T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1+ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1+ transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1+ may extend survival of porcine pancreatic islet and organ xenografts.

Genetic engineering of pigs for xenotransplantation to overcome immune rejection and physiological incompatibilities: The first clinical steps

“…To inhibit direct NK cell cytotoxicity, Weiss et al generated hHLA-E/hB2M transgenic pigs and found that pig endothelium derived from these animals was protected against human NK cytotoxicity and inhibited NKsecreted IFN-g (24). Rao et al inserted an HLA-G1 transgene, encoding a non-classical MHC-I protein, to the porcine ROSA26 locus, and created GGTA1 -/-/hHLA-G1 pigs (25). In fibroblasts, transplantable organs, and islets, the positive expression of HLA-G1 plays a central role in immune suppression by lowering IFN-g production via T cells and proliferation of CD4 + and CD8 + T cells, B cells, and NK cells (25).…”

“…Rao et al inserted an HLA-G1 transgene, encoding a non-classical MHC-I protein, to the porcine ROSA26 locus, and created GGTA1 -/-/hHLA-G1 pigs (25). In fibroblasts, transplantable organs, and islets, the positive expression of HLA-G1 plays a central role in immune suppression by lowering IFN-g production via T cells and proliferation of CD4 + and CD8 + T cells, B cells, and NK cells (25). To reduce xenoantigen expression and thus reduce the recipient's immune response, Fu et al generated GGTA1 -/-/B2M -/-/major histocompatibility complex class II transactivator (CIITA -/-) triple-knockout pigs, named GBC-21, by CRISPR/Cas technology and found that the resulting elimination of swine leukocyte antigen class I could effectively alleviate xenogeneic immune responses and prolong pig organ survival (26).…”

“…inserted an HLA-G1 transgene, encoding a non-classical MHC-I protein, to the porcine ROSA26 locus, and created GGTA1 -/- / hHLA-G1 pigs ( 25 ). In fibroblasts, transplantable organs, and islets, the positive expression of HLA-G1 plays a central role in immune suppression by lowering IFN-γ production via T cells and proliferation of CD4 + and CD8 + T cells, B cells, and NK cells ( 25 ). To reduce xenoantigen expression and thus reduce the recipient’s immune response, Fu et al.…”

Cellular Immune Responses in Islet Xenograft Rejection

“…This includes the expression of Human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein that pays an important role in the maintenance of maternal-fetal tolerance. GKTO/HLAG1+ pigs have been developed and used as islet cell donors with the objective of extending xenograft survival and function in both preclinical NHP models and future clinical trials ( 128 ). GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4 + and CD8 + T cells, B cells and NK cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression.…”

“…GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4 + and CD8 + T cells, B cells and NK cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1+ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes ( 128 ). Using CRISPR/Cas9, it is possible to target the cellular immune system at multiple levels.…”