We have previously reported that co-transplantation of the kidney with vascularized donor thymus from α-1,3-galactosyltransferase gene knockout pigs with an anti-CD154 with rituximab-based regimen led to improved xenograft survival in baboons with donor-specific unresponsiveness. However, nephrotic syndrome emerged as a complication in which the glomeruli showed mild mesangial expansion with similarities to minimal change disease (MCD) in humans. Since MCD is associated with CD80 expression in glomeruli and elevated urinary excretion, we evaluated a potential role for CD80 in xenograft nephropathy. Study 1 confirmed high urinary CD80 excretion in nephrotic animals with renal xenografts showing CD80 expression in glomeruli. In Study 2, baboons receiving xenografts received CTLA4-Ig once a week from the second postoperative week or no CTLA4-Ig. The non-CTLA4-Ig group developed severe proteinuria with modest mesangial expansion with high urinary excretion of CD80 and documented CD80 expression in glomerular podocytes. All of the recipients in non-CTLA4-Ig groups had to be euthanized before POD 60. In contrast, CTLA4-Ig group showed a marked reduction in proteinuria and survived significantly longer, up to 193 days. These results demonstrate that anti-CD80 targeted therapy represents a promising strategy for reduction of proteinuria following renal xeno-transplantation with improved survival.

Background We have recently demonstrated that human‐CD47 (hCD47) expressed on endothelial cells of porcine lung xenografts extended median graft survival from 3.5 days to 8.7 days in baboons. Intra‐bone bone marrow transplantation (IBBMTx) in a pig‐to‐baboon model was previously shown to markedly prolong the duration of macrochimerism up to 21 days from 1 to 4 days by intravenous BMTx. We now examined whether the use of hCD47 transgenic (Tg) BM further prolonged the duration of chimerism following IBBMTx. We then tested if lung xenograft survival was prolonged following IBBMTx. Methods Baboons received GalTKO‐hCD47/hCD55Tg (n = 5) or ‐hCD55Tg (n = 1) or ‐hCD46/HLA‐E Tg (n = 1) pig IBBMTx. Macrochimerism, anti‐pig T cells and antibody responses were assessed. Animals received lung xenografts from either hCD47+ or hCD47‐ porcine lungs 1‐3 months later. Results All baboons that received hCD47Tg porcine IBBM maintained durable macrochimerism >30 days, and two maintained chimerism for >8 weeks. Notably, anti‐pig antibody levels decreased over time and anti‐pig cellular unresponsiveness developed following IBBMTx. Lungs from hCD47Tg IBBMTx matched pigs were transplanted at day 33 or day 49 after IBBMTx. These animals showed extended survival up to 13 and 14 days, while animals that received lungs from hCD47 negative pigs displayed no prolonged survival (1‐4 days). Conclusion This is the first report demonstrating durable macrochimerism beyond 8 weeks, as well as evidence for B cell tolerance in large animal xenotransplantation. Using hCD47Tg pigs as both IBBMTx and lung donors prolongs lung xenograft survival. However, additional strategies are required to control the acute loss of lung xenografts.

Background Our initial studies utilizing a galactosyl‐α1‐3‐galactosyltransferase gene knockout (GalTKO) pig‐to‐baboon renal transplant model demonstrated that the early development of nephrotic syndrome has been a significant obstacle to the long‐term survival of baboon recipients. We have recently documented that sphingomyelin phosphodiesterase‐3 (SMPDL3b) and CD80 expressed on podocytes in porcine kidney grafts contribute to this complication. We have hypothesized that one regulator of immune function is CD47 and that incompatibilities in CD47 between pig and baboon could potentially affect macrophage function, increasing the susceptibility of the kidney grafts to immunologically induced injury. Methods In order to address this hypothesis in vitro, we isolated and cultured porcine podocytes and ECs from GalTKO alone, human CD47 (hCD47)/hCD55 expressing transgenic (Tg) GalTKO swine, and GalTKO hCD46/hCD55 Tg swine along with baboon or human macrophages. Results We found that baboon macrophages phagocytosed porcine ECs in a similar manner to human macrophages, and this response was significantly reduced when porcine ECs and podocytes expressed hCD47/hCD55 but not hCD46/hCD55 without hCD47. Furthermore, masking hCD47 by anti‐hCD47 antibody on hCD47/hCD55Tg ECs restored phagocytosis. These results are consistent with the hypothesis that CD47 incompatibility plays an important role in promoting macrophage phagocytosis of endogenous cells from the transplanted kidney. Conclusions The similar levels of phagocytosis of porcine cells by baboon and human macrophages suggest that the expression of hCD47Tg on glomerular cells in donor porcine kidneys may prove to be a key strategy for preventing proteinuria following kidney xenotransplantation in a pig‐to‐human as well as a pig‐to‐baboon model.

Background Nephrotic syndrome is a common complication of pig‐to‐baboon kidney xenotransplantation (KXTx) that adversely affects outcomes. We have reported that upregulation of CD80 and down‐regulation of SMPDL‐3b in glomeruli have an important role in the development of proteinuria following pig‐to‐baboon KXTx. Recently we found induced expression of human CD47 (hCD47) on endothelial cells and podocytes isolated from hCD47 transgenic (Tg) swine markedly reduced phagocytosis by baboon and human macrophages. These observations led us to hypothesize that transplanting hCD47 Tg porcine kidneys could overcome the incompatibility of the porcine CD47‐baboon SIRPα interspecies ligand‐receptor interaction and prevent the development of proteinuria following KXTx. Methods Ten baboons received pig kidneys with vascularized thymic grafts (n = 8) or intra‐bone bone marrow transplants (n = 2). Baboons were divided into three groups (A, B, and C) based on the transgenic expression of hCD47 in GalT‐KO pigs. Baboons in Group A received kidney grafts with expression of hCD47 restricted to glomerular cells (n = 2). Baboons in Group B received kidney grafts with high expression of hCD47 on both glomerular and tubular cells of the kidneys (n = 4). Baboons in Group C received kidney grafts with low/no glomerular expression of hCD47, and high expression of hCD47 on renal tubular cells (n = 4). Results Consistent with this hypothesis, GalT‐KO/hCD47 kidney grafts with high expression of hCD47 on glomerular cells developed minimal proteinuria. However, high hCD47 expression in all renal cells including renal tubular cells induced an apparent destructive inflammatory response associated with upregulated thrombospondin‐1. This response could be avoided by a short course of weekly anti‐IL6R antibody administration, resulting in prolonged survival without proteinuria (mean 170.5 days from 47.8 days). Conclusion Data showed that transgenic expression of hCD47 on glomerular cells in the GalT‐KO donor kidneys can prevent xenograft nephropathy, a significant barrier for therapeutic applications of xenotransplantation. The ability to prevent nephrotic syndrome following KXTx overcomes a critical barrier for future clinical applications of KXTx.

Background Recent advances in gene editing technology have enabled the production of multi‐knockout (KO) and transgenic pigs in order to overcome immunologic barriers in xenotransplantation (XTx). However, the genetic manipulations required to produce these changes may have the unintended consequence of producing or revealing neoantigens reactive with natural antibodies present in baboons. In this study, we examined whether the neoantigens that develop in multi‐transgenic (mTg) GalT, Cytidine monophospho‐N‐acetylneuraminic acid hydroxylase (CMAH), β‐1,4‐N‐acetyl‐galactosaminyl transferase 2 (B4) KO pigs can cause rejection of xenografts in baboons. Methods Five baboons that had <35% cytotoxicity against GalT‐KO peripheral blood mononuclear cells (PBMCs) in a pre‐screening assay received pig kidneys and vascularized thymic grafts (VT + K) from multi‐transgenic hCD47, human thrombomodulin (hTBM), human endothelial protein C receptor (EPCR) with/without hCD46 and hCD55 with GalT‐KO/NeuGC‐KO/B4‐KO (mTg Tri‐KO) swine. In order to further examine the effects of anti‐donor non‐Gal natural antibody (nAb), anti‐pig preformed IgM and IgG nAb binding against the GalT‐KO PBMCs was compared with the donor‐type PBMCs using donor pretransplant sera as well as 5 additional naïve baboon sera by flow cytometric analysis. Results Five baboons that received VT + K grafts had stable renal function in the first 11 days (serum creatinine < 1.5 mg/dL). Two of the five baboons had higher binding of preformed IgG to mTg Tri‐KO PBMCs than to GalT‐KO PBMCs (mTg Tri‐KO > GalT‐KO), and they rejected their grafts at POD 20. In contrast, the other three baboons demonstrated either mTg Tri‐KO = GalT‐KO or mTg Tri‐KO < GalT‐KO, and they maintained renal function 43, 52, and 154 days without rejection. Among 10 baboon sera, two had less antibody binding against PBMCs that were syngeneic to the mTg Tri‐KO than against GalT‐KO PBMCs (mTg Tri‐KO < GalT‐KO); three had similar binding to mTg Tri‐KO and GalT‐KO PBMCs (mTg Tri‐KO = GalT‐KO); and five had higher binding to m Tg Tri‐KO than to GalT‐KO PBMCs (mTg Tri‐KO > GalT‐KO). Conclusions These data suggest that neoantigens associated with mTg Tri‐KO promote acute xenograft rejection in a pig‐to‐baboon VT + K XTx model. The screening assays may be useful to select “safe” recipients to receive mTg Tri‐KO kidneys.