Xenogeneic Rejection Research Articles

The hybrid CL-SP-D molecule has the potential to regulate xenogeneic rejection by human neutrophils more efficiently than CD47

ObjectiveInnate immunity plays a vital role in xenotransplantation. A CD47 molecule, binding to the SIRPα expressed on monocyte/macrophage cells, can suppress cytotoxicity. Particularly, the SIRPα contains ITIM, which delivers a negative signal. Our previous study demonstrated that the binding between CL-P1 and surfactant protein-D hybrid (CL-SP-D) with SIRPα regulates macrophages' phagocytic activity. In this study, we examined the effects of human CD47 and CL-SP-D expression on the inhibition of xenograft rejection by neutrophils in swine endothelial cells (SECs). MethodsWe first examined SIRPα expression on HL-60 cells, a neutrophil-like cell line, and neutrophils isolated from peripheral blood. CD47-expressing SECs or CL-SP-D-expressing SECs were generated through plasmid transfection. Subsequently, these SECs were co-cultured with HL-60 cells or neutrophils. After co-culture, the degree of cytotoxicity was calculated using the WST-8 assay. The suppressive function of CL-SP-D on neutrophils was subsequently examined, and the results were compared with those of CD47 using naïve SECs as controls. Additionally, we assessed ROS production and neutrophil NETosis. ResultsIn initial experiments, the expression of SIRPα on HL-60 and neutrophils was confirmed. Exposure to CL-SP-D significantly suppressed the cytotoxicity in HL-60 (p = 0.0038) and neutrophils (p = 0.00003). Furthermore, engagement with CD47 showed a suppressive effect on neutrophils obtained from peripheral blood (p = 0.0236) but not on HL-60 (p = 0.4244). The results of the ROS assays also indicated a significant downregulation of SEC by CD47 (p = 0.0077) or CL-SP-D (p = 0.0018). Additionally, the suppression of NETosis was confirmed (p = 0.0125) in neutrophils co-cultured with S/CL-SP-D. ConclusionThese results indicate that CL-SP-D is highly effective on neutrophils in xenogeneic rejection. Furthermore, CL-SP-D was more effective than CD47 at inhibiting neutrophil-mediated xenograft rejection.

216.5: The hybrid molecule CL-SP-D has the potential to regulate xenogeneic rejection by human neutrophils

216.5: The hybrid molecule CL-SP-D has the potential to regulate xenogeneic rejection by human neutrophils

Advances in Innate Immunity to Overcome Immune Rejection during Xenotransplantation

Transplantation is an effective approach for treating end-stage organ failure. There has been a long-standing interest in xenotransplantation as a means of increasing the number of available organs. In the past decade, there has been tremendous progress in xenotransplantation accelerated by the development of rapid gene-editing tools and immunosuppressive therapy. Recently, the heart and kidney from pigs were transplanted into the recipients, which suggests that xenotransplantation has entered a new era. The genetic discrepancy and molecular incompatibility between pigs and primates results in barriers to xenotransplantation. An increasing body of evidence suggests that innate immune responses play an important role in all aspects of the xenogeneic rejection. Simultaneously, the role of important cellular components like macrophages, natural killer (NK) cells, and neutrophils, suggests that the innate immune response in the xenogeneic rejection should not be underestimated. Here, we summarize the current knowledge about the innate immune system in xenotransplantation and highlight the key issues for future investigations. A better understanding of the innate immune responses in xenotransplantation may help to control the xenograft rejection and design optimal combination therapies.

P17.06: Escape From Macrophage-Mediated Rejection by Human Surfactant Protein (SP)-A

Introduction: Macrophage-mediated xenogeneic rejection is one of the important immunological obstructions that need to be overcome. Because macrophage is one of the main sources of proinflammatory cytokines and the fact that proinflammatory cytokines orchestrate a variety of inflammatory responses, the regulation of macrophages could result in solving the problem of xenogeneic rejection. We recently reported that membrane-type surfactant D (SP-D) on swine endothelial cells (SECs) suppresses macrophage-mediated rejection. Similar to SP-D, the carbohydrate recognition domain of surfactant protein-A (SP-A) induces inhibitory signals in effector cells. In this study, we examined the suppressive effect of SP-A on macrophage-mediated xenogeneic rejection. Methods: Naïve SEC and SP-A-transfected SEC (SEC/SP-A) were co-cultured with THP-1 cells and cytotoxicity was evaluated. To investigate the effect on phagocytosis, human macrophages were co-cultured with SEC or SEC/SP-A, and the extent of phagocytosis and production of reactive oxygen species (ROS) were assessed by flow cytometry. The mRNA expression of inflammatory cytokines in macrophages was determined using RT-PCR. In addition, THP-1 cell with a luciferase reporter gene driven by an NF-kB response element (THP-1 Luc NF-kB) cells was used to verify their effects on the NF-κB transcription factors. Results: The cytotoxicity of SEC/SP-A was significantly reduced compared to those of naïve SEC (40.7 vs 22.9%, p=0.025). The phagocytosis by macrophages against SEC was also significantly suppressed by SP-A on SEC (75.8 vs 33.0%, n=5, p=0.0010). Co-culture of human macrophages with SEC/SP-A decreased ROS production (%MFI: 99.9 vs 79.5, p=0.013). The mRNA expression of TNFα was reduced in macrophages (1.160 vs 0.009, p=0.0022), whereas that of IL-10 was increased (1.357 vs 10.20, p=0.0007). The balance between iNOS and Arg-1 was significantly suppressed by CL-SPA (1.002 vs 0.4792, p = 0.0027), indicating that CL-SPA on porcine cells inhibits the differentiation of peripheral blood monocytes into inflammatory M1 macrophages. The NF-κB transcription was decreased in THP-1 Luc NF-kB which was co-cultured with SEC/SP-A compared to that in THP-1 Luc NF-kB with SEC (RLU: 1160 vs 983.8, p=0.0258). Conclusion: The ectopic expression of human SP-A in porcine cells represents an attractive method for suppressing macrophage-mediated cytotoxicity. To further investigate the effects of SP-A on xenogeneic innate immune response in more detail, in vivo studies should be performed in the future.

Suppression of xenogeneic innate immune response by a membrane-type human surfactant protein-A.

Macrophage-mediated xenogeneic rejection is a major immunological obstacle. We recently reported that membrane-type surfactant protein-D (SP-D) on swine endothelial cells (SECs) suppressed macrophage-mediated rejection. Similar to SP-D, the carbohydrate recognition domain of surfactant protein-A (SP-A) can induce inhibitory signals in effector cells. The present study aimed to examine the suppressive effect of SP-A on macrophage-mediated xenogeneic rejection. Naive SECs and SPA-transfected SECs (SEC/SP-A) were co-cultured with THP-1 cells and cytotoxicity was evaluated. To investigate the effect of SP-A on phagocytosis, human macrophages were co-cultured with SEC or SEC/SP-A, and the extent of phagocytosis and production of reactive oxygen species were assessed via flow cytometry. The mRNA expression levels of inflammatory cytokines in macrophages were determined using reverse transcription-PCR. Additionally, the effects of THP-1-Lucia NF-κB cells on transcription factors were evaluated. The cytotoxicity and phagocytosis of SEC/SP-A were significantly decreased compared with those of naive SEC. Furthermore, the co-culture of human macrophages with SEC/SP-A decreased reactive oxygen species production, and the mRNA expression levels of TNFα were decreased in macrophages, whereas those of IL-10 were increased. In addition, NF-κB transcription was decreased in SEC/SP-A compared with that in SEC. In conclusion, the ectopic expression of human SP-A in porcine cells represents an attractive method for suppressing macrophage-mediated cytotoxicity.

Suppression of macrophage-mediated xenogeneic rejection by the ectopic expression of human CD177

Cellular xenogeneic rejection by the innate immune system is a major immunological obstruction that needs to be overcome for the successful clinical use of xenografts. Our focus has been on macrophage-mediated xenogeneic rejection, since suppressing macrophage function has considerable potential for practical applications in the area of xenotransplantation. We report herein on an investigation of the suppressive effect of human CD177 (hCD177) against macrophage-mediated xenogeneic rejection. Wild type swine aortic endothelial cell (SEC) and an SEC transfectant with hCD177 (SEC/hCD177) were co-cultured with macrophages, and the degree of cytotoxicity was evaluated by WST-8 assays, and phagocytosis was examined using Calcein-AM labeling methods. The expression of anti/pro-inflammatory cytokines was evaluated by RT-qPCR and the phosphorylation of SHP-1 on macrophages in co-culture was evaluated by Western blotting. The result of cytotoxicity assays indicated that hCD177 suppressed M1 macrophage-mediated xenogeneic rejection (vs. SEC, p < 0.0001). Similarly, the result of phagocytosis assays indicated that hCD177 suppressed it (vs. SEC, p < 0.05). In addition, hCD177 significantly suppressed the expression of IL-1β, a pro-inflammatory cytokine, in M1 macrophages (vs. SEC, p < 0.01). Luciferase assays using THP1-Lucia NF-kB also showed a significant difference in NF-kB activation (vs. SEC, p < 0.001). In addition, hCD177 was found to induce the phosphorylation of SHP-1 in M1 macrophages (vs. SEC, p < 0.05). These findings indicate that hCD177 suppresses M1 macrophage-mediated xenogeneic rejection, at least in part via in the phosphorylation of SHP-1.

FC041: Regeneration of Nephrons and Renal Stroma using the Renal Developmental Environment of Animal Fetuses

Abstract BACKGROUND AND AIMS The kidney is derived from three types of progenitor cells: nephron progenitor cells (NPCs), stromal progenitor cells (SPCs) and ureteric buds (UBs). Extensive research in regenerative medicine has made it possible to induce pluripotent stem cells (PSCs) to develop into NPCs and UBs in vitro and generate the organoids of nephrons and collecting ducts. Renal stroma, including mesangial cells and erythropoietin- and renin-producing cells, is essential for maintaining biological functions. However, to our knowledge, the induction of SPC differentiation from PSCs has not been established yet. Moreover, the renal stroma cannot mature in the in vitro environment, making it difficult to generate the whole kidney, including the renal stroma. In this study, we attempted to regenerate the renal stroma in the in vivo environment by transplanting rat SPCs into the nephrogenic zone of mouse fetuses to provide rat SPCs with the renal developmental mechanism of heterologous mouse fetuses. Furthermore, we developed an interspecies dual replacement of progenitor (i-DROP) system wherein two progenitor cell lines, NPCs and SPCs, were genetically modified and replaced with transplanted progenitor cells and examined the feasibility of the regeneration of kidneys, including nephrons and renal stroma. METHOD We crossed Foxd1GC/+ mice with R26RDTR/DTR mice to generate Foxd1GC/+; R26RDTR/+ mice (Foxd–DTR mice) can eliminate Foxd1-positive SPCs. A group of SPC-rich cells extracted from green fluorescent protein (GFP) rat metanephros targeting platelet-derived growth factor receptor alpha (PDGFRa) was transplanted into the kidneys of Foxd1–DTR mouse fetuses. Subsequently, the organ-cultured metanephros or the transplanted metanephros in immunodeficient NOD/Shi- scid IL2rγnull (NOG) mice or Sprague–Dawley (SD) rats were evaluated using immunostaining. Next, we assessed whether the i-DROP system could regenerate interspecies nephrons and stroma. We crossed Six2TGC/+; Foxd1GC/+ mice obtained from Six2TGC/+ mice and Foxd1GC/+ mice with R26RDTR/DTR mice to generate Six2TGC/+; Foxd1GC/+; and R26RDTR/+ mice (Six2/Foxd1–DTR mice) that can simultaneously eliminate Six2-positive NPCs and Foxd1-positive SPCs. The SPCs targeted to PDGFRa+ and NPCs targeted to PDGFRa−/integrin alpha 8 (Itga8)+ from GFP rat metanephros were simultaneously transferred to Six2/Foxd1–DTR mouse metanephros and evaluated similarly. RESULTS In the organ culture of Foxd1–DTR mouse metanephros, SPCs around the cap mesenchyme replaced from mouse to GFP rat. We achieved extensive rat stromal cell regeneration in mouse kidneys, including endocrine functions such as erythropoietin production in the rat in response to anemia in the host mouse. Next, in the organ culture of the hind kidney of Six2/Foxd1–DTR mice equipped with the i-DROP system, the NPCs and SPCs of GFP rats aggregated around the mouse UB and the cap mesenchyme reconstituted. In vivo, rat nephrons and renal stroma were regenerated simultaneously in the mouse kidneys. The newly generated glomeruli were composed of GFP-positive rat cells, both podocytes and mesangial cells of different lineages (Figure 1). Intravenous injection of fluorescently labeled dextran into host mice revealed that the regenerated nephrons had glomerular filtration and tubular reabsorption capabilities. Furthermore, taking the xenotransplantation of mouse metanephros into SD rats as a control, the transplantation of regenerated rat kidneys into SD rats using our method resulted in reduced xenogenic immune rejection. CONCLUSION The newly developed i-DROP system regenerated multifunctional kidneys, composed of nephrons and renal stroma originating from different species, with urine production and endocrine functions. Functional human kidneys could be regenerated in the future using the renal developmental mechanism of large heterologous animals, such as pigs.

The Innate Cellular Immune Response in Xenotransplantation.

Xenotransplantation is very attractive strategy for addressing the shortage of donors. While hyper acute rejection (HAR) caused by natural antibodies and complement has been well defined, this is not the case for innate cellular xenogeneic rejection. An increasing body of evidence suggests that innate cellular immune responses contribute to xenogeneic rejection. Various molecular incompatibilities between receptors and their ligands across different species typically have an impact on graft outcome. NK cells are activated by direct interaction as well as by antigen dependent cellular cytotoxicity (ADCC) mechanisms. Macrophages are activated through various mechanisms in xenogeneic conditions. Macrophages recognize CD47 as a “marker of self” through binding to SIRPα. A number of studies have shown that incompatibility of porcine CD47 against human SIRPα contributes to the rejection of xenogeneic target cells by macrophages. Neutrophils are an early responder cell that infiltrates xenogeneic grafts. It has also been reported that neutrophil extracellular traps (NETs) activate macrophages as damage-associated pattern molecules (DAMPs). In this review, we summarize recent insights into innate cellular xenogeneic rejection.

Proteomics Analysis of Aqueous Humor and Rejected Graft in Pig-to-Non-Human Primate Corneal Xenotransplantation.

Although pig-to-non-human primate (NHP) corneal xenotransplantation has shown long-term graft survival, xenogeneic antigen-related immune responses are still stronger than allogeneic antigen-associated responses. Therefore, there is an unmet need to investigate major rejection pathways in corneal xenotransplantation, even with immunosuppression. This study aimed to identify biomarkers in aqueous humor for predicting rejection and to investigate rejection-related pathways in grafts from NHPs transplanted with porcine corneas following the administration of steroids combined with tacrolimus/rituximab. NHPs who had received corneas from wild-type (WT) or α-1,3-galactosyltransferase gene-knockout (GTKO) pigs were divided into groups with or without rejection according to clinical examinations. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the proteomes of corneal tissues or aqueous humor. The biological functions of differentially expressed proteins (DEPs) were assessed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathways and protein–protein interaction network analysis. Among the 66 DEPs in aqueous humor, complement proteins (C3, C5, and C9) and cholesterol metabolic proteins (APOA1 and APOA2) were related to xenogeneic rejection as biomarkers, and alternative pathways of the complement system seemed to be important in xenogeneic graft rejection. Among the 416 DEPs of the cornea, NF-κB1 and proteosomes (PSMD7, PSMA5, and PSMD3) seemed to be related to xenogeneic graft rejection. Additionally, oxidative phosphorylation and leukocyte activation-related pathways are involved in rejection. Overall, our proteomic approach highlights the important role of NF-κB1, proteosomes, oxidative phosphorylation, and leukocyte activation-related inflammation in the cornea and the relevance of complement pathways of the aqueous humor as a predictive biomarker of xenogeneic rejection.

Development and characterization of Gal KO porcine bone marrow-derived mesenchymal stem cells.

We demonstrated that neonatal porcine bone marrow-derived mesenchymal stem cell (npBM-MSCs) could improve a critical ischemic limb disease in rat model more efficiently compared with human MSCs. However, since porcine MSC presents galactosyl-alpha 1,3-galactose antigen (Gal antigen), MSC could be eliminated by the xenogeneic rejection. Recently, we established Gal knockout (KO) pigs by a technique of the electroporation of the CRISPR/Cas9 system into vitro-fertilized zygotes. In this study, we hypothesized that MSC from the established Gal KO pigs could further improve the efficacy. Before examining the hypothesis, in this study, we have established and characterized bone marrow-derived MSC from the Gal KO adult pigs (apBM-MSCs). Mononuclear cells (MNCs) were isolated from bone marrow cells of both Gal KO adult pigs and wild-type (WT) adult pigs. MNCs were further manipulated to create Gal KO apBM-MSCs and WT apBM-MSCs. Both MSCs were assessed by their surface markers, the capability of differentiation into adipocytes, osteocytes and chondrocytes, grow speed and colony-forming assay. To assess the efficacy of Gal KO apBM-MSCs, angiogenesis-related genes and immunosuppression-related genes were assessed by cytokine stimulation. Gal KO apBM-MSC showed no Gal antigen on their cell surfaces. Both Gal KO apBM-MSCs and WT apBM-MSCs, presented little or no negative surface markers of MSCs, while they presented positive surface markers of MSCs. Furthermore, Gal KO apBM-MSCs were able to differentiate into adipocytes, osteocytes, and chondrocytes as well as WT apBM-MSCs. There was no difference in doubling time between Gal KO apBM-MSCs and WT apBM-MSCs. Interestingly, the colony-forming efficiency of Gal KO apBM-MSCs was about half that of WT apBM-MSC. However, angiogenesis and immunosuppression-related genes were equally upregulated in both Gal KO apBM-MSCs and WT apBM-MSCs by cytokine stimulation. We created and characterized Gal KO apBM-MSCs which showed similar characteristics and cytokine-induced gene upregulation to the WT apBM-MSCs.

The Regulation of Neutrophil Extracellular Trap-induced Tissue Damage by Human CD177.

Neutrophil-induced tissue damage contributes to the rejection in xenotransplantation. Therefore, suppressing neutrophil function could be effective in suppressing xenogeneic rejection. In a previous study, we demonstrated that the ectopic expression of human cluster of differentiation 31 (CD31) on porcine endothelial cells (PEC) significantly suppressed neutrophil-mediated cytotoxicity through the homophilic binding of CD31. Cluster of differentiation 177 (CD177) was recently reported to be a high-affinity heterophilic binding partner for CD31 on endothelial cells. Thus, we hypothesized that human CD177 on PEC might induce a stronger suppression in neutrophil-mediated cytotoxicity compared with CD31. In this study, the inhibitory function of human CD177 on PEC in neutrophil-mediated cytotoxicity was investigated. PEC were transfected with a cloning plasmid containing cDNA inserts that encoded for hCD177 and hCD31 genes. Neutrophil-induced cytotoxicity was evaluated by flow cytometry after coculturing with PEC or PEC/CD177 in the presence of phorbol 12-myristate 13-acetate. To elucidate the mechanisms responsible for hCD177-induced suppression, the phosphorylation of src homology region 2 domain containing phosphatase 1 was measured by immunoblot analysis. Human CD177 on PEC induced a significant reduction in neutrophil-induced cytotoxicity. In addition, CD177 on PEC induced a significant increase in the phosphorylation of src homology region 2 domain-containing phosphatase 1 in neutrophils and suppressed NETosis. These findings suggest that human CD177 suppresses neutrophil-mediated cytotoxicity through the inhibition of NETosis.

XENOGENEIC LUNG PERFUSION USING GALTKO.HCD46 LUNGS EXPRESSING HTFPI AND HCD47

Purpose: To address known xenogeneic rejection pathways, various genetic modifications have been introduced into donor pig lines. Here we evaluated whether tissue factor pathway inhibitor (TFPI), a protease inhibitor which blocks initial steps of the extrinsic coagulation cascade, and human CD47, a “don’t eat me” immunoregulatory protein, lessen inflammation and promote GalTKO.hCD46 lung xenograft survival. Methods: In a well-established ex vivo lung perfusion model, 8 GalTKO.hCD46 and 9 GalTKO.hCD46.hTFPI.hCD47 lungs were perfused with freshly collected heparinized human blood. All lungs were treated with an anti-GPIb Fab, 1-BIA, a thromboxane synthase inhibitor and Famotidine; and donor pigs were pre-treated with Desmopressin to reduce vWF expression. In both groups, 3 or 4 lungs in addition received selectin and integrin blockers. Organs were perfused for up to 8 hours or until the lung failed. Results: All except two GalTKO.hCD46 organs (183, 440 min) ‘survived’ until elective termination after 8 hours of perfusion. While the pulmonary vascular resistance (PVR) remained low with both genotypes, additional selectin and integrin blockade lowered PVRs in both groups. Neutrophil but not platelet sequestration and activation were reduced during the first 2 hours in association with hTFPI.hCD47 expression; this trend was more pronounced in the absence of selectin and integrin blockade, reaching significance at the 1 and 2 hour time point. Further biochemical analyses are underway. Conclusion: While the expression of hTFPI and hCD47 showed a trend toward protective effects (improved survival, delayed neutrophil sequestration), overall lung physiology and hematological analyses did not demonstrate significant improvements. Lack of improvement of parameters other than PVR with addition of selectin and integrin blockade suggests that other injury pathways (e.g. non-gal antibody; cytokines; coagulation pathway dysregulation; GPIIb/IIIa-driven platelet activation) contribute to xenolung injury. Identifying and addressing these factors may be necessary to reveal effects attributable to hTFPI and hCD47.NIH U19 AI090959. Unrestricted educational gifts from United Therapeutics/ Lung Biotechnology LLC.

Corneal xenotransplantation: Where are we standing?

The search for alternatives to allotransplants is driven by the shortage of corneal donors and is demanding because of the limitations of the alternatives. Indeed, current progress in genetically engineered (GE) pigs, the introduction of gene-editing technology by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, and advanced immunosuppressants have made xenotransplantation a possible option for a human trial. Porcine corneal xenotransplantation is considered applicable because the eye is regarded as an immune-privileged site. Furthermore, recent non-human primate studies have shown long-term survival of porcine xenotransplants in keratoplasty. Herein, corneal immune privilege is briefly introduced, and xenogeneic reactions are compared with allogeneic reactions in corneal transplantation. This review describes the current knowledge on special issues of xenotransplantation, xenogeneic rejection mechanisms, current immunosuppressive regimens of corneal xenotransplantation, preclinical efficacy and safety data of corneal xenotransplantation, and updates of the regulatory framework to conduct a clinical trial on corneal xenotransplantation. We also discuss barriers that might prevent xenotransplantation from becoming common practice, such as ethical dilemmas, public concerns on xenotransplantation, and the possible risk of xenozoonosis. Given that the legal definition of decellularized porcine cornea (DPC) lies somewhere between a medical device and a xenotransplant, the preclinical efficacy and clinical trial data using DPC are included. The review finally provides perspectives on the current standpoint of corneal xenotransplantation in the fields of regenerative medicine.

Human CD200 Suppresses the HL-60 Mediated Xenocytotoxicity

BackgroundNeutrophils play an important role in xenogeneic rejection and represent a major obstacle in clinical application of xenografts. CD200 and its receptor CD200R are both type-1 membrane glycoproteins, which are members of the immunoglobulin superfamily (IgSF) and the ligation of CD200 with CD200R induces inhibitory NPXY signaling. The expression of CD200R appears in myeloid cells such as macrophages and granulocytes. Thus, we hypothesized that human CD200 expression on porcine cells might suppress the xenogeneic neutrophil-mediated cytotoxicity against porcine cells. MethodsTo prove our hypothesis, the suppressive effect of human CD200 in neutrophil-like human cell line 60 (HL-60)–mediated xenogeneic cytotoxicity against swine endothelial cells (SECs) was examined. Cytotoxicity was assessed with water-soluble tetrazolium salt 8 (WST-8) assay. ResultsHL-60 cells differentiated into CD66b+ CD200R+ neutrophil-like cells in the presence of dimethyl sulfoxide (DMSO). HL-60-mediated cytotoxicity against SECs was significantly suppressed by human CD200 on SECs. ConclusionsThe findings in this study indicate that human CD200 may suppress neutrophil-mediated xenogeneic rejection.

Human CD31 on Swine Endothelial Cells Induces SHP-1 Phosphorylation in Macrophages

BackgroundInnate immunity by natural killer (NK) cells, macrophages, and neutrophils cause severe rejections in xenotransplantation. Therefore, the development of strategies for suppressing macrophages has considerable potential in practical applications of xenotransplantation. Recently, we found that human CD31 on swine endothelial cells (SECs) suppresses neutrophil-mediated xenogeneic rejection through homophilic binding. Since a significant amount of CD31 is expressed not only on neutrophils but also on macrophages, we studied the function of human CD31 in macrophage-mediated cytotoxicity. MethodsSECs and hCD31-transfected SECs (SEC/hCD31) were co-cultured with macrophages and cytotoxicity by macrophages was evaluated with water-soluble tetrazolium salt, or WST-8, assay. To confirm whether or not inhibitory signals are induced by hCD31 homophilic binding, the phosphorylation of the enzyme SHP-1 was investigated with Western blotting. ResultsNo suppression of cytotoxicity was induced in macrophages that had been co-cultured with SEC/CD31. However, phosphorylation of SHP-1 was induced in macrophages that had been co-cultured with SEC/hCD31. ConclusionsHuman CD31 on SEC may induce not only inhibitory signals but also activation signals via the binding to other receptors for hCD31.

The effect of SLA expression on genetically-engineered pig renal cells on the human T cell xenoresponse

Abstract Background Xenotransplantation using genetically-engineered (GE) pig organs offers an unlimited supply of organs for patients with end-stage organ failure. Disrupting the porcine GGTA1/CMAH/β4GalNT2 genes (TKO) markedly reduces binding of human natural anti-pig antibodies in human to carbohydrate antigens expressed on the pig organ. However, more evident have demonstrated that T cell responses play a major role in xenogeneic rejection, and that immunosuppression alone is likely incapable of completely suppressing these responses. The object of this study is at an organ-specific level to evaluate human T cell-mediated response against GE pig renal microvascular endothelial cells (RMEC) with or without swine leukocyte antigen (SLA) class I or II. Methods Primary RMEC were isolated from the GGTA1/CMAH knockout (DKO) pigs, and immortalized using Lenti-SV40 T. The gene editing was performed to create specific TKO RMEC cell lines expressing SLA class II. Stimulation of human T cell activation or proliferation by RMEC was performed flow cytometry-based mixed lymphocyte reaction (MLR). Results The specific GE cells lacking carbohydrates or expressing SLA class I or II were confirmed. MLR data shows that human CD4+ T cell proliferation by CD31+ RMEC was higher than that by fibroblast-like renal cells (RC), and was significantly inhibited by immunosuppressive drugs. Furthermore, both of CD69+ and CD25+ T cells were significantly enhanced by RMEC with expression of SLA class I and II compared with SLA class I alone. Conclusion Porcine RMEC express SLA class I/II and known costimulatory molecules and have the ability to simulate human T cell proliferation. RMEC will be a useful reagent for the further study of xenotransplantation.

A Strategy for Suppressing Macrophage-mediated Rejection in Xenotransplantation.

Although xenografts are one of the most attractive strategies for overcoming the shortage of organ donors, cellular rejection by macrophages is a substantial impediment to this procedure. It is well known that macrophages mediate robust immune responses in xenografts. Macrophages also express various inhibitory receptors that regulate their immunological function. Recent studies have shown that the overexpression of inhibitory ligands on porcine target cells results in the phosphorylation of tyrosine residues on intracellular immunoreceptor tyrosine-based inhibitory motifs on macrophages, leading to the suppression of xenogenic rejection by macrophages. It has also been reported that myeloid-derived suppressor cells, a heterogeneous population of immature myeloid cells, suppress not only NK and cytotoxic T lymphocyte cytotoxicity but also macrophage-mediated cytotoxicity. This review is focused on the recent findings regarding strategies for inhibiting xenogenic rejection by macrophages.

Rejection of xenogeneic porcine islets in humanized mice is characterized by graft-infiltrating Th17 cells and activated B cells.

Xenogeneic porcine islet transplantation is a promising potential therapy for type 1 diabetes (T1D). Understanding human immune responses against porcine islets is crucial for the design of optimal immunomodulatory regimens for effective control of xenogeneic rejection of porcine islets in humans. Humanized mice are a valuable tool for studying human immune responses and therefore present an attractive alternative to human subject research. Here, by using a pig-to-humanized mouse model of xenogeneic islet transplantation, we described the human immune response to transplanted porcine islets, a process characterized by dense islet xenograft infiltration of human CD45+ cells comprising activated human B cells, CD4+ CD44+ IL-17+ Th17 cells, and CD68+ macrophages. In addition, we tested an experimental immunomodulatory regimen in promoting long-term islet xenograft survival, a triple therapy consisting of donor splenocytes treated with ethylcarbodiimide (ECDI-SP), and peri-transplant rituximab and rapamycin. We observed that the triple therapy effectively inhibited graft infiltration of T and B cells as well as macrophages, promoted transitional B cells both in the periphery and in the islet xenografts, and provided a superior islet xenograft protection. Our study therefore indicates an advantage of donor ECDI-SP treatment in controlling human immune cells in promoting long-term islet xenograft survival.

Immortalization of porcine hepatocytes with a α-1,3-galactosyltransferase knockout background.

In vivo pig liver xenotransplantation preclinical trials appear to have poor efficiency compared to heart or kidney xenotransplantation because of xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia. In contrast, ex vivo pig liver (wild type) perfusion systems have been proven to be effective in "bridging" liver failure patients until subsequent liver allotransplantation, and transgenic (human CD55/CD59) modifications have even prolonged the duration of pig liver perfusion. Despite the fact that hepatocyte cell lines have also been proposed for extracorporeal blood circulation in conditions of acute liver failure, porcine hepatocyte cell lines, and the GalT-KO background in particular, have not been developed and applied in this field. Herein, we established immortalized wild-type and GalT-KO porcine hepatocyte cell lines, which can be used for artificial liver support systems, cell transplantation, and even in vitro studies of xenotransplantation. Primary hepatocytes extracted from GalT-KO and wild-type pigs were transfected with SV40 LT lentivirus to establish immortalized GalT-KO porcine hepatocytes (GalT-KO-hep) and wild-type porcine hepatocytes (WT). Hepatocyte biomarkers and function-related genes were assessed by immunofluorescence, periodic acid-Schiff staining, indocyanine green (ICG) uptake, biochemical analysis, ELISA, and RT-PCR. Furthermore, the tumorigenicity of immortalized cells was detected. In addition, a complement-dependent cytotoxicity (CDC) assay was performed with GalT-KO-hep and WT cells. Cell death and viability rates were assessed by flow cytometry and CCK-8 assay. GalT-KO and wild-type porcine hepatocytes were successfully immortalized and maintained the characteristics of primary porcine hepatocytes, including albumin secretion, ICG uptake, urea and glycogen production, and expression of hepatocyte marker proteins and specific metabolic enzymes. GalT-KO-hep and WT cells were confirmed as having no tumorigenicity. In addition, GalT-KO-hep cells showed less apoptosis and more viability than WT cells when exposed to complement and xenogeneic serum. Two types of immortalized cell lines of porcine hepatocytes with GalT-KO and wild-type backgrounds were successfully established. GalT-KO-hep cells exhibited higher viability and injury resistance against a xenogeneic immune response.

Human TIGIT on porcine aortic endothelial cells suppresses xenogeneic macrophage-mediated cytotoxicity

PurposeThe delayed rejection caused by strong cell-mediated innate and adaptive xenogeneic immune responses continues to be a major obstacle.Therefore, suppressing macrophage function could be effective in avoiding this type of rejection. In this study, the suppression of T-cell immunoglobulin and ITIM domain (TIGIT) function against macrophage-mediated xenogeneic rejection was investigated. Material and methodsNaïve porcine aortic endothelial cell (PAEC) and PAEC transfectant with TIGIT (PAEC/TIGIT) were co-cultured with M1 macrophages, and the degree of cytotoxicity was determined by a counting beads assay. The anti/pro-inflammatory gene expression was determined by RT-PCR and the phosphorylated SHP-1 in the macrophages after co-culturing with PAEC or PAEC/TIGIT was evaluated by western blotting. ResultsCD155 was expressed at essentially equal levels on both M1 and M2 macrophages, whereas TIGIT was highly expressed on M2 macrophages but not in M1 macrophages. TIGIT on PAEC significantly reduced the cytotoxicity of M1 macrophages but no significant suppression of phagocytosis was detected. TIGIT also caused a decrease in the expression of pro-inflammatory cytokines, namely TNFα, IL-1β and IL-12 in M1 macrophages. Furthermore, PAEC/TIGIT caused a significant increase in phosphorylated SHP-1 in M1 macrophages compared to PAEC. ConclusionThe findings of this study indicate that TIGIT suppresses xenogeneic M1 macrophage-induced cytotoxicity, probably at least in part, via the phosphorylation of SHP-1. In addition, the reduced expression of some pro-inflammatory cytokines, namely TNFα, IL-1β and IL-12, was observed in M1 macrophages that had been cultured with PAEC/TIGIT.