How should cardiac xenotransplantation be initiated in Japan?

The world's first clinical cardiac xenotransplantation, using a genetically engineered pig heart with 10 gene modifications, prolonged the life of a 57-year-old man with no other life-saving options, by 60 days. It is foreseeable that xenotransplantation will be introduced in clinical practice in the United States. However, little clinical or regulatory progress has been made in the field of xenotransplantation in Japan in recent years. Japan seems to be heading toward a "device lag", and the over-importation of medical devices and technology in the medical field is becoming problematic. In this review, we discuss the concept of pig-heart xenotransplantation, including the pathobiological aspects related to immune rejection, coagulation dysregulation, and detrimental heart overgrowth, as well as genetic modification strategies in pigs to prevent or minimize these problems. Moreover, we summarize the necessity for and current status of xenotransplantation worldwide, and future prospects in Japan, with the aim of initiating xenotransplantation in Japan using genetically modified pigs without a global delay. It is imperative that this study prompts the initiation of preclinical xenotransplantation research using non-human primates and leads to clinical studies.

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

OBJECTIVE: Innate 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). METHODS: We 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. RESULTS: In 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. CONCLUSION: These 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.

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-1ß, 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.

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.

Effect of a C5a receptor antagonist on macrophage function in an intestinal transplant rat model.

BACKGROUND: C5a promotes alloreactivity via the C5a receptor 1 (C5aR1) on immune cells, but this has not been confirmed in the case of small intestine transplantation immunity. In the present study, we examined the effect of C5aR1 antagonist (PMX53) on macrophage function in small intestinal transplantation. METHODS: The model was created by heterotopic intestinal transplantation using donor Dark Agouti and recipient Lewis rats. PMX53 was administered starting on the day of operation until postoperative day 7. The graft survivals were compared, and HE staining of grafts, lymphocyte mixed reaction test (MLR, mixed culture of T cells from lymph nodes and spleen cells from donors), and changes in macrophage and T cell accumulation in grafts on day 6 after transplantation were evaluated. In addition, the effect of PMX53 on macrophage differentiation and activation was assessed using macrophages derived from bone marrow (BMDM). RESULTS: Graft survival was significantly prolonged in the therapeutic group compared to the untreated group. Histological evaluation showed that PMX53 inhibited the shortening of the graft villus, and the stimulation index of MLR was significantly lower in the therapeutic group compared to the untreated group. In the therapeutic group, the accumulation of macrophages in intestinal graft and monocyte in blood were reduced, compared with the untreated group. PMX53 decreased the differentiation in BMDM and the mRNA expression of IL-1ß and TNF-α in activated BMDM. CONCLUSION: Inhibition of C5a/C5aR1 signaling appears to regulate macrophage differentiation and suppress rejection in small intestine transplantation immunity.

Elevation of microRNA-214 is associated with progression of liver fibrosis in patients with biliary atresia.

BACKGROUND: MicroRNAs (miRNAs) play an important role in regulating fibrogenesis in the liver. The current study examined the ability of microRNA-214 (miR-214) level in liver and serum samples obtained from patients with BA to predict progressive liver fibrosis in patients with biliary atresia (BA). METHODS: We examined miR-214 level in relation to conventional markers of liver fibrosis, with liver and serum samples from BA patients. Fifty-two patients with BA who underwent Kasai portoenterostomy and four control patients underwent liver biopsy. In 28 patients with BA, blood samples were collected to analyze circulating serum miR-214. RESULTS: MiR-214 levels in liver tissue were significantly upregulated in patients with BA who had severe liver fibrosis (F3-4) compared to those with none to mild fibrosis (F0-2), whereas suppressors-of-fused homolog (Sufu) mRNA levels were significantly suppressed in F3-4. Serum miR-214 levels were significantly higher in patients with F3-4 compared with F0-2. Area under the curve analysis showed that the serum miR-214 cut-off level for predicting F3-4 was 0.805 (p = 0.0046). CONCLUSION: Hepatic overexpression of miR-214 is associated with progression of liver fibrosis in patients with BA, and the circulating miR-214 level may serve as a non-invasive predictor of liver fibrosis.

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

BACKGROUND: Innate 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. METHODS: SECs 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. RESULTS: No 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. CONCLUSIONS: Human CD31 on SEC may induce not only inhibitory signals but also activation signals via the binding to other receptors for hCD31.

Human CD200 Suppresses the HL-60 Mediated Xenocytotoxicity.

BACKGROUND: Neutrophils 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. METHODS: To 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. RESULTS: HL-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. CONCLUSIONS: The findings in this study indicate that human CD200 may suppress neutrophil-mediated xenogeneic rejection.

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.

The effect of a novel immunosuppressive drug, a PAK-2 inhibitor, on macrophage differentiation/polarization in a rat small intestinal transplantation model.

OBJECTIVE: PQA-18 (Prenylated quinolinecarboxylic acid-18) has been reported to be a novel immunosuppressant that attenuates the production of various cytokines, and the differentiation of macrophages by inhibiting PAK2. In this study, we investigated the function of this drug mainly on macrophages using a rat small intestinal transplant model. METHODS: Male Dark Agouti (DA) and Lewis rats (LEW), 7-9 weeks of age, were used as donor and recipient, respectively. Approximately 15 cm intestinal grafts were heterotopically transplanted to the recipient rats. The recipient rat was treated with PQA-18 (4 mg/kg/day) by intraperitoneal injection (ip) from postoperative day 1 for 2 weeks. The in vivo effects of this drug were evaluated based on changes in body weight, and the population of each type of blood cell. Mixed lymphocyte reaction (MLR) was also assessed, using the T cells from intestinal mesenteric lymph nodes (MLN) of the grafts on POD6. Total cells from MLN and graft Payer's patch (PP) were next collected on POD6, and the number of infiltrated macrophages was determined. RESULTS: While the survival time was 7.0 ±â€¯0.77 days for the control group (n = 9), that for the PQA-18 group was 10.7 ±â€¯1.26 days (n = 10) (p < .001). Histological examinations showed a relatively clear difference in the grafts for both groups. In addition, the MLR response was significantly lower in recipients treated with PQA-18, suggesting PQA-18 well suppressed the T cells. Moreover, while a significant increase of both MHC class II and CD11b/c positive cells, estimated as differentiated/polarized macrophages, in MLN & PP was observed in the control group, PQA-18-administration significantly suppressed the differentiation of macrophages in the MLN & PP. CONCLUSION: PQA-18 significantly prolonged the survival of the rats with intestinal grafts, and also suppressed the infiltration of lymphocytes, and macrophages to the grafts.

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

PURPOSE: The 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 METHODS: Naïve 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. RESULTS: CD155 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-1ß and IL-12 in M1 macrophages. Furthermore, PAEC/TIGIT caused a significant increase in phosphorylated SHP-1 in M1 macrophages compared to PAEC. CONCLUSION: The 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-1ß and IL-12, was observed in M1 macrophages that had been cultured with PAEC/TIGIT.

The novel immunosuppressant prenylated quinolinecarboxylic acid-18 (PQA-18) suppresses macrophage differentiation and cytotoxicity in xenotransplantation.

Innate immunity plays a major role in xenograft rejection. However, the majority of immunosuppressants focus on inhibiting acquired immunity and not innate immunity. Therefore, a novel immunosuppressant suitable for use in conjunction with xenografts continues to be needed. It has been reported that prenylated quinolinecarboxylic acid-18 (PQA-18), a p21-activated kinase 2 (PAK2) inhibitor, exerts an immunosuppressive function on T cells. Hence, the possibility exists that PQA-18 might be used in conjunction with xenografts, which prompted us to investigate the efficacy of PQA-18 on macrophages compared with Tofacitinib, a janus kinase (JAK) inhibitor. Initial experiments confirmed that PQA-18 is non-toxic to swine endothelial cells (SECs) and human monocytes. Both PQA-18 and Tofacitinib suppressed macrophage-mediated cytotoxicity in both the differentiation and effector phases. Both PQA-18 and tofacitinib suppressed the expression of HLA-ABC by macrophages. However, contrary to Tofacitinib, PQA-18 also significantly suppressed the expression of CD11b, HLA-DR and CD40 on macrophages. PQA-18 significantly suppressed CCR7 expression on day 3 and on day 6, but Tofacitinib-induced suppression only on day 6. In a mixed lymphocyte reaction (MLR) assay, PQA-18 was found to suppress Interleukin-2 (IL-2)-stimulated T cell proliferation to a lesser extent than Tofacitinib. However, PQA-18 suppressed xenogeneic-induced T cell proliferation more strongly than Tofacitinib on day 3 and the suppression was similar on day 7. In conclusion, PQA-18 has the potential to function as an immunosuppressant for xenotransplantation.

Human CD31 on porcine cells suppress xenogeneic neutrophil-mediated cytotoxicity via the inhibition of NETosis.

BACKGROUND: Xenotransplantation is one of the promising strategies for overcoming the shortage of organs available for transplant. However, many immunological obstructions need to be overcome for practical use. Increasing evidence suggests that neutrophils contribute to xenogeneic cellular rejection. Neutrophils are regulated by activation and inhibitory signals to induce appropriate immune reactions and to avoid unnecessary immune reactivity. Therefore, we hypothesized that the development of neutrophil-targeted therapies may have the potential for increased graft survival in xenotransplantation. METHODS: A plasmid containing a cDNA insert encoding the human CD31 gene was transfected into swine endothelial cells (SEC). HL-60 cells were differentiated into neutrophil-like cells by culturing them in the presence of 1.3% dimethyl sulfoxide for 48 hours. The cytotoxicity of the differentiated HL-60 cells (dHL-60) and peripheral blood-derived neutrophils was evaluated by WST-8 assays. To investigate the mechanism responsible for hCD31-induced immunosuppression, citrullinated histone 3 (cit-H3) and phosphorylation of SHP-1 were detected by a cit-H3 enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. RESULTS: A significant decrease in dHL-60 and neutrophil-mediated cytotoxicity in SEC/hCD31 compared with SEC was seen, as evidenced by a cytotoxicity assay. Furthermore, the suppression of NETosis and the induction of SHP-1 phosphorylation in neutrophils that had been co-cultured with SEC/CD31 were confirmed by cit-H3 ELISA and Western blotting with an anti-phosphorylated SHP-1. CONCLUSION: These data suggest that human CD31 suppresses neutrophil-mediated xenogenic cytotoxicity via the inhibition of NETosis. As CD31 is widely expressed in a variety of inflammatory cells, human CD31-induced suppression may cover the entire xenogeneic cellular rejection, thus making the generation of human CD31 transgenic pigs very attractive for use in xenografts.

A membrane-type surfactant protein D (SP-D) suppresses macrophage-mediated cytotoxicity in swine endothelial cells.

OBJECTIVE: Surfactant protein D (SP-D), which is secreted mainly in the lung, is an oligometric C type lectin that promotes phagocytosis by binding to carbohydrates on microbial surfaces. SP-D can also bind SIRPα, leading to a decrease in cytokine production by monocytes/macrophages. In the present study, we examined the possibility that SP-D suppresses macrophage-mediated xenogeneic cytotoxicity, by creating a membrane-type SP-D. METHODS: The cDNA for the carbohydrate recognition domain (CRD) of human SP-D was switched to that of a membrane-type protein, collectin placenta 1 (CL-P1), with a Flag-tag. The cDNA of CD47 was prepared as a control. The suppressive function of the membrane-type protein of the hybrid molecule, CL-SP-D, to monocytes/macrophages was then studied and the results compared with that for CD47. RESULTS: The expression of Flag-tagged CL-SP-D on the transfected SECs and the SIRPα on monocyte-like cells, THP-1 cells, was confirmed by FACS using anti-Flag Ab and anti-CD172a, respectively. The molecular size of the hybrid protein was next assessed by western blot. While significant cytotoxicity against SEC was induced in differentiated THP-1 cells, CL-SP-D significantly reduced THP-1-mediated cytotoxicity. In addition, phosphorylated SHP-1 was clearly detected in SEC/CL-SP-D in western blots. Moreover, IL-10 production was upregulated and IL-1ß production was suppressed in the case of THP-1 and SEC/CL-SP-D, compared with naïve SEC. Next, the cytotoxicity caused by the in vitro generated macrophage was assessed under the same conditions as were used for THP-1. CL-SP-D also showed the significant down-regulation on the macrophage. In addition, changes in IL-10 production by the macrophage confirmed the results. CONCLUSIONS: These findings indicate that the membrane-type SP-D serve as an effective therapeutic strategy for inhibiting macrophage-mediated xenograft rejection in xenotransplantation.

Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis.

PURPOSE: Various strategies, such as the generation of alpha-1,3-galactosyltransferase knocked-out pigs and CD55 transgenic pigs, have been investigated to inhibit pig to human xenogeneic rejection. Our aim is to develop strategies to overcome the hurdle of not only hyper acute rejection, but also that of cellular xenogeneic rejection (CXR). Although macrophages have been well known to play a critical role in CXR, monocyte/macrophage-mediated xenogeneic rejection has not been well studied. In this study, we evaluated the effect of CD200 in xenogeneic rejection by macrophages. METHODS: Naïve swine endothelial cells (SEC) and SEC/CD200 were co-cultured with M0 macrophages and the cytotoxicity was measured by a WST-8 assay. The phagocytosis of SEC and SEC/CD200 by macrophages was analyzed by flow cytometry. RESULTS: While CD200 failed to suppress a significant amount of cytotoxicity against SEC by monocytes, M0 macrophage-mediated cytotoxicity was significantly suppressed by human CD200. The phagocytosis by M0 macrophages was also tested. The phagocytosis assay revealed that human CD200 suppresses M0 macrophage-mediated phagocytosis. CONCLUSIONS: Our findings indicate that human CD200 suppresses the xenogeneic rejection by CD200R+ macrophages and that the generation of hCD200 transgenic pigs for use in xenografts is very attractive for preventing the macrophage-mediated rejection.

Correction to: Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis.

In the original publication, the fifth author name was erroneously published as "Patmika Jiaravuthiasan". The correct author name should read as, "Patmika Jiaravuthisan". The original article was corrected.

Studies of innate immune systems against human cells.

BACKGROUND: Pigs are frequently used as animal models for experiments in the surgical field, including allo- and xeno-transplantation. Regeneration studies, including studies dealing with human- and monkey-induced pluripotent stem cells (iPSC), have gradually progressed, with pigs sometimes being used as the scaffold. However, the immunological response of pigs against humans, especially innate immunities, remain unclear. This study reports on a comprehensive study of pig innate immunity against humans. METHODS: Hemolytic complement activity of pig serum was measured using a microtitration technique. The pig natural anti-human antibody (Ab) was examined using human peripheral blood mononuclear cells (PBMC). The reaction of pig natural killer (NK) cells and monocytes/macrophages against human cells was also assessed. RESULTS: Most of the pig complement titers were measured based on methods used in human complement assays. The alternative pathway for pig complement reacts with human cells, indicating that pig complement can react with human cells. Pig serum contains relatively high levels of natural antibodies, IgM and IgG, to human PBMC. Furthermore, the killing of NK cells- and monocyte/macrophage-mediated human cells was clearly confirmed. CONCLUSION: The collective findings indicate that the pig innate immunological systems, not only serum but also cellular factors, are able to recognize and injure human cells.

Cardiomyocytes Derived from MHC-Homozygous Induced Pluripotent Stem Cells Exhibit Reduced Allogeneic Immunogenicity in MHC-Matched Non-human Primates.

Induced pluripotent stem cells (iPSCs) can serve as a source of cardiomyocytes (CMs) to treat end-stage heart failure; however, transplantation of genetically dissimilar iPSCs even within species (allogeneic) can induce immune rejection. We hypothesized that this might be limited by matching the major histocompatibility complex (MHC) antigens between the donor and the recipient. We therefore transplanted fluorescence-labeled (GFP) iPSC-CMs donated from a macaque with homozygous MHC haplotypes into the subcutaneous tissue and hearts of macaques having heterozygous MHC haplotypes (MHC-matched; group I) or without identical MHC alleles (group II) in conjunction with immune suppression. Group I displayed a higher GFP intensity and less immune-cell infiltration in the graft than group II. However, MHC-matched transplantation with single or no immune-suppressive drugs still induced a substantial host immune response to the graft. Thus, the immunogenicity of allogeneic iPSC-CMs was reduced by MHC-matched transplantation although a requirement for appropriate immune suppression was retained for successful engraftment.

Monocytic MDSCs regulate macrophage-mediated xenogenic cytotoxicity.

BACKGROUND: Xenotransplantation is considered to be one of the most attractive strategies for overcoming the worldwide shortage of organs. However, many obstructions need to be overcome before it will achieve clinical use in patients. One such obstacle is the development of an effective immunosuppressive strategy. We previously reported that myeloid-derived suppressor cells (MDSCs), a heterogeneous population of progenitor and immature myeloid cells, suppress xenogenic CTL-mediated cytotoxicity. Because of their heterogeneous nature, MDSC can function via several suppressive mechanisms that disrupt both innate and adaptive immunity. Since macrophages play a pivotal role in the rejection of a xenograft, in this study, we evaluated the suppressive effects of MDSC against macrophage-mediated xenogenic rejection. MATERIALS AND METHODS: To evaluate the effect of monocyte-derived MDSCs on xenogenic immune reactions, a CFSE(carboxyfluorescein diacetate, succinimidyl ester)assay was employed to assess cytotoxicity. RESULTS: While, in the absence of activation, primed MDSCs had no detectable effect on macrophage-induced cytotoxicity against SEC cells, LPS-activated MDSCs were found to significantly suppress xenogenic cytotoxicity. A CFSE cytotoxicity assay revealed that MDSCs significantly suppressed macrophage-induced cytotoxicity. Furthermore, an indoleamine 2,3 dioxygenase (IDO) inhibitor, 1-methyl tryptophan (1-MT), abolished the MDSC-induced suppression of macrophage-mediated xeno-rejection, indicating that MDSCs may suppress macrophage-mediated cytotoxicity in an IDO-dependent manner. CONCLUSION: These findings indicate that MDSCs have great potential for immunosuppressing macrophage-mediated xeno-rejection.

Generation of a felinized swine endothelial cell line by expression of feline decay-accelerating factor.

Embryonic stem cell research has facilitated the generation of many cell types for the production of tissues and organs for both humans and companion animals. Because ≥30% of pet cats suffer from chronic kidney disease (CKD), xenotransplantation between pigs and cats has been studied. For a successful pig to cat xenotransplant, the immune reaction must be overcome, especially hyperacute rejection. In this study, we isolated the gene for feline decay-accelerating factor (fDAF), an inhibitor of complement proteins, and transfected a swine endothelial cell line with fDAF to "felinize" the pig cells. These fDAF-expressing cells were resistant to feline serum containing anti-pig antibodies, suggesting that felinized pig cells were resistant to hyperacute rejection. Our results suggest that a "felinized" pig kidney can be generated for the treatment of CKD in cats in the future.