Publications by authors named "Shuji Miyagawa"

77 Publications

Reactions to Porcine Cells With or Without β4GalNT2.

Transplant Proc 2020 Jul - Aug;52(6):1916-1918. Epub 2020 May 30.

Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; International Institute for Bio-Resource Research, Meiji University, Kawasaki, Japan; Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. Electronic address:

β-1,4-acetyl-galactosaminyltransferase 2 (β4GalNT2)-knockout (KO) pigs have been produced and reveal less antigenicity to both humans and nonhuman primates (NHP). In this study, we checked the antibody response of human and NHP sera to pig cells with or without this gene. The β4GalNT2-KO porcine endothelial cell (PEC), clone #11, was first established using the plasmid pX330 expressing hCas9 and sgRNA for β4GalNT2. The glycoantigen feature on the PEC was then studied. The Sd antigen, synthesized by β4GalNT2, was slightly ascertained on wild-type (WT)-PEC, and it became null in clone #11. The PEC response to lectins was also assessed, such as Dolichos biflorus agglutinin, soybean agglutinin, and Wisteria floribunda agglutinin. All of these lectins reduced the binding reaction to clone #11 as compared with WT-PEC. Next, several human and cynomolgus sera were checked for their natural antibody reaction to both WT-PEC and clone #11. In addition, human monocyte-mediated PEC phagocytosis was assessed. However, the reduction in phagocytosis to clone #11 was not significant. Human sera showed less reactivity to the changes in antigenicity of PEC by knocking out the β4GalNT2 than cynomolgus sera.
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http://dx.doi.org/10.1016/j.transproceed.2020.01.154DOI Listing
November 2020

Human CD200 Suppresses the HL-60 Mediated Xenocytotoxicity.

Transplant Proc 2020 Jul - Aug;52(6):1910-1912. Epub 2020 May 19.

Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Osaka, Japan.

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.
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http://dx.doi.org/10.1016/j.transproceed.2020.01.141DOI Listing
November 2020

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

Transplant Proc 2020 Jul - Aug;52(6):1913-1915. Epub 2020 May 10.

Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

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.
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http://dx.doi.org/10.1016/j.transproceed.2020.01.140DOI Listing
November 2020

A Strategy for Suppressing Macrophage-mediated Rejection in Xenotransplantation.

Transplantation 2020 04;104(4):675-681

Department of Surgery, Osaka University Graduate School of Medicine, Osaka, Japan.

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.
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http://dx.doi.org/10.1097/TP.0000000000003024DOI Listing
April 2020

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

Transpl Immunol 2019 12 14;57:101246. Epub 2019 Sep 14.

Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Japan.

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 ± 0.77 days for the control group (n = 9), that for the PQA-18 group was 10.7 ± 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.
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http://dx.doi.org/10.1016/j.trim.2019.101246DOI Listing
December 2019

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

Immunobiology 2019 09 3;224(5):605-613. Epub 2019 Aug 3.

Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Osaka, Japan; Meiji University International Institute for Bio-Resource Research, Kanagawa, Japan.

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-1β 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-1β and IL-12, was observed in M1 macrophages that had been cultured with PAEC/TIGIT.
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http://dx.doi.org/10.1016/j.imbio.2019.07.008DOI Listing
September 2019

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

Immunobiology 2019 07 2;224(4):575-584. Epub 2019 Apr 2.

Department of Surgery, Osaka University Graduate School of Medicine Japan.

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.
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http://dx.doi.org/10.1016/j.imbio.2019.04.003DOI Listing
July 2019

Current activity of xenotransplantation in Japan.

Xenotransplantation 2019 01 15;26(1):e12487. Epub 2019 Feb 15.

Japanese Society for Xenotransplantation, Nagakute, Japan.

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http://dx.doi.org/10.1111/xen.12487DOI Listing
January 2019

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

Xenotransplantation 2018 09 8;25(5):e12396. Epub 2018 Apr 8.

Department of Surgery, Osaka University Graduate School of Medicine, Suita, Japan.

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.
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http://dx.doi.org/10.1111/xen.12396DOI Listing
September 2018

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

Transpl Immunol 2018 04 6;47:44-48. Epub 2018 Feb 6.

Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Electronic address:

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-1β 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.
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http://dx.doi.org/10.1016/j.trim.2018.02.003DOI Listing
April 2018

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

Surg Today 2018 02;48(2):252

Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

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.
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http://dx.doi.org/10.1007/s00595-017-1604-9DOI Listing
February 2018

Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis.

Surg Today 2018 Jan 1;48(1):119-126. Epub 2017 Jun 1.

Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

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.
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http://dx.doi.org/10.1007/s00595-017-1546-2DOI Listing
January 2018

Studies of innate immune systems against human cells.

Transpl Immunol 2017 02 7;40:66-71. Epub 2016 Dec 7.

Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.trim.2016.12.002DOI Listing
February 2017

Depression of Complement Regulatory Factors in Rat and Human Renal Grafts Is Associated with the Progress of Acute T-Cell Mediated Rejection.

PLoS One 2016 29;11(2):e0148881. Epub 2016 Feb 29.

Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

Background: The association of complement with the progression of acute T cell mediated rejection (ATCMR) is not well understood. We investigated the production of complement components and the expression of complement regulatory proteins (Cregs) in acute T-cell mediated rejection using rat and human renal allografts.

Methods: We prepared rat allograft and syngeneic graft models of renal transplantation. The expression of Complement components and Cregs was assessed in the rat grafts using quantitative real-time PCR (qRT-PCR) and immunofluorescent staining. We also administered anti-Crry and anti-CD59 antibodies to the rat allograft model. Further, we assessed the relationship between the expression of membrane cofactor protein (MCP) by immunohistochemical staining in human renal grafts and their clinical course.

Results: qRT-PCR results showed that the expression of Cregs, CD59 and rodent-specific complement regulator complement receptor 1-related gene/protein-y (Crry), was diminished in the rat allograft model especially on day 5 after transplantation in comparison with the syngeneic model. In contrast, the expression of complement components and receptors: C3, C3a receptor, C5a receptor, Factor B, C9, C1q, was increased, but not the expression of C4 and C5, indicating a possible activation of the alternative pathway. When anti-Crry and anti-CD59 mAbs were administered to the allograft, the survival period for each group was shortened. In the human ATCMR cases, the group with higher MCP expression in the grafts showed improved serum creatinine levels after the ATCMR treatment as well as a better 5-year graft survival rate.

Conclusions: We conclude that the expression of Cregs in allografts is connected with ATCMR. Our results suggest that controlling complement activation in renal grafts can be a new strategy for the treatment of ATCMR.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148881PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4771804PMC
July 2016

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

Stem Cell Reports 2016 Mar 18;6(3):312-20. Epub 2016 Feb 18.

Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stemcr.2016.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788782PMC
March 2016

Structural Changes in N-Glycans on Induced Pluripotent Stem Cells Differentiating Toward Cardiomyocytes.

Stem Cells Transl Med 2015 Nov 16;4(11):1258-64. Epub 2015 Sep 16.

Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan

Unlabelled: Cell-surface glycans vary widely, depending on cell properties. Previously, we reported that the pattern of N-glycan expression on murine induced pluripotent stem cells (iPSCs) changed toward that of the cardiac tissue during cardiomyogenic differentiation. In this study, N-glycans were isolated from human iPSCs, iPSC-derived cardiomyocytes (iPSC-CMs), and human cardiomyocytes (hCMCs). Their structures were analyzed by a mapping technique based on high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometric data. Of 52 isolated N-glycans, the structures of 38 were clearly identified. In addition, 11 structures were partially identified because the binding style and fucose binding site at the nonreduced terminal could not be identified. Quantitation of each type of N-glycan, based on the terminal glycosylation process, revealed that the exposed N-acetylglucosamine (GlcNAc) and the nonreduced terminal fucose types decreased, whereas the exposed galactose or the α2-3 NeuAc types increased in the iPSCs during cardiomyogenic differentiation. However, the bisecting GlcNAc and the triantennary structures were found in relative abundance in the iPSC-CMs in comparison with hCMCs or iPSCs. Expression of MGAT3, a glycosyltransferase-encoding gene that produces the bisecting GlcNAc structures, was higher in iPSCs and iPSC-CMs than in hCMCs. These findings will prove useful in understanding the directional precision of cardiomyogenic differentiation in vitro.

Significance: This study focused on N-glycans produced in human induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes to investigate their change on cardiomyogenic differentiation in vitro. This shows that the expression pattern of N-glycans in human iPSCs changed toward the pattern observed in human cardiomyocytes upon cardiomyogenic differentiation. Structural differences were also observed in the bisecting N-acetylglucosamine and the triantennary structures upon cardiomyogenic differentiation. The findings of this study will help in understanding the directional precision of cardiomyogenic differentiation in vitro.
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http://dx.doi.org/10.5966/sctm.2015-0029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622404PMC
November 2015

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

J Reprod Dev 2015 26;61(5):449-57. Epub 2015 Jul 26.

Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.
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http://dx.doi.org/10.1262/jrd.2015-058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4623151PMC
August 2016

Monocytic MDSCs regulate macrophage-mediated xenogenic cytotoxicity.

Transpl Immunol 2015 Oct 21;33(2):140-5. Epub 2015 Jul 21.

Department of Surgery, Osaka University Graduate School of Medicine, Osaka, Japan.

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.
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http://dx.doi.org/10.1016/j.trim.2015.07.002DOI Listing
October 2015

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

PLoS One 2015 11;10(2):e0117682. Epub 2015 Feb 11.

Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117682PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4324824PMC
January 2016

Suppression of human macrophage-mediated cytotoxicity by transgenic swine endothelial cell expression of HLA-G.

Transpl Immunol 2015 Mar 3;32(2):109-15. Epub 2015 Jan 3.

Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Electronic address:

Background: Xenotransplantation is an appealing alternative to human allotransplantation because of a worldwide shortage of organs. One of the obstacles for xenografts is cellular rejection by the innate immune system, comprised of NK cells, monocytes, and macrophages. In this study the inhibitory function of HLA-G1, a MHC Ib molecule, on macrophage-mediated cytotoxicity was examined. Furthermore, this study also evaluates the suppressive effect of cytokine production by macrophages.

Methods: The expression of inhibitory receptors that interact with HLA-G1, immunoglobulin-like transcript 2 (ILT2), ILT4 and KIR2DL4 (CD158d) on in vitro generated macrophages were examined by flow cytometry. Complementary DNA (cDNA) of HLA-G1, HLA-E and human β2-microglobulin (hβ2m) were prepared and transfected into swine endothelial cells (SECs). The expression of the transgenic genes was evaluated by flow cytometry, and macrophage-mediated SEC cytolysis was assessed using the macrophages.

Results: In vitro generated macrophages expressed not only ILT2 and ILT4 but CD158d as well. The transgenic HLA-G1 on SECs indicated significant suppression in macrophage-mediated cytotoxicity, which was equivalent to that of transgenic HLA-E. Furthermore, the results on real time PCR and ELISA revealed that transgenic HLA-G1 induces the anti-inflammatory cytokines, such as IL-10 and TGF-β, and suppresses iNOS mRNA expression, indicating that transgenic HLA-G1 has suppressive effects in a broad range of transplant rejection.

Conclusion: These results indicate that generating HLA-G1 transgenic pigs can protect porcine grafts from macrophage-mediated cytotoxicity.
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http://dx.doi.org/10.1016/j.trim.2014.12.004DOI Listing
March 2015

N-glycans: phenotypic homology and structural differences between myocardial cells and induced pluripotent stem cell-derived cardiomyocytes.

PLoS One 2014 30;9(10):e111064. Epub 2014 Oct 30.

Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

Cell surface glycans vary widely, depending on cell properties. We hypothesized that glycan expression on induced pluripotent stem cells (iPSCs) might change during cardiomyogenic differentiation toward the myocardial phenotype. N-glycans were isolated from iPSCs, iPSC-derived cardiomyocytes (iPSC-CM), and original C57BL/6 mouse myocardium (Heart). Their structures were analyzed by a mapping technique based on HPLC elution times and MALDI-TOF/MS spectra. Sixty-eight different N-glycans were isolated; the structures of 60 of these N-glycans were identified. The quantity of high-mannose type (immature) N-glycans on the iPSCs decreased with cardiomyogenic differentiation, but did not reach the low levels observed in the heart. We observed a similar reduction in neutral N-glycans and an increase in fucosylated or sialyl N-glycans. Some structural differences were detected between iPSC-CM and Heart. No N-glycolyl neuraminic acid (NeuGc) structures were detected in iPSC-CM, whereas the heart contained numerous NeuGc structures, corresponding to the expression of cytidine monophosphate-N-acetylneuraminic acid hydroxylase. Furthermore, several glycans containing Galα1-6 Gal, rarely identified in the other cells, were detected in the iPSC-CM. The expression of N-glycan on murine iPSCs changed toward the myocardial phenotype during cardiomyogenic differentiation, leaving the structural differences of NeuGc content or Galα1-6 Gal structures. Further studies will be warranted to reveal the meaning of the difference of N-glycans between the iPSC-CM and the myocardium.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111064PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214687PMC
June 2015

A structural analysis of N-glycans of neonatal porcine islet-like cell clusters (NPCC).

Transpl Immunol 2014 Jun 24;31(1):48-53. Epub 2014 May 24.

Department of Life Science, Meiji University, Higashisanda 1-1-1, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.

Background: N-glycans isolated from neonatal porcine islet-like cell clusters (NPCCs) were analyzed by a mapping technique, to examine the differences in glycosylation and antigenicity between adult pig islets (APIs) and NPCCs.

Methods: NPCCs were isolated from 1-to-3 day-old neonatal wild-type pigs and cultured for 9 days, using the technique described by Korbutt et al. The extract was proteolyzed by treatment with a chymotrypsin and trypsin mixture and further digested with glycoamidase A to release the N-glycans. After the removal of the peptide materials, the reducing ends of the N-glycans were derivatized with 2-aminopyridine. This mixture was applied to DEAE, amide and ODS columns. PA-oligosaccharides were also subjected to MALDI TOF-MS analysis.

Results: The NPCC glycans were comprised of 14 neutral, 5 mono-sialyl and 5 di-sialyl glycans. As a feature of the N-glycans of NPCC, NPCC contained large amounts of high mannose structures. On the other hand, all of the hybrid and complex types contained a Fucα1-6GlcNAc structure, but were not modified with sulfate residues. Among them, the NPCC preparation contained five neutral and two mono-sialyl glycans and two di-sialyl glycans that were not typically found in adult islets, and seven of these nine were not detected in human islets. Moreover, most of the structures could be clearly identified in this study.

Conclusions: The data herein will be helpful for future studies of the glycoantigen associated with NPCC.
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http://dx.doi.org/10.1016/j.trim.2014.05.003DOI Listing
June 2014

Monocytic suppressor cells derived from human peripheral blood suppress xenogenic immune reactions.

Xenotransplantation 2014 Jan-Feb;21(1):46-56. Epub 2013 Dec 24.

Department of Surgery, Osaka University Graduate School of Medicine, Osaka, Japan.

Background: Myeloid-derived suppressor cells (MDSC) were initially found to contribute to the immunosuppression in tumor patients and have recently been recognized as a subset of innate immune cells that are capable of regulating adaptive immunity. A variety of innate immune stimuli such as Lipopolysaccharide (LPS), which act as a double-edged sword, induce both the maturation of dendritic cells (DC) and the expansion of MDSCs.

Methods: In this study, we isolated MDSCs from peripheral blood mononuclear cells and examined the suppressive effect of MDSCs against cytotoxic T lymphocyte (CTL)-mediated xenocytotoxicity.

Results: Peripheral blood monocytes cultured in the presence of GM-CSF and IL-4 were stimulated with polyiosinic-polycytidylic acid [poly (I:C)] or LPS. Flow cytometric analyses revealed that LPS and poly I:C stimulation allows the CD33(+) CD14(+) HLA-DR(-) subset to be significantly increased. To assess the suppressive capacity of MDSCs in xenotoxicity, CTL assay was performed. Poly (I:C)-activated MDSCs dramatically suppressed the CTL xenocytotoxicity. Phagocytosis assays revealed that activated MDSCs aggressively phagocytose the xenogenic CTLs. Characterization of MDSCs by real-time PCR revealed that poly (I:C) and LPS-stimulated MDSCs expressed significant amounts of mRNA for indolamine 2,3-dioxygenase (IDO) compared to untreated MDSCs. Furthermore, when MDSCs were incubated with the IDO inhibitor, the MDSC-induced suppression of xenocytotoxicity was abolished. Taken together, the possibility that activated MDSCs could induce apoptosis in xenogenic CTLs via an IDO-dependent manner and aggressively phagocytose apoptotic CTLs cannot be excluded.

Conclusion: These findings indicate that MDSCs have a great deal of potential as a therapeutic strategy for dealing with xenograft rejection. Further investigations of the underlying mechanisms will facilitate the development of this therapeutic strategy.
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http://dx.doi.org/10.1111/xen.12067DOI Listing
July 2015

A comparison of the main structures of N-glycans of porcine islets with those from humans.

Glycobiology 2014 Feb 7;24(2):125-38. Epub 2013 Oct 7.

Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, Osaka, Japan.

After producing α1-3-galactosyltransferase knockout (GKO) pigs, most of the organs of these pigs showed less antigenicity to the human body. However, wild-type adult pig islets (API) that originally contained negligible levels of α-galactosidase now showed a clear antigenicity to human serum. In this study, N-glycans were isolated from both APIs and human islets. Their structures were then analyzed by a mapping technique based on their high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric data. Both preparations contained substantial amounts of high-mannose structures. The N-glycans from human islets were separated into 17 neutral, 8 mono-sialyl and 4 di-sialyl glycans, and the API glycans were comprised of 11 neutral, 8 mono-sialyl, 3 di-sialyl, 2 mono-sulfated, 3 mono-sialyl-mono-sulfated and 1 di-sulfated glycans. Among them, the API preparation contained one neutral, five mono-sialyl glycans and six sulfated glycans that were not detected in human islets. The structures of 9 of these 12 could be clearly determined. In addition, a study of the sulfate-depleted API suggests that sulfate residues could be antigenic to humans. The data herein will be helpful for future studies of the antigenicity associated with API.
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http://dx.doi.org/10.1093/glycob/cwt088DOI Listing
February 2014

The suppression of inflammatory macrophage-mediated cytotoxicity and proinflammatory cytokine production by transgenic expression of HLA-E.

Transpl Immunol 2013 Dec 29;29(1-4):76-81. Epub 2013 Aug 29.

Department of Surgery, Osaka University Graduate School of Medicine, Osaka, Japan. Electronic address:

Background: Macrophages participate in xenogenic rejection and represent a major biological obstacle to successful xenotransplantation. The signal inhibitory regulatory protein α (SIRPα) receptor was reported to be a negative regulator of macrophage phagocytic activity via interaction with CD47, its ligand. Because a majority of human macrophages express the inhibitory receptor CD94/NKG2A, which binds specifically to the human leukocyte antigen (HLA)-E and contains immunoreceptor tyrosine-based inhibition motifs (ITIMs), the inhibitory function of HLA class I molecules, HLA-E, on macrophage-mediated cytolysis was examined. The suppressive effect against proinflammatory cytokine production by macrophages was also examined.

Methods: Complementary DNA (cDNA) of HLA-E, and CD47 were prepared and transfected into swine endothelial cells (SEC). The expression of the modified genes was evaluated by flow cytometry and macrophage-mediated cytolysis was assessed using in vitro generated macrophages.

Results: Transgenic expression of HLA-E significantly suppressed the macrophage-mediated cytotoxicity. HLA-E transgenic expression demonstrated a significant suppression equivalent to CD47 transgenic expression. Furthermore, transgenic HLA-E suppressed the production of pro-inflammatory cytokines by inflammatory macrophages.

Conclusions: These results indicate that generating transgenic HLA-E pigs might protect porcine grafts from, not only NK cytotoxicity, but also macrophage-mediated cytotoxicity.
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http://dx.doi.org/10.1016/j.trim.2013.08.001DOI Listing
December 2013

A lectin microarray study of glycoantigens in neonatal porcine islet-like cell clusters.

J Surg Res 2013 Jul 8;183(1):412-8. Epub 2013 Jan 8.

Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

Background: Besides α-Gal expression, the differences of glycosylation and antigenicity between adult pig islets (APIs) and neonatal porcine islet-like cell clusters (NPCCs) are altogether unclear. In this study, lectin microarray analyses of NPCCs were performed and the results compared with the corresponding values for wild-type APIs and NPCCs from α-Gal transferase knockout (GalT-KO) pig.

Methods: NPCCs were isolated from 1-3-d-old neonatal wild-type pigs and cultured for 1 d, 5 d, and 9 d, using a previously described technique. Alternatively, the isoration of APIs were isolated based on the method for human islets.

Results: In a comparison between NPCCs and APIs, all of the NPCCs showed higher signals for Sambucus nigra, Sambucus sieboldiana, and Trichosanthes japonica I and the binding of α2,6 sialc acid, whereas the APIs showed stronger signals for Lotus tetragonolobus, Aleuria aurantia, Narcissus pseudonarcissus, and Galanthus nivalis, suggesting that APIs contain high levels of high-mannose forms. Among the NPCCs, NPCC (day1) appeared to be richer than the others in Lotus tetragonolobus, Narcissus pseudonarcissus, Galanthus nivalis, and Urtica dioica, implying the presence of high-mannose forms. However, as a whole, the signals for many lectins for NPCCs were very similar. The NPCCs from a GalT-KO pig indicated not only the downregulation of α-Gal expression but α-GalNAc as well, and α2-6 sialic acid was upregulated.

Conclusions: The results reported herein contain useful information for the future production of immunomodified pigs with less antigenicity than GalT-KO pigs toward clinical applications of NPCCs.
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http://dx.doi.org/10.1016/j.jss.2012.12.037DOI Listing
July 2013

Lectin array analysis for wild-type and α-Gal-knockout pig islets versus healthy human islets.

Surg Today 2013 Dec 3;43(12):1439-47. Epub 2013 Apr 3.

Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan,

Purpose: We performed lectin microarray analyses of islets from wild-type (WT) pigs and α1-3galactosyltransferase gene knockout (GKO) pigs and compared the results with the corresponding values for islets from healthy humans.

Methods: Islets were isolated from the pancreas. After sonication and centrifugation, the proteins in the supernatant from each islet were labeled with Cy3 and applied to a lectin array.

Results: Despite negligible expression of the Gal antigen on the adult pig islets (APIs), GKO-islets showed weaker signals, not only for GS-I-B4 but also for PNA, WFA, PTL-I, and GS-I-A4, than the WT islets, indicating reduced contents of α-linked GalNAc and Galβ1-3GalNAc. In comparing the islets of pigs vs. humans, human islets showed stronger signals for UEA-I, AAL, TJA-II, EEL, WFA, HPA, DBA, SBA and PTL-I, indicating that besides ABO blood type antigens, high levels of fucose and α-linked GalNAc are present. On the other hand, the high mannose form was very rich in the APIs.

Conclusion: GKO reduced alpha-linked GalNAc, despite negligible expression of the Gal antigen on WT-API. On the other hand, the high-mannose form was richer in both APIs than in healthy human islets. These results provide useful information for future studies.
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http://dx.doi.org/10.1007/s00595-013-0569-6DOI Listing
December 2013

Ontogeny and localization of the cells produce IL-2 in healthy animals.

Cytokine 2013 Mar 16;61(3):831-41. Epub 2013 Jan 16.

Department of Microbiology and Immunology, Loyola University Chicago, 2160 South First Ave., Maywood, IL 60153, USA.

IL-2 is a growth factor for activated T cells and is required for maintenance of naturally arising regulatory T cells (nTregs). Mice defective in IL-2/IL-2 receptor signaling pathways have impaired nTregs and suffer from lymphoproliferative disorders, suggesting that IL-2 is present and functional in healthy animals. However, the cellular source of IL-2 is currently unknown. To determine which cells produce IL-2 in healthy animals, we established mice carrying cre gene knock in at the il-2 locus (termed IL-2(cre)). When IL-2(cre) mice were crossed with EGFP reporter mice, EGFP was exclusively expressed by a fraction of CD4 T cells present in both lymphoid and non-lymphoid tissues. Live imaging of IL-2(cre) mice that carry the luciferase reporter showed concentrated localization of luciferase(+) cells in Peyer's patches. These cells were not observed in new born mice but appeared within 3days after birth. Reduction of antigen receptor repertoire by transgene expression reduced their number, indicating that recognition of environmental antigens is necessary for generation of these IL-2 producers in healthy animals. A substantial fraction of EGFP(+) cells also produce IL-10 and IFN-γ, a characteristic profile of type 1 regulatory T cells (Tr1). The data suggest that a group of Tr1 cells have addition roles in immune homeostasis by producing IL-2 along with other cytokines and help maintaining Tregs.
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http://dx.doi.org/10.1016/j.cyto.2012.11.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3595346PMC
March 2013

The blocking of CXCR3 and CCR5 suppresses the infiltration of T lymphocytes in rat renal ischemia reperfusion.

Nephrol Dial Transplant 2012 Oct 17;27(10):3799-806. Epub 2012 Aug 17.

Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

Background: Recent studies have identified T cells and natural killer T (NKT) cells as important mediators in renal ischemia-reperfusion (I/R) injury. The recruitment of these cells is induced by chemotaxis factors. We investigated the effects of blocking CXCR3 and CCR5 by an antagonist (TAK) using a rat renal I/R injury model.

Methods: The Sprague-Dawley rats were either subjected to sham operation or left renal occlusion for 45 min followed by reperfusion and contralateral nephrectomy. The control or TAK groups were, respectively, injected phosphate-buffered saline or TAK at 30 min prior to clamp. Serum creatinine, tubular injury, chemokines expression and infiltrating cells were assessed.

Results: TAK treatment significantly suppressed the elevation in serum creatinine (sham 0.40 ± 0.05 mg/dL, control 2.86 ± 0.67 mg/dL, TAK 1.60 ± 0.73 mg/dL) and resulted in a lower tubular injury score compared with the control group (sham 0, control 4.8 ± 0.3, TAK 3.3 ± 1). The mRNA expression of chemokines that bind to CXCR3 and CCR5 in the post-ischemic kidneys was elevated at 1 h after reperfusion in each group. Moreover, the infiltration of CD4+ T cells and CD8+ NKT cells in the control group increased compared with the sham group and TAK injection significantly suppressed the number of CD4+ T cells (sham 13.5 ± 3.5 × 10(4) cells, control 28.9 ± 15.4 × 10(4) cells, TAK 11.8 ± 3.5 × 10(4) cells) and the number of CD8+ NKT cells (sham 11.7 ± 5.4 × 10(4) cells, control 30.1 ± 8.6 × 10(4) cells, TAK 11.8 ± 2.9 × 10(4) cells).

Conclusions: These findings suggest that the blocking of CXCR3 and CCR5 suppress the infiltration of T cells and NKT cells and have a protective effect on kidneys that are injured by I/R.
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http://dx.doi.org/10.1093/ndt/gfs360DOI Listing
October 2012