Noritaka Koga

Blockade of the Interaction Between PD-1 and PD-L1 Accelerates Graft Arterial Disease in Cardiac Allografts

Background—Programmed death-1 (PD-1), a member of the CD28 family, has been identified. PD-1 is involved in the negative regulation of some immune responses. We evaluated the role of PD-ligand 1 (PD-L1) in graft arterial disease (GAD) of cardiac allografts and in smooth muscle cells (SMCs). Methods and Results—C57BL/6 murine hearts were transplanted into B6.C-H2KhEg mice for examination of GAD. PD-L1 was expressed in SMCs of the thickened intima in the graft coronary arteries, and administration of anti–PD-L1 monoclonal antibody (mAb) enhanced the progression of GAD (luminal occlusion: 55±5.0% versus 9.8±4.3%, P<0.05). The expressions of interferon γ (IFN-γ) and tumor necrosis factor α of cardiac allografts were upregulated in response to anti–PD-L1 mAb treatment. In vitro, PD-L1 expression was induced in SMCs in response to IFN-γ stimulation. Sensitized splenocytes increased SMC proliferation, and anti–PD-L1 mAb in combination with IFN-γ stimulation increased this proliferation. Conclusions—The PD-L1 pathway regulates both the proliferation of SMCs and GAD. Thus, control of this interaction is a promising strategy for suppression of GAD.

Induction of immunologic tolerance to cardiac allograft by simultaneous blockade of inducible co-stimulator and cytotoxic T-lymphocyte antigen 4 pathway.

Background. Inducible co-stimulator (ICOS) is one of the most recently described members of the CD28 family, and it plays an important role in immune responses. To investigate the role of ICOS in allograft rejection, the authors studied graft survival after cardiac transplantation in mice. Methods. Hearts from BALB/c mice were transplanted into C3H/He mice. Immunohistochemical staining and flow cytometry were performed. Monoclonal antibody to ICOS or ICOS-immunoglobulin (Ig) was injected intraperitoneally. The authors performed mixed lymphocyte reaction (MLR). Results. ICOS was expressed strongly by graft-infiltrating cells during rejection of the allograft. Blockade of the ICOS pathway with anti-ICOS antibody and ICOSIg significantly prolonged graft survival time relative to that in untreated mice; however, all cardiac allografts were eventually rejected by a single treatment. Treatment with both ICOSIg and cytotoxic T-lymphocyte antigen 4 (CTLA4) Ig induced not only long-term acceptance of the cardiac allograft but also donor-specific tolerance, which was shown by acceptance of donor but not third-party skin. Graft arterial intimal hyperplasia in these cardiac allografts was remarkably less than that in cardiac allografts treated with tacrolimus. Addition of anti-ICOS antibody or ICOSIg to MLR resulted in inhibition of T-cell proliferation. Conclusions. Inhibition of T-cell proliferation with ICOSIg and CTLA4Ig was more effective than that with ICOSIg alone. Thus, ICOS appears to be an important regulator of T-cell activation, and may be an effective therapy in clinical cardiac transplantation.

Hepatocyte Growth Factor Ameliorates the Progression of Experimental Autoimmune Myocarditis. A Potential Role for Induction of T Helper 2 Cytokines

Hepatocyte growth factor (HGF) plays a role in cell protection, antiapoptosis, antifibrosis, and angiogenesis. However, the role of HGF in the immune system is not well defined. We examined the influence of HGF on T cells and the effects of HGF therapy in acute myocarditis. Lewis rats were immunized on day 0 with cardiac myosin to establish experimental autoimmune myocarditis (EAM). Human HGF gene with hemagglutinating virus of the Japan-envelope vector was injected directly into the myocardium on day 0 or on day 14 (two groups of treated rats). Rats were killed on day 21. Expression of c-Met/HGF receptor in splenocytes and myocardial infiltrating cells was confirmed by immunohistochemical staining or FACS analysis. Myocarditis-affected areas were smaller in the treated rats than in control rats. Cardiac function in the treated rats was markedly improved. An antigen-specific T cell proliferation assay was done with CD4-positive T cells isolated from control rats stimulated with cardiac myosin. HGF suppressed T cell proliferation and production of IFN-γ and increased production of IL-4 and IL-10 secreted from CD4-positive T cells in vitro. Additionally, TUNEL assay revealed that HGF reduced apoptosis in cardiomyocytes. HGF reduced the severity of EAM by inducing T helper 2 cytokines and suppressing apoptosis of cardiomyocytes. HGF has potential as a new therapy for myocarditis.

Pioglitazone Prevents Acute and Chronic Cardiac Allograft Rejection

Background— Peroxisome proliferator–activated receptor-&ggr; plays an important role in regulating inflammation. Although cardiac transplantation is an established therapy for patients with end-stage heart disease, allograft rejection is a major concern for long-term survival. We investigated the role of pioglitazone in acute and chronic rejection in a murine cardiac transplantation model. Methods and Results— We performed heterotopic murine cardiac transplantation in total allomismatch or major histocompatibility complex class II–mismatched combinations. Recipient mice were given standard chow or chow containing pioglitazone (3 mg · kg−1 · d−1) beginning 1 day before cardiac transplantation. In acute rejection, animals given pioglitazone showed significantly longer cardiac allograft survival than control mice (mean survival time, 34.6±7.8 versus 8.4±0.4 days; P<0.003). Treatment with pioglitazone significantly suppressed graft expression of interferon-&ggr; and monocyte chemoattractant protein-1. In chronic rejection, neointimal hyperplasia was significantly lower in allografts from mice treated with pioglitazone (luminal occlusion, 25.1±8.8%) than in those from control mice (65.8±7.3%, P<0.001). Pioglitazone-treated allografts showed significantly reduced expression of interferon-&ggr;, interleukin-10, and monocyte chemoattractant protein-1. We performed mixed lymphocyte reactions and in vitro proliferation assays of smooth muscle cells. Addition of pioglitazone to mixed lymphocyte reactions inhibited proliferation of T cells. Smooth muscle cells showed significant proliferation when cocultured with activated splenocytes. This proliferation was significantly inhibited by the addition of pioglitazone (1 &mgr;mol/L). Conclusions— Pioglitazone prolongs allograft survival and attenuates neointimal hyperplasia through the suppression of proliferation of smooth muscle cells. Pioglitazone may be a novel means to prevent acute and chronic allograft rejection.

Attenuation of Graft Arterial Disease by Manipulation of the LIGHT Pathway

Objective—The tumor necrosis factor (TNF) superfamily member LIGHT, which binds herpes virus entry mediator (HVEM) and lymphotoxin β receptor (LTβR), plays important roles in regulating the immune response. To clarify the mechanism underlying graft arterial disease (GAD), we investigated the role of the LIGHT pathway in the progression of GAD. Methods and Results—Hearts from Bm12 mice were transplanted into C57BL/6 (B/6) mice (class II mismatch). Recipients were injected intraperitoneally with HVEMIg (100 μg per treatment) every 7 days for 8 weeks. Treatment with HVEMIg significantly attenuated GAD (luminal occlusion=16.5±7.7% versus control allograft=62.6±12.1%, P <0.05), and significantly decreased intragraft IL-4, IL-6, and interferon-γ (IFN-γ) mRNA expression compared with controls. LTβR was expressed in smooth muscle cells (SMCs) with or without cytokine stimulation, whereas HVEM was detected in SMCs stimulated by IFN-γ. Coculture of SMCs with T cells after transplantation induced SMC proliferation, and addition of HVEMIg resulted in inhibition of SMC proliferation. Conclusions—These results indicate that the LIGHT pathway plays important roles in the regulation not only of T-cell activation but also of SMC proliferation. Blockade of the LIGHT pathway is a promising avenue for the prevention of GAD.

Pitavastatin suppresses acute and chronic rejection in murine cardiac allografts.

Introduction. HMG-CoA reductase inhibitors play several roles in the maintenance of organ transplants. We investigated the role of pitavastatin, a potent and newly developed HMG-CoA reductase inhibitor, in cardiac allograft rejection and mechanism of graft arterial disease (GAD) suppression. Methods. Balb/c mice hearts were transplanted into C3H/He mice (a full allomismatch combination) to assess acute rejection or C57BL/6 hearts into B6.C-H2KhEg (a class II mismatch combination) to examine the extent of GAD. Pitavastatin was administered orally to mice everyday (3 mg/kg/day). To assess the effect in acute rejection, mixed lymphocyte reaction was performed and cytokine mRNA expression was examined with ribonuclease protection assay. Results. Pitavastatin significantly prolonged allograft survival. Lymphocyte proliferation was inhibited by pitavastatin, and RPA showed down-regulation of interleukin-6 in pitavastatin-treated cardiac allografts. Allografts in the pitavastatin-treated group after 8 weeks showed less GAD compared with the control group. In vitro, pitavastatin suppressed the smooth muscle cell proliferation in response to activated T cells and inhibited extracellular signal-regulated kinase 1/2 activation. Conclusion. Pitavastatin could be effective in the suppression of acute rejection and GAD development in cardiac transplantation.

Critical Role of Inducible Costimulator Signaling in the Development of Arteriosclerosis

Objective—Proliferation and migration of smooth muscle cells (SMCs) and migration and accumulation of monocytes and T cells are landmark events in the development of arteriosclerosis. SMC proliferation in the intima induces interruption of blood flow and results in ischemia and graft rejection. Inducible costimulator (ICOS) is a major costimulator of T cell activation. However, the effect of costimulatory molecules on the formation of neointimal hyperplasia has not been fully elucidated. We examined the role of the ICOS pathway in SMC proliferation. Methods and Results—ICOS ligand (ICOSL) was detected in SMCs stimulated by interleukin (IL)-1&bgr;, and coculture of stimulated SMCs and activated T cells induced SMC proliferation. Inhibition of the ICOS pathway resulted in inhibition of SMC proliferation. In models of transplantation and vascular injury, ICOSL was induced in SMCs in the neointima. Expression of IL-1&bgr;, a key inducer of ICOSL expression, was significantly reduced in mice treated with anti-ICOS antibody or soluble form of ICOS (ICOSIg) and in ICOS-deficient mice. Inhibition of the ICOS pathway significantly suppressed neointimal thickening. Conclusions—These results indicate that ICOS on activated T cells contributes to neointimal formation through the regulation of SMC proliferation. These findings provide insights into new therapeutic strategies for arteriosclerosis.

Gene Therapy for Heart Transplantation-Associated Acute Rejection, Ischemia / Reperfusion Injury and Coronary Arteriosclerosis

Acute cardiac allograft rejection is still a major complication after heart transplantation. Acute rejection usually responds to conventional immunosuppressants, however, the nonspecific nature of the immunosuppression and the toxicities of the drugs can be life threatening and may compromise the recipients quality of life. In addition, cardiac allograft arteriosclerosis or chronic rejection limits the long-term survival of recipients. Such conditions cannot be prevented with conventional therapies. To overcome acute and chronic rejection of cardiac allograft as well as ischemia/reperfusion injury associated with organ preservation many novel approaches have been proposed. Gene transfection of the donor organ during organ preservation is an attractive method, because the transfected genes would not affect recipients and treatment could be delivered specifically to the site of inflammation. This method could be useful to prevent graft failure without systemic adverse effects. Here we shall review current advances in gene therapies to prevent and treat organ failure of transplanted allografts.