Ryo Gotoh

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.

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.

E-Selectin Blockade Decreases Adventitial Inflammation and Attenuates Intimal Hyperplasia in Rat Carotid Arteries After Balloon Injury

Objective—Inflammation is one of the initial repair processes after vascular injury. E-selectin facilitates adherence of leukocytes to vascular endothelium at the site of inflammation. Because the role of E-selectin in this process is not fully understood, we studied the role of E-selectin in vascular injury with a flow chamber model and a rat model of carotid artery injury. Methods and Results—We established a rat aortic endothelial cell (RAEC) culture system from the aortas of adult male rats. When rat myelomonocytes were suspended in a flow chamber, rolling and adhesion to lipopolysaccharide (LPS)-stimulated RAECs were observed. Cell rolling and adhesion were greatly reduced by addition of anti–E-selectin monoclonal antibody (mAb). We then induced balloon injury in the left carotid arteries of rats. E-selectin expression was enhanced in endothelial cells at adventitial small vessels 7 days after injury. Rats with balloon injury were injected intraperitoneally with anti–E-selectin mAb for 8 days. Inflammatory cell infiltration was reduced by anti–E-selectin mAb treatment at the adventitia at 7 days after injury. This reduction was associated with attenuation of intimal hyperplasia in the rats treated with the mAb. Conclusions—These data suggest that E-selectin regulates adventitial inflammation through leukocyte adhesion and contributes to the process of intimal hyperplasia after balloon injury.