Ko Okumura

Role of Fas-Fas ligand interactions in the immunorejection of allogeneic mouse corneal transplants.

BACKGROUNDnThe expression of Fas ligand (FasL) in the eye has been proposed to be an important component of ocular immune privilege. Since the unusually favorable outcome of corneal transplantation is thought to result from the immune privilege of the eye, examination of the function of FasL on corneal allografts would be a test of that hypothesis.nnnMETHODSnTo investigate the role of Fas-FasL interaction in corneal allografts, orthotopic corneal transplantation was performed using C57BL/6 (B6, FasL+) and B6-gld (FasL-) mice as cornea donors and BALB/c mice as recipients. The rejection rate of B6-gld grafts (FasL- group) was compared with that of normal B6 control corneas.nnnRESULTSnThe rejection rate at the final observation (8 weeks) in the FasL- group (89%) was significantly higher than in the FasL+ control group (47%). FasL expression was found on the corneal endothelium by staining with anti-FasL monoclonal antibodies. The TdT-mediated dUTP nick-end labeling assay revealed that apoptotic cells were attached to the endothelium in the control group but not in the FasL- groups.nnnCONCLUSIONSnApoptosis of infiltrating cells on the corneal endothelium resulting from Fas-FasL interaction plays an important role in the high success rate of corneal transplantation.

Preferential elimination of CD28+ T cells in systemic lupus erythematosus (SLE) and the relation with activation-induced apoptosis

CD28 on T cells provides a potent costimulatory signal for T cell activation. Down‐regulation of CD28 on peripheral T cells has been reported in certain clinical conditions, but full studies on the mechanism and biological significance have not been performed. Our extensive phenotype analysis of peripheral blood lymphocytes (PBL) from SLE patients revealed that the absolute number of CD28+ T cells of both CD4 and CD8 phenotypes was selectively decreased, while that of CD28− T cells was maintained. CD28+ T cells from SLE patients exhibited mostly normal proliferative responses to both CD28‐dependent and ‐independent stimulations. In contrast, CD28− T cells were hyporesponsive to anti‐CD3 stimulation in both SLE and normal controls. These results implied that the selective decrease of CD28+ T cells in SLE does not result from a hyporesponsiveness of CD28+ T cells. To investigate the reason for the selective loss of CD28+ T cells, we determined the appearance of apoptotic cells in culture with or without anti‐CD3 stimulation. Apoptotic cells defined by merocyanine (MC)540 were gradually increased from 12u2003h to 24u2003h. Anti‐CD3‐induced apoptosis of CD28+ T cells was significantly accelerated in SLE, whereas apoptosis of CD28− T cells was hardly detected in both SLE and normal controls. Comparative analysis between CD28+ and CD28− T cells on CD95 (Fas) and Bcl‐2 expression, which are related to activation‐induced cell death (AICD), did not show a major difference, although CTLA4, which has been demonstrated to transmit an apoptosis‐inducing signal, was expressed only on CD28+ T cells. Our results suggest that CD28‐mediated costimulation influences T cell susceptibility to AICD and may be involved in T cell lymphopenia in SLE.

Fas-mediated cytotoxicity--a new immunoregulatory and pathogenic function of Th1 CD4+ T cells.

T cell-mediated cytotoxicity not only constitutes an important component of specific effector mechanisms in immune surveillance against virus-infected or transformed cells but also has been implicated in the tissue damage associated with some autoimmune diseases and graft-versus-host disease (GVHD). The molecular mechanisms of T cell cytotoxicity had not been fully understood and were a subject for controversy until quite recently So far, two models have been proposed the granule exocytosis model and the induced suicide model (Henkart 1985, Young et al. 1988, Yagita et al. 1992). The effector molecules in the former, such as perforin and granzymes, have been identified recently and now it has been established by gene-targeting studies that the perforin-dependent mechanism constitutes one primary pathway of T cell cytotoxicity (Kagi et al. 1994a., Lowin et al. 1994a, Walsh et al. 1994b). However, such studies also revealed the presence of a perforin-independent mechanism, which now turns out to be mediated by the Fas molecule on the target cells and its ligand (FasL) on the effector T cells (Kagi et al. 1994b, Lowin et al. 1994b. Kojima et al. 1994). This review describes the expression of FasL in T cell subpopulations and discuss the physiological role of Fas-mediated cytotoxicity in the immune system.

Protection of islet allografts transplanted together with Fas ligand expressing testicular allografts

Summary Fas ligand (FasL) is highly expressed in testicular tissues and thought to be responsible for protection from allograft rejection by inducing apoptosis of anti-graft activated T cells. FasL-expressing islets have been shown to induce a granulocyte-mediated inflammatory reaction. We investigated whether a graft can be protected from alloimmune responses by manipulating the Fas/FasL-system. We transplanted allogeneic islets under the kidney capsule of streptozotocin-induced diabetic mice together with testicular tissue. Significant prolongation of survival of C3H islet allograft was observed in C57BL/6 (B6) recipients transplanted with C3H testicular tissue, but not in those transplanted with C3H-gld testicular tissue expressing non-functional FasL. No significant prolongation was observed in B6-lpr recipients expressing non-functional Fas. Immunohistochemical staining of C3H testicular tissue in the composite graft showed a high expression of FasL, but not that of the C3H-gld testicular tissue. In situ terminal deoxynucleotidyl transferase-mediated dUDP-biotin catalysed DNA nick-end labelling (TUNEL) staining of a composite graft of C3H islet and testicular tissue in B6 recipients demonstrated extensive apoptosis of infiltrating mononuclear cells around the graft. The protective effect of C3H testicular tissue was abrogated when anti-FasL monoclonal antibody was administered i. p. postoperatively. Our results suggest that FasL-positive testicular allografts protect composite islet allografts and indicate that manipulation of Fas/FasL mediated apoptosis is a suitable strategy for controlling rejection of islet allografts. [Diabetologia (1998) 41: 315–321]

Expression of Costimulatory Molecule CD40 in Murine Heart With Acute Myocarditis and Reduction of Inflammation by Treatment With Anti-CD40L/B7-1 Monoclonal Antibodies

Evidence has accumulated that antigen-specific T cells infiltrate the heart and play an important role in the pathogenesis of viral myocarditis. Costimulatory molecules such as B7s and CD40 expressed on antigen-presenting cells are known to play a critical role for antigen-specific T-cell activation to occur. To investigate the role for a costimulatory molecule, CD40, in the development of acute viral myocarditis, we first analyzed the expression of CD40 in the hearts of mice with acute viral myocarditis induced by Coxsackievirus B3. We also evaluated the induction of CD40 in cultured cardiac myocytes treated with interferon gamma in vitro. Second, we analyzed the cytokine production by cultured cardiac myocytes by stimulation with anti-CD40 monoclonal antibody (mAb) in vitro. Third, we examined the effects of in vivo administration of anti-CD40L/B7-1 mAbs on the development of acute viral myocarditis. We found that Coxsackievirus B3-induced murine acute myocarditis results in enhanced expression of CD40 on cardiac myocytes. The expression of CD40 on cardiac myocytes could be induced by interferon gamma in vitro. We also found that the production of interleukin-6 by cardiac myocytes was stimulated with anti-CD40 mAb and that in vivo anti-CD40L/B7-1 mAb treatment significantly decreased the myocardial inflammation. Our findings strongly suggest that CD40 plays an important role in the development of acute viral myocarditis and raise the possibility of immunotherapy with anti-CD40L/B7-1 mAbs to prevent T cell-mediated myocardial damage in viral myocarditis.

Expression of Costimulatory Molecules B7–1, B7–2, and CD40 in the Heart of Patients With Acute Myocarditis and Dilated Cardiomyopathy

BACKGROUNDnIn patients with acute myocarditis and dilated cardiomyopathy (DCM), we previously reported that antigen-specific T cells infiltrate the heart and play an important role in the myocardial damage involved. For antigen-specific T-cell activation to occur, it is necessary for T cells to receive a costimulatory signal provided by costimulatory molecules expressed on antigen-presenting cells (APCs) as well as the main signal provided by binding of T-cell receptors to the antigen.nnnMETHODS AND RESULTSnTo investigate the roles of the costimulatory molecules B7-1, B7-2, and CD40 in the development of acute myocarditis and DCM, we analyzed the expression of these antigens in the myocardial tissues of patients with acute myocarditis and DCM. We also examined the expression of a cytolytic factor, perforin, in the infiltrating cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, because both killer lymphocytes are thought to damage B7-1-expressing APCs. We found that B7-1, B7-2, and CD40 were moderately to strongly expressed in the cardiac myocytes of patients with acute myocarditis. Weak to moderate expression of these antigens was also found in the cardiac myocytes of patients with DCM. There was infiltration of perforin-expressing CTLs and NK cells in the myocardial tissues of patients with acute myocarditis and DCM.nnnCONCLUSIONSnOur findings strongly suggest that expression of B7-1, B7-2, and CD40 antigens on cardiac myocytes may make them APCs for CTLs and NK cells and that they may play an important role in the direct myocardial damage by these killer cells in acute myocarditis and DCM.

Cell Surface Antigen Marking the Stages of Murine T Cell Ontogeny and its Functional Subsets

The gene products expressed on the murine T lymphocyte, such as Tla, Thy-1 and Lyt antigens are known to be extremely useful markers in classifying functional T cell subsets and studying the T cell differentiation pathway. Since the discovery of the T cell antigens, which are selectively expressed on certain T cell subsets, attempts to discover whether the T cell antigens are involved in the immune response as receptor molecules have been extensive. However, we still have no definite knowledge of the surface antigens which link to the T cell receptor for foreign antigens and self-MHC molecules, although recent molecular genetic study is clarifying the long-term mystery of the diversification of the T cell receptor. In the past two decades, the T cell antigens as markers have broadly contributed to dissect the cellular regulatory network by T cell subpopulations in the immune response, indicating that terminal inductive or suppressive signals of T cells are required via the complicated processes of a series of cellular interactions. In addition to the investigation of functional T ceil subsets in immune regulation, we have been studying differentiation and maturation pathways of the T cell lineage in the thymus by using surface antigens expressed on the T cell or pre-T cell as markers. We shall review two different series of studies in which the T cell antigens are applied as markers; firstly, for the analysis of T cell differentiation in the fetal thymus, and secondly for the functional analysis of the mature T cell in the immune response.

A crucial role of host CD80 and CD86 in rat cardiac xenograft rejection in mice.

BACKGROUNDnGraft rejection can be initiated by two primary pathways of antigen presentation: (a) direct activation of host T cells by donor-derived antigen presenting cells (APC) and (b) indirect presentation of processed graft antigens by host APC.nnnMETHODSnWe investigated the differential roles for direct and indirect antigen presentation by preventing the CD28 costimulatory pathway with monoclonal antibodies to rat or mouse CD80 and CD86 in a rat-to-mouse cardiac transplantation model.nnnRESULTSnAlthough the mouse anti-rat monoclonal antibodies to CD80 and CD86 did not significantly prolong the survival of rat cardiac xenografts in mice, the rat anti-mouse monoclonal antibodies to CD80 and CD86 did prolong the survival. Development of the anti-donor antibodies was inhibited, and the deposition of C3, IgM, and IgG on endothelium in the xenografts was mild in the anti-mouse CD80/CD86-treated mice. Infiltration of macrophages, neutrophils, and lymphocytes expressing perforin and interferon-gamma was decreased by the anti-mouse CD80/CD86 treatment.nnnCONCLUSIONSnThese findings suggest that the indirect antigen presentation, which is mediated by CD80 and CD86 pathway on host APC, plays a crucial role in concordant cardiac xenograft rejection.

Augmented Lung Adenocarcinoma Cytotoxicity by the Combination of a Genetically Modified Anti-Lewis Y Antibody and Antibodies to Complement Regulatory Proteins

Complement‐dependent cytotoxicity (CDC) mediated by a chimeric anti‐Lewis Y monoclonal antibody (cH18A; human IgGl) was investigated in this study. Human lung adenocarcinoma cell lines (PC7, PC9, and PC 14) were used as the target cells. PC7 and PC9 cells, expressed Lewis Y antigen and were lysed by cH18A as effectively as by the parent mouse anti‐Lewis Y antibodies (mH18A) in a concentration‐dependent manner. PC14 cells did not express Lewis Y antigen and were not lysed by either cH18A or mH18A. cH18A mediated CDC activity against PC7 and PC9 cells was enhanced by the combined use of monoclonal antibodies directed against CD46(MCP), CD55(DAF), and CD59. These molecules are complement‐regulatory proteins which protect host cells from CDC. PC7 and PC9 cells, showed high levels of surface expression of these proteins, PC7 cells were more susceptible to cH ISA‐mediated CDC than PC9 cells. Use of multiple blocking antibodies to the complement‐regulatory proteins produced more enhancement of cH18A‐mediated CDC than a single antibody. Moreover, expression of CD55 and CD59 by PC7 and PC9 cells was decreased after treatment with Pl‐PLC, resulting in increased susceptibility to cHISA‐mediated CDC. Although the reason is unknown, PC7 cells became more susceptible to CDC than PC9 cells after PI‐PLC treatment even in the absence of cH18A. These data suggest that chimeric monoclonal antibodies can be used to induce CDC against lung adenocarcinoma, and that such CDC is potentiated by a variety of antibodies blocking compliment‐regulatory proteins on the tumour cell surface.

Lack of cognate help by CD4+ T cells and anergy of CD8+ T cells are the principal mechanisms for anti-leukocyte function-associated antigen-1/intercellular adhesion molecule-1-induced cardiac allograft tolerance.

Combined treatment with anti-leukocyte function-associated antigen-1 (LFA-1) and anti-intercellular adhesion molecule-1 (ICAM-1) monoclonal antibodies leads to allograft tolerance in murine cardiac transplantation. In the present study, we analyzed the mechanisms for this tolerance induction. In the tolerant mice, proliferative response of splenic T cells against donor-type cardiac myocytes and of CD8+ T cells against donor-type alloantigens was impaired as compared with responses in naive or rejected mice, but was completely restored with exogenous interleukin 2. This suggests that class I-restricted CD8+ T cells of tolerant mice were rendered anergic against donor-type alloantigens in the periphery. In contrast, proliferative response of CD4+ T cells against donor-type alloantigens in vitro was comparable between tolerant and naive mice. When heart and skin grafts from the same donor (BALB/c [H2d]) were simultaneously transplanted to C3H mice (H2k), both were rejected within 29 days, even though the mice were similarly treated with anti-LFA-1 and anti-ICAM-1 monoclonal antibodies. In contrast, when heart graft from BALB/c and skin graft from third-party donor (C57BL/6 [H2b]) were simultaneously transplanted to C3H mice under the same condition, the heart graft was accepted indefinitely and the skin graft was rejected. These findings suggest that the peripheral tolerance against cardiac allografts could be induced by selective inactivation of alloreactive CD8+ T cells resulting from the lack of cognate help by CD4+ T cells.