Absence of Activation-induced Cytidine Deaminase, a Regulator of Class Switch Recombination and Hypermutation in B Cells, Suppresses Aorta Allograft Vasculopathy in Mice.

BACKGROUND: Antibody-mediated rejection is caused in part by increasing circulation/production of donor-specific antibody (DSA). Activation-induced cytidine deaminase (AID) is a key regulator of class switch recombination and somatic hypermutation of immunoglobulin in B cells, yet its role in antibody-mediated transplant rejection remains unclear. We show here that AID deficiency in mice enables suppression of allograft vasculopathy (AV) after aorta transplantation, a DSA-mediated process. METHODS: Splenocytes from C57BL/6 J (B6) AID(−/−) mice were used for determining in vitro proliferation responses, alloreactivity, cell surface marker expression, and antibody production. BALB/c mouse aortas were transplanted into B6 AID(−/−) mice with or without FK506 treatment. Blood and aorta grafts were harvested on day 30 after transplantation and were subjected to DSA, histological, and immunohistological analyses. RESULTS: The AID(−/−) splenocytes were comparable to wild type splenocytes in proliferation responses, alloreactivity, and expression of cell surface markers in vitro. However, they completely failed to produce immunoglobulin G, although they were not impaired in immunoglobulin M production relative to controls. Furthermore, BALB/c aorta grafts from B6 AID(−/−) recipient mice on day 30 after transplantation showed reduced signs of AV compared to the grafts from B6 wild type recipient mice which had severe vascular intimal hyperplasia, interstitial fibrosis, and inflammation. Treatment with FK506 produced a synergistic effect in the grafts from AID(−/−) recipients with further reduction of intimal hyperplasia and fibrosis scores. CONCLUSIONS: The AID deficiency inhibits DSA-mediated AV after aorta transplantation in mice. We propose that AID could be a novel molecular target for controlling antibody-mediated rejection in organ transplantation.

In vitro and in vivo characterization of AS2643361, a novel and highly potent inosine 5'-monophosphate dehydrogenase inhibitor.

Inosine 5'-monophosphate (IMP) dehydrogenase is a critical target in solid organ transplantation. To this end, the development of mycophenolate mofetil (MMF) represents a major advance in transplant medicine. Here, we investigated the in vitro and in vivo pharmacological effects of a novel IMP dehydrogenase inhibitor, AS2643361, in several immunological and non-immunological models. The in vitro inhibitory activity of AS2643361 on immune cell and endothelial cell proliferation and on antibody production from lipopolysaccharide-stimulated B cells, was significantly more potent than that of mycophenolic acid, the active form of MMF, despite the similar potency of these compounds on IMP dehydrogenase. In a rat heterotopic cardiac transplant model, monotherapy using orally administered AS2643361 at 10 or 20mg/kg/day prolonged the median graft survival time from 6 to 16 and 19days, respectively. In dinitrophenol-lipopolysaccharide stimulated rats, oral administration of AS2643361 at 2.5, 5 or 10mg/kg/day resulted in suppression of antibody production. In vivo antibody production against alloantigen was also suppressed by AS2643361 treatment at 5 or 10mg/kg/day. Furthermore, treatment with AS2543361 effectively inhibited balloon injury induced-intimal thickening, which is a major cause of late allograft loss. Overall, the in vivo activity of AS2643361 was over two-fold more potent than that of MMF. In addition, gastrointestinal toxicity, considered a dose-limiting factor for MMF, was reduced with AS2643361 treatment. These results suggest AS2643361 has higher potency and less toxicity than MMF, making it a potential candidate for treatment of acute and chronic rejection in transplant medicine.

Enhancement of T-cell-mediated anti-tumour immunity via the ectopically expressed glucocorticoid-induced tumour necrosis factor receptor-related receptor ligand (GITRL) on tumours.

Glucocorticoid-induced tumour necrosis factor receptor-related receptor (GITR) costimulates functions of both effector and regulatory T cells. The administration of agonistic anti-GITR monoclonal antibodies efficiently enhances various T-cell-mediated immune responses; however, it is unknown to what extent the ligand of GITR (GITRL) contributes to T-cell responses. We investigated the involvement of endogenously expressed GITRL on dendritic cells and ectopically expressed GITRL on tumours in T-cell-mediated immunity. Expression of GITRL on dendritic cells in secondary lymphoid organs was limited, and treatment with anti-GITRL monoclonal antibodies did not substantially affect T-cell-mediated immunity to alloantigens, a specific protein antigen (ovalbumin), or tumour antigens. The introduction of GITRL promoted anti-tumour immunity in four tumour models. Tumour-associated GITRL greatly augmented the effector function of CD8(+) T cells and enhanced the contribution of CD8(+) T cells. These events reduced the crucial contribution of CD25(+) CD4(+) regulatory T cells, which were found to inhibit immunity against tumours lacking GITRL. Peritumoral injection of GITRL tumour vaccine efficiently inhibited the growth of established tumours. Our results suggest that the ectopic expression of GITRL in tumour cells enhances anti-tumour immunity at peripheral tumour sites. Consequently, the combined use of a GITRL tumour vaccine with methods aimed at enhancing the activation of host antigen-presenting cells in secondary lymphoid tissues may be a promising strategy for tumour immunotherapy.

FR255734, a humanized, Fc-Silent, Anti-CD28 antibody, improves psoriasis in the SCID mouse-psoriasis xenograft model.

In psoriasis, CD28/B7 costimulatory molecules are well characterized. Here, using the severe combined immunodeficient (SCID) mouse-psoriasis xenograft model, we report therapeutic efficacy of a humanized anti-CD28 monoclonal antibody (FR255734; Astellas Pharmaceuticals Inc., Tokyo, Japan). Transplanted psoriasis plaques on the SCID mouse were treated weekly for 4 weeks with intraperitoneal injections of FR255734 at 10, 3, and 1-mg kg(-1) doses. Groups treated with doses of 10 and 3 mg kg(-1) had significant thinning of the epidermis and reduced HLA-DR-positive lymphocytic infiltrates. The length of the rete pegs changed from 415.2+/-59.6 to 231.4+/-40.4 microm (P<0.005) in the 10-mg kg(-1) group, and from 323.4+/-69.6 to 237.5+/-73.6 microm in the 3-mg kg(-1) group (P=0.002). Positive controls treated with CTLA4-Ig and cyclosporine had significant histological improvement, whereas plaques treated with saline and isotype controls (human and mouse IgG2) remained unchanged. In vitro studies have shown that FR255734 effectively blocked T-cell proliferation and proinflammatory cytokine production. These observations warrant studies to evaluate the efficacy of FR255734 in human autoimmune diseases.

Immunomodulatory properties of FK734, a humanized anti-CD28 monoclonal antibody with agonistic and antagonistic activities.

BACKGROUND: We describe immunomodulatory effects of FK734, a humanized version of a mouse anti-human CD28 mAb (clone TN228), in vitro and in a chimeric human-mouse model of allograft rejection. METHODS: Cytokine production and proliferation were assessed in a mixed lymphocyte reaction containing FK734, human T cells, and endothelial cells or monocytes. FK734 was also administered to SCID mice engrafted with human skin and adoptively transferred with human peripheral blood mononuclear cells allogeneic to the skin graft. RESULTS: In vitro, FK734 enhanced secretion of interleukin-2 and interferon-gamma as well as proliferation of CD4+ and CD8+ T cells stimulated by allogeneic human leukocyte antigen (HLA)-DR+ human umbilical vein endothelial cells (which lack B7 molecules and FcgammaRs) or by blood monocytes (which express low levels of B7 molecules and FcgammaRs) compared with control mAb, but these effects were significantly smaller than those provided by mAb 28.2, a stimulatory mouse anti-human CD28 mAb, at comparable concentrations. However, FK734 generally inhibited cytokine secretion and T cell proliferation in cocultures with human umbilical vein endothelial cells transduced to express CD86. In vivo using SCID/beige mice bearing human skin with adoptively transferred peripheral blood mononuclear cells, administration of FK734 protected human endothelial cell-lined microvessels, significantly but incompletely reducing endothelial cell injury and T cell infiltration into the graft one or two weeks later. CONCLUSIONS: FK734 is a partial agonist of CD28 signaling that can reduce human T cell alloresponses in the presence of strong costimulation by B7 molecules in vitro and can reduce T cell-mediated skin allograft rejection in vivo.

Dimeric but not monomeric soluble CD40 prolongs allograft survival and generates regulatory T cells that inhibit CTL function.

BACKGROUND: We previously reported that adenovirus mediated CD40Ig gene therapy (AdCD40Ig) induced long-term acceptance of fully allogeneic rat cardiac allografts, however, the underlying mechanism has not been fully clarified. To address this we have compared the ability of dimeric and monomeric soluble CD40 to prolong allograft survival in vivo and generate regulatory T cells in vitro. METHODS: The ability of CD40Ig (soluble dimmer, containing an Fc region) or CD40/Myc/His (soluble monomer, lacking an Fc region) therapy to generate CD4CD25 regulatory T cells in vitro and to prevent rejection of rat cardiac allografts (ACI to LEWIS) was compared. Immunoregulatory capacity of regulatory T cells generated was determined by suppression of alloantigen specific proliferation and cytotoxicity. RESULTS: Dimeric soluble CD40Ig did not inhibit CD4 T cell proliferation but rather promoted IL-2 production and the generation of CD4CD25 T cells, which regulated alloantigen-specific cytotoxic T lymphocyte activity. Treatment with either AdCD40Ig or purified soluble CD40Ig prolonged the survival of rat cardiac allografts. In contrast, although monomeric soluble CD40/Myc/His suppressed IL-12 production in a similar manner to that achieved by CD40Ig, it did not augment IL-2 production. Moreover, while CD40/Myc/His also generated CD4CD25 T cells, they did not exhibit regulatory activity and administration of soluble CD40/Myc/His failed to prolong cardiac allograft survival. CONCLUSIONS: These results suggest signaling through CD154 in addition to blocking of CD154-CD40 interaction is important for the immunomodulatory effects of soluble CD40Ig. Taken together, our results provide new insight into the mechanism of immunomodulation by soluble CD40 constructs.

FK506 suppresses E-selectin, ICAM-1 and VCAM-1 expression on vascular endothelial cells by inhibiting tumor necrosis factor alpha secretion from peripheral blood mononuclear cells.

FK506 suppresses activation of T cells; however, it down-regulates E-selectin, ICAM-1 and VCAM-1 expression in inflamed tissues. In this study, we investigated the effect of FK506 on expression of those adhesion molecules on human vascular endothelial cells (HMVEC). Culture supernatant from peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 plus anti-CD2 antibodies effectively induced the expression of E-selectin, ICAM-1 and VCAM-1 on HMVEC, and treatment with FK506 down-regulated their expression. Culture supernatant contained tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta, which effectively induced adhesion molecules, and FK506 suppressed both cytokine secretions. TNFalpha content in culture supernatant was parallel to the induction of adhesion molecules by the culture supernatant. IL-1beta content was not enough to induce those adhesion molecules. Anti-TNFalpha antibody completely inhibited those expressions. FK506 did not inhibit either TNFalpha- or IL-1beta-induced expression of adhesion molecules, or viability of HMVEC. These results indicate that FK506 suppresses migration of inflammatory cells through the inhibition of TNFalpha secretion from leukocytes.

Gene therapy-mediated CD40L and CD28 co-stimulatory signaling blockade plus transient anti-xenograft antibody suppression induces long-term acceptance of cardiac xenografts.

BACKGROUND: The authors have previously demonstrated that inhibition of CD28 and CD40 ligand (CD40L) co-stimulatory signals by adenovirus-mediated cytotoxic T-lymphocyte-associated (CTL) antigen 4 (A4) immunoglobulin (Ig) and CD40Ig gene therapies induces tolerance or long-term acceptance in rat liver and heart allograft transplantation. In this study, the authors examined whether co-stimulation blockade with a brief course treatment of FK779, a novel leflunomide derivative, would be an ideal strategy for controlling xenograft rejection. METHODS: Hamster hearts were transplanted into Lewis rats. Adenovirus vector coding (Ad) CD40Ig, CTLA4Ig, or LacZ gene (1 x 10(9) plaque-forming units) was administered intravenously to recipient rats 2 days before or immediately after xenografting. FK779 (10 mg/kg/day) was administered orally to recipients for 7 days beginning on day -1. Graft survival, graft histology, and xenoreactive antibodies were examined. RESULTS: : Both untreated and AdLacZ-treated control rats rejected cardiac xenografts, with a median survival time (MST) of 3 days. Co-stimulatory blockade alone by AdCTLA4Ig, AdCD40Ig, or both could not overcome such delayed xenograft rejection (DXR) (MST, 3-4 days). Under a short-course FK779 treatment that suppressed T-cell-independent xenoreactive antibodies, administration of AdCD40Ig (MST, 30.5 days) but not AdCTLA4Ig (MST, 9 days) significantly prolonged xenograft survival as compared with the FK779 monotherapy (MST, 7 days). In contrast, DXR and cellular rejection were controlled successfully and all xenografts were accepted for over 100 days when AdCTLA4Ig and AdCD40Ig were administered under FK779 induction therapy. However, chronic rejection was present in all long-term surviving xenografts. CONCLUSIONS: : Gene therapy-based co-stimulation blockade with FK779 induction treatment seems to be an attractive strategy with which to control xenograft rejection.

Stromal cell-derived factor-1 and CXCR4 interaction is critical for development of transplant arteriosclerosis.

BACKGROUND: Posttransplant chronic allograft deterioration associated with development of transplant arteriosclerosis (TA) remains an unresolved problem. Recent studies suggest that the smooth muscle cells (SMCs) constituting the neointima are derived from recipient hematopoietic stem cells (HSCs). However, the underlying mechanisms of the process are not yet fully elucidated. METHODS AND RESULTS: We examined the genes expressed in allografts at different stages of TA development using a mice aortic transplantation model. Genes were analyzed by a differential mRNA display technique. We show that stromal cell-derived factor-1alpha (SDF-1alpha) is a critical molecular target for the treatment of TA. During the course of TA, intragraft SDF-1alpha expression was upregulated with time, and the circulating HSCs expressing its counterreceptor CXCR4 increased in the recipients receiving allografts. CXCR4-positive HSCs, derived from transplant recipients, migrated into allografts via microvessels in the adventitia and then toward the luminal side. The HSCs differentiated into SMC-like cells, contributing to the in situ formation of the neointima. In support of a functional role for these molecules, in vivo neutralization of SDF-1alpha inhibited HSC mobilization and significantly attenuated neointimal formation. CONCLUSIONS: Interaction between SDF-1alpha and CXCR4 plays a key role in TA development. Blockade of SDF-1alpha may become a new therapeutic modality for TA.

Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies.

BACKGROUND: We have previously demonstrated that blockade of either CD80/86-CD28 or CD40-CD154 costimulatory pathways by using adenovirus vector coding CTLA4Ig (AdCTLA4Ig) or CD40Ig (AdCD40Ig) genes induced donor-specific tolerance in rat liver transplantation. In this study, we asked whether these gene-therapy-based costimulation blockade would induce tolerance in cardiac transplantation. METHODS: Heterotopic heart transplantation was performed in a full major histocompatibility complex (MHC) barrier combination of ACI (RT1avl) to Lewis (LEW, RT1l) rats. Vector (1 x 10(9) plaque forming unit [PFU]), AdLacZ, AdCTLA4Ig, or AdCD40Ig, was administered intravenously to recipient animals immediately after grafting, and graft survival, serum CTLA4Ig/CD40Ig levels, and graft histology were assessed. Tolerance was determined by secondary skin-graft challenging. RESULTS: Allografts of both untreated and AdLacZ controls were promptly rejected within 7 days, whereas a single treatment with AdCTLA4Ig or AdCD40Ig significantly prolonged median graft survival to 55.5 and 28.5 days, respectively. In contrast, the combined AdCTLA4Ig and AdCD40Ig gene therapy maintained high CTLA4Ig and CD40Ig levels through the posttransplant period and allowed long-term cardiac allograft survival for more than 270 days. However, both donor and third-party skin grafts were rejected in the animals who harbored cardiac grafts over 150 days. Also, typical features of chronic rejection were evident in the long-term surviving grafts. CONCLUSION: Simultaneous blockade of CD28 and CD154 pathways by AdCTLA4Ig plus AdCD40Ig induces a strong immunosuppression that allows long-term acceptance of full MHC mismatched cardiac graft in rats. This strategy, however, was not enough to induce tolerance to skin grafts and to avoid chronic rejection, as shown in the liver-transplantation model.

Critical function of T cell death-associated gene 8 in glucocorticoid-induced thymocyte apoptosis.

Transcriptional expression of a gene or genes is absolutely required for induction of glucocorticoid-induced thymocyte apoptosis. We have previously shown that expression of T cell death-associated gene 8 (TDAG8) is quickly induced exclusively in the thymus after dexamethasone (DEX) treatment. Here, we present data that TDAG8 expression is induced prior to induction of DEX-mediated apoptosis. In contrast, TDAG8 expression in thymocytes was not induced in the process of gamma-irradiation-mediated apoptosis. TDAG8 expression accelerated only DEX-induced, but not TCR-mediated or gamma-irradiation-induced, thymocyte apoptosis in transgenic mice overexpressing TDAG8. Interestingly, these effects were specifically detected in CD4(+)CD8(+) double-positive thymocytes. Moreover, activation of caspase-3, -8 and -9 was enhanced in thymocytes of TDAG8 transgenic mice after DEX stimulation. In conclusion, TDAG8 expression is involved in glucocorticoid-induced signals to activate caspase-9, -8 and -3 for subsequent apoptosis induction in CD4(+)CD8(+) double-positive thymocytes.