Autologous dendritic cells prolong allograft survival through Tmem176b-dependent antigen cross-presentation.

The administration of autologous (recipient-derived) tolerogenic dendritic cells (ATDCs) is under clinical evaluation. However, the molecular mechanisms by which these cells prolong graft survival in a donor-specific manner is unknown. Here, we tested mouse ATDCs for their therapeutic potential in a skin transplantation model. ATDC injection in combination with anti-CD3 treatment induced the accumulation of CD8(+) CD11c(+) T cells and significantly prolonged allograft survival. TMEM176B is an intracellular protein expressed in ATDCs and initially identified in allograft tolerance. We show that Tmem176b(-/-) ATDCs completely failed to trigger both phenomena but recovered their effect when loaded with donor peptides before injection. These results strongly suggested that ATDCs require TMEM176B to cross-present antigens in a tolerogenic fashion. In agreement with this, Tmem176b(-/-) ATDCs specifically failed to cross-present male antigens or ovalbumin to CD8(+) T cells. Finally, we observed that a Tmem176b-dependent cation current controls phagosomal pH, a critical parameter in cross-presentation. Thus, ATDCs require TMEM176B to cross-present donor antigens to induce donor-specific CD8(+) CD11c(+) T cells with regulatory properties and prolong graft survival.

Cell therapy with autologous tolerogenic dendritic cells induces allograft tolerance through interferon-gamma and epstein-barr virus-induced gene 3.

Innovative therapeutic strategies are needed to diminish the impact of harmful immunosuppression in transplantation. Dendritic cell (DC)-based therapy is a promising approach for induction of antigen-specific tolerance. Using a heart allograft model in rats, we analyzed the immunoregulatory mechanisms by which injection of autologous tolerogenic DCs (ATDCs) plus suboptimal immunosuppression promotes indefinite graft survival. Surprisingly, we determined that Interferon-gamma (IFNG), a cytokine expected to be propathogenic, was threefold increased in the spleen of tolerant rats. Importantly, its blockade led to allograft rejection [Mean Survival Time (MST) = 25.6 ± 4 days], showing that IFNG plays a critical role in immunoregulatory mechanisms triggered by ATDCs. IFNG was expressed by TCRαß(+) CD3(+) CD4(-) CD8(-) NKRP1(-) cells (double negative T cells, DNT), which accumulated in the spleen of tolerant rats. Interestingly, ATDCs specifically induced IFNG production by DNT cells. ATDCs expressed the cytokinic chain Epstein-Barr virus-induced gene 3 (EBI3), an IL-12 family member. EBI3 blockade or knock-down through siRNA completely abolished IFNG expression in DNT cells. Finally, EBI3 blockade in vivo led to allograft rejection (MST = 36.8 ± 19.7 days), demonstrating for the first time a role for EBI3 in transplantation tolerance. Taken together our results have important implications in the rationalization of DC-based therapy in transplantation as well as in the patient immunomonitoring follow-up.

Long-term allograft tolerance is characterized by the accumulation of B cells exhibiting an inhibited profile.

Numerous reports have highlighted the central role of regulatory T cells in long-term allograft tolerance, but few studies have investigated the B-cell aspect. We analyzed the B-cell response in a rat model of long-term cardiac allograft tolerance induced by a short-term immunosuppression. We observed that tolerated allografts are infiltrated by numerous B cells organized in germinal centers that are strongly regulated in their IgG alloantibody response. Moreover, alloantibodies from tolerant recipients exhibit a deviation toward a Th2 isotype and do not activate in vitro donor-type endothelial cells in a pro-inflammatory way but maintained expression of cytoprotective molecules. Interestingly, this inhibition of the B-cell response is characterized by the progressive accumulation in the graft and in the blood of B cells blocked at the IgM to IgG switch recombination process and overexpressing BANK-1 and the inhibitory receptor Fcgr2b. Importantly, B cells from tolerant recipients are able to transfer allograft tolerance. Taken together, these results demonstrate a strong regulation of the alloantibody response in tolerant recipients and the accumulation of B cells exhibiting an inhibited and regulatory profile. These mechanisms of regulation of the B-cell response could be instrumental to develop new strategies to promote tolerance.

Tolerogenic dendritic cells actively inhibit T cells through heme oxygenase-1 in rodents and in nonhuman primates.

Clinical translation of dendritic cell (DC)-based cell therapy requires preclinical studies in nonhuman primates (NHPs). The aim of this work was to establish the in vitro conditions for generation of NHP tolerogenic DCs (Tol-DCs), as well as to analyze the molecular mechanisms by which these cells could control an immune response. Two populations of NHP bone marrow-derived DCs (BMDCs) were obtained: adherent and nonadherent. Although both populations displayed a quite similar phenotype, they were very different functionally. We characterized the adherent BMDCs as Tol-DCs that were poor stimulators of T cells and actively inhibited T-cell proliferation, whereas the nonadherent population displayed immunogenic properties in vitro. Interestingly, the anti-inflammatory and immunosuppressive enzyme heme oxygenase-1 (HO-1) was up-regulated in Tol-DCs, compared to the immunogenic BMDCs. We demonstrated that HO-1 mediates the immunosuppressive properties of Tol-DCs in vitro (in NHPs and rats) and that HO-1 is involved in the in vivo tolerogenic effect of Tol-DCs in a rat model of allotransplantation. In conclusion, here we characterized the in vitro generation of NHP Tol-DCs. Furthermore, we showed for the first time that HO-1 plays a role in the active inhibition of T-cell responses by rat and NHP Tol-DCs.

An immunomodulatory role for follistatin-like 1 in heart allograft transplantation.

Donor-specific tolerance to heart allografts in the rat can be achieved by donor-specific blood transfusions (DST) before transplantation. We have previously reported that this tolerance is associated with strong leukocyte infiltration, and that host CD8(+) T cells and TGFbeta are required. In order to identify new molecules involved in the induction phase of tolerance, we compared tolerated and rejected heart allografts (suppressive subtractive hybridization) 5 days after transplantation. We identified overexpression of Follistatin-like 1 (FSTL1) transcript in tolerated allografts compared to rejected allografts or syngeneic grafts. We show that FSTL1 is overexpressed during both the induction and maintenance phase of tolerance, and appears to be specific to the tolerance model induced by DST. Analysis of graft-infiltrating cells revealed predominant expression of FSTL1 in CD8(+) T cells from tolerated grafts, and depletion of these cells prior to transplantation abrogated FSTL1 expression and heart allograft survival. Moreover, overexpression of FSTL1 by adenovirus gene transfer in vivo significantly prolonged allograft survival in association with inhibition of the proinflammatory cytokines, IL6, IL17 A and IFNgamma. Taken together, these results suggest that FSTL1 could be an active component of the mechanisms mediating heart allograft tolerance.

Impaired Notch4 activity elicits endothelial cell activation and apoptosis: implication for transplant arteriosclerosis.

OBJECTIVE: Notch signaling pathway controls key functions in vascular and endothelial cells (EC). However, little is known about the role of Notch in allografted vessels during the development of transplant arteriosclerosis (TA). This study investigated regulation of the Notch pathway on cardiac allograft arteriosclerosis and further examined its implication in EC dysfunction. METHODS AND RESULTS: Here we show that, among Notch receptors, Notch2, -3, and -4 transcript levels were markedly downregulated in TA compared to tolerant and syngeneic allografts. TA correlates with high levels of tumor necrosis factor (TNF), transforming growth factor (TGF)beta, and IL10, which consistently decrease Notch4 expression in transplants and cultured ECs. We found that inhibition of Notch activity, reflected by both a reduced CBF1 activity and Hes1 expression, parallels the downregulation of Notch4 expression mediated by TNF in ECs. Notch4 and Hes1 knockdown enhances vascular cell adhesion molecule-1 expression and promotes EC apoptosis. Silencing Notch4 or Hes1 also drastically inhibits repair of endothelial injury. Overall, our results suggest that Notch4 and basal Notch activity are required to maintain EC quiescence and for optimal survival and repair in response to injury. CONCLUSIONS: Together, our findings indicate that impaired Notch4 activity in graft ECs is a key event associated with TA by triggering EC activation and apoptosis.

Role of IFNgamma in allograft tolerance mediated by CD4+CD25+ regulatory T cells by induction of IDO in endothelial cells.

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3(-) CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFNgamma-dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNgamma or IDO, and anti-IFNgamma reduces the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNgamma and IDO in this process.

Anti-TCR Vbeta-specific DNA vaccination prolongs heart allograft survival in adult rats.

Allospecific T cells are known to play a central role in the process of allograft rejection. Recently, it has been shown that T cell function could be specifically targeted using DNA vaccination. In our model, PCR analysis of the TCR-beta chain repertoire of T cells infiltrating rejected allografts showed specific expansions of the Vbeta13 and Vbeta2 families. In this study, we tested the effect on allograft survival of DNA vaccination against a specific TCR Vbeta, in a model of heart allograft rejection in adult rats. Our results showed that anti-TCR Vbeta13 DNA vaccination lead to a significant prolongation of allograft survival compared to vaccination against other Vbeta families or untreated recipients. The prolongation of allograft survival correlated in vitro with a decrease in anti-donor reactivity of spleen cells from Vbeta13-vaccinated rats. These results show that, in a transplantation model, DNA vaccination could be used as a method to specifically manipulate a T cell response and thus prolong allograft survival.

Anti-TCR-specific DNA vaccination demonstrates a role for a CD8+ T cell clone in the induction of allograft tolerance by donor-specific blood transfusion.

Donor-specific allograft tolerance can be induced in the adult rat by pregraft donor-specific blood transfusion (DST). This tolerance appeared to be mediated by regulatory cells and to the production of the suppressive cytokine TGF-beta1. A potential immunoregulatory CD8+ clone bearing a Vbeta18-Dbeta1-Jbeta2.7 TCR gene rearrangement was previously identified in DST-treated recipients. To assess the functional role of this T cell clone in the induction of tolerance by DST, we have vaccinated DST-treated recipients with a plasmid construct encoding for the Vbeta18-Dbeta1-Jbeta2.7 TCR beta-chain. DST-induced allograft tolerance was abolished by anti-TCR Vbeta18-Dbeta1-Jbeta2.7 DNA vaccination in six of seven recipients, whereas vaccination with the vector alone, or with the construct encoding a TCR Vbeta13 beta-chain, had no effect. However, the transcript number of the Vbeta18-Dbeta1-Jbeta2.7 chain was unchanged in allografts from vaccinated DST-treated rats, suggesting that this clone was not depleted by vaccination, but rather was altered in its function. Moreover, TCR Vbeta18-Dbeta1-Jbeta2.7 DNA vaccination restored the anti-donor alloantibody production, partially restore the capacity of spleen cells from tolerized recipients to proliferate in vitro against donor cells, and decreased the inhibitory effect of TGF-beta1, seen in DST-treated recipients, in spleen cells from vaccinated DST-treated ones. This study strongly suggests that this CD8+ TCR Vbeta18-Dbeta1-Jbeta2.7 T cell clone has an effective immunoregulatory function in allograft tolerance induced by DST.

New insight into mechanisms of allograft transplantation in the rat by differential display: macrophage scavenger receptor-A brings to light.

BACKGROUND: Donor specific tolerance to heart allografts is induced in LEW.1A rat recipient by two donor LEW.1W blood transfusions prior engraftment. Although the tolerant allograft is infiltrated by leukocytes, graft infiltrating cells are only expressing low levels of the Th1- or Th2-related cytokines suggesting that induction of tolerance is an active phenomenon in which the mechanisms remain to be elucidated. MATERIALS AND METHODS: Differential display (DD) method was applied on RNAs extracted from graft infiltrating cells (GIC) derived from allografts either from rejecting untreated rats or donor-specific blood transfusion treated tolerant rats. Quantitative RT/PCR was performed to confirm mRNA expressions of the selected genes. RESULTS: Among the six differentially displayed DNAs (ddDNA) overexpressed in GIC from rejected allografts, the macrophage scavenger receptor-A (A:D13265) was identified; it exhibited a stricking induction of mRNA expression from day 1 to 7 after transplantation. Among the seven ddDNAs overexpressed in GIC from tolerant allografts, the 3-hydroxy-3-methyl glutaryl coenzyme-A reductase (A:M29249) and an "unknown gene" (ddDNA EC9) were identified and both were confirmed to be up-regulated by quantitative RT/PCR. CONCLUSIONS: The relevance of these genes in transplantation has not yet been reported and must therefore be elucidated; they represent possible targets for immunointervention. Nevertheless, our data demonstrate that the DD is a powerful tool to identify new genes involved in organ transplantation.

Reassessment of the role of CD8+ T cells in the induction of allograft tolerance by donor-specific blood transfusion.

Donor-specific allograft tolerance can be induced in adult rats by pregraft donor-specific blood transfusion (DST). We have previously shown that DST elicits in recipients the expansion of a donor-specific CD8+ T cell clone displaying the Vbeta18-Dbeta1-Jbeta2.7 TCR rearrangement, which rapidly infiltrates allografts after transplantation, suggesting a regulatory function for this clone in DST-induced tolerance. In this study, recipients were pretreated before grafting, using an anti-CD8 monoclonal antibody to deplete CD8+ T cells. CD8 depletion before DST and transplantation abrogated allograft tolerance, and the CD8+ T cell clone was absent from allografts. These effects were not observed when CD8 depletion was performed after DST but before transplantation. We conclude that CD8+ T cells play a role in the induction of allograft tolerance by DST.