Elise Chiffoleau

CD40Ig treatment results in allograft acceptance mediated by CD8+CD45RClow T cells, IFN-γ, and indoleamine 2,3-dioxygenase

Treatment with CD40Ig results in indefinite allograft survival in a complete MHC-mismatched heart allograft model in the rat. Here we show that serial second, third, and fourth adoptive transfers of total splenocytes from CD40Ig-treated recipients into secondary recipients led to indefinite donor-specific allograft acceptance. Purification of splenocyte subpopulations from CD40Ig-treated recipients demonstrated that only the adoptively transferred CD8(+)CD45RC(low) subset resulted in donor-specific long-term survival, whereas CD8(+)CD45RC(low) T cells from naive animals did not. Accepted grafts displayed increased indoleamine 2,3-dioxygenase (IDO) expression restricted in the graft to ECs. Coculture of donor ECs with CD8(+)CD45RC(low) T cells purified from CD40Ig-treated animals resulted in donor-specific IDO expression dependent on IFN-gamma. Neutralization of IFN-gamma or IDO triggered acute allograft rejection in both CD40Ig-treated and adoptively transferred recipients. This study demonstrates for what we believe to be the first time that interference in CD40-CD40 ligand (CD40-CD40L) interactions induces allospecific CD8(+) Tregs that maintain allograft survival. CD8(+)CD45RC(low) T cells act through IFN-gamma production, which in turn induces IDO expression by graft ECs. Thus, donor alloantigen-specific CD8(+) Tregs may promote local graft immune privilege through IDO expression.

Tribbles-1 as a Novel Biomarker of Chronic Antibody-Mediated Rejection

Diagnosis of the specific cause of late allograft injury is necessary if more personalized and efficient immunosuppressive regimens are to be introduced. This study sought previously unrecognized biomarkers for specific histologic diagnoses of late graft scarring by comparison of gene sets from published microarray studies. Tribbles-1 (TRIB1), a human homolog of Drosophila tribbles, was identified to be a potentially informative biomarker. For testing this, mRNA expression in 76 graft biopsies, 71 blood samples, and 11 urine samples were profiled from independent cohorts of renal transplant patients with different histologic diagnoses recruited at two European centers. TRIB1 but not TRIB2 or TRIB3 was found to be a potential blood and tissue biomarker of chronic antibody-mediated rejection, an active immune-mediated form of chronic allograft failure associated with a poor prognosis. TRIB1 mRNA levels in peripheral blood mononuclear cells discriminated patients with chronic antibody-mediated rejection from those with other types of late allograft injury with high sensitivity and specificity. TRIB1 was also upregulated in a rodent model of chronic cardiac vasculopathy, suggesting that this biomarker may be useful in other solid-organ transplants and across species. It was determined that TRIB1 is expressed primarily by antigen-presenting cells and activated endothelial cells. Overall, these data support the potential use of TRIB1 as a biomarker of chronic antibody-mediated allograft failure.

Role of IFNγ 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 IFNγ‐dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNγ or IDO, and anti‐IFNγ 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 IFNγ and IDO in this process.

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 TCRαβ+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.

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 TGFβ 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 IFNγ. Taken together, these results suggest that FSTL1 could be an active component of the mechanisms mediating heart allograft tolerance.

New evidence for a role of allograft accommodation in long-term tolerance.

Background. Progressively better therapies have largely prevented or at least effectively treated acute allograft rejection. Consequently, the long-term survival of solid organ transplants has increasingly become limited primarily by the development of chronic allograft rejection. The mechanisms of chronic rejection remain largely unknown and the induction of specific tolerance would be the ultimate achievement in transplant immunology. We previously demonstrated, in a fully major histocompatibility complex (MHC)-mismatched rat cardiac allograft combination, that a 20-day treatment with a deoxyspergualin (DSG) analogue, LF15-0195, induces allograft tolerance with the development of potent CD4+CD25+ regulatory T cells. In order to better characterize the mechanisms involved in allograft tolerance, we compared long-term tolerated allografts with allografts exhibiting signs of chronic rejection induced by donor-specific blood transfusion. Methods. We analyzed both types of allografts for infiltration, alloantibody production and gene expression by histology, exhaustive microarray and quantitative reverse-transcriptase polymerase chain reaction. Results. Interestingly, we observed in tolerated allografts an infiltrate as dense as the one observed in chronically rejected allografts and alloantibody deposits on graft endothelial cells. Prominent gene expression of many putative proinflammatory cytokines and genes related to cell activation or cytotoxicity were observed in tolerated allografts. However, we observed a specific upregulation of cytoprotective genes such as nitric oxide synthase, BclXL, and indoleamine 2,3 dioxygenase, and a poor in situ expression of immunoglobulin chain gene. Conclusions. This study demonstrates a state of accommodation of tolerated allografts and suggests the importance of early control of humoral immunity for the prevention of chronic rejection and the maintenance of long-term tolerance.

Tmem176B and Tmem176A are associated with the immature state of dendritic cells

DCs play a central role in the development of innate and adaptive immunity but also in the induction and maintenance of immune tolerance. Identification of factors that govern DC activation, their maturation state, and their capacity to induce proinflammatory or tolerogeneic responses therefore represents a crucial aim of research. We previously identified a new molecule, Tmem176B (which we named TORID initially), as highly expressed in a model of allograft tolerance in the rat. We showed that its overexpression in rat DCs blocked their maturation, suggesting a role for this molecule in the maturation process. To characterize the function of Tmem176B further, we used a split‐ubiquitin yeast, two‐hybrid system to identify interacting partners and found that Tmem176B associated with itself but also with Tmem176A, a membrane protein similar to Tmem176B. Interestingly, these two molecules showed similar mRNA expression patterns among various murine tissues and immune cells and were both down‐regulated following DC maturation. In addition, we showed that in using RNAi, these molecules are both involved in the maintenance of the immature state of the DCs. Taken together, these data suggest that Tmem176B and Tmem176A associate to form multimers and restrain DC maturation. Therefore, these two molecules may represent valid targets to regulate DC function.

Accumulation of T cells with potent regulatory properties and restricted Vbeta7-TCR rearrangements in tolerated allografts.

Background. We have previously demonstrated that a short-course treatment with LF15-0195, a 15-deoxyspergualin analogue, induces donor-specific tolerance of cardiac allografts in rats and expansion of splenic CD4+CD25+ regulatory T cells. Methods. To further characterize long-term tolerance in this model, we have analyzed the phenotype, regulatory properties and TCR-V&bgr; usage of the T cells infiltrating the tolerated allografts. Results. We demonstrate that the tolerated allografts express high levels of FoxP3 transcripts and contain a large number of CD4+ T cells, half of which express CD25. Moreover, T cells from these tolerated allografts are very powerful at transferring tolerance to a subsequent allograft recipient, demonstrating the presence of potent regulatory T cells at the site of the graft. Interestingly, the T cells infiltrating the tolerated allografts systematically display restricted V&bgr;7 TCR rearrangements. Conclusion. These results demonstrate in this model of tolerance, a specific accumulation of T cells with potent regulatory properties and exhibiting restricted V&bgr;7-TCR rearrangements at the graft site.

Superiority of bone marrow-derived dendritic cells over monocyte-derived ones for the expansion of regulatory T cells in the macaque.

Regulatory T cells (Treg) have been identified as playing a pivotal role in the control of tolerance and in the suppression of pathologic immune responses in autoimmune diseases, transplantation, and graft-versus-host disease. Treg expanded ex vivo by dendritic cells could be potential reagents to promote antigen-specific tolerance in vivo. However, in vivo studies have been carried out mostly in rodents and will need validation in primates before clinical application. We characterized macaque dendritic cell derived either from bone marrow with and without prior CD34+ cell selection (BMDC), or from CD14+ peripheral blood mononuclear cells (Mo-DC). We demonstrate that with a semi-mature phenotype, BMDC are superior to Mo-DC in their capacity to expand freshly isolated allogeneic macaque CD4+ CD25+ CD127- Foxp3+ Treg in vitro in the presence of interleukin-2. Moreover, the expanded Treg maintain their phenotype and suppressive activity. These data provide a step toward the use of macaque dendritic cell to expand Treg for future preclinical testing.

Immunoregulatory function of IL-27 and TGF-β1 in cardiac allograft transplantation.

Background Deciphering the mechanisms of tolerance represents a crucial aim of research in transplantation. We previously identified by DNA chip interleukin (IL)-27 p28 and transforming growth factor (TGF)–&bgr;1 as overexpressed in a model of rat cardiac allograft tolerance mediated by regulatory CD4+CD25+ T cells. The role of these two molecules on the control of the inflammatory response remains controversial. However, both are involved in the regulation of the T helper 17/Treg axis, suggesting their involvement in tolerance. Methods We analyzed regulation of IL-27 and TGF-&bgr;1 expression in allograft response and their role in tolerance by using blocking anti–TGF-&bgr; antibody and by generating an adeno-associated virus encoding IL-27. Results Here, we confirmed the overexpression of IL-27 and TGF-&bgr;1 in tolerated cardiac allografts in two different rodent models. We observed that their expression correlates with inhibition of T helper 17 differentiation and with expansion of regulatory CD4+CD25+ T cells. We showed in a rat model that anti–TGF-&bgr; treatment abrogates infectious tolerance mediated by the transfer of regulatory CD4+CD25+ T cells. Moreover, overexpression of IL-27 by adeno-associated virus administration in combination with a short-term immunosuppression allows prolongation of cardiac allograft survival and one tolerant recipient. We found that IL-27 overexpression did not induce Foxp3+CD4+CD25+ T-cell expansion but rather IL-10–expressing CD4+ T cells in the tolerant recipient. Conclusions Taken together, these data suggest that both TGF-&bgr;1 and IL-27 play a role in the mechanisms of tolerance. However, in contrast to TGF-&bgr;1, IL-27 seems not to be involved in regulatory CD4+CD25+ T-cell expansion but rather in their mode of action.