Polyfunctional HCV-specific T-cell responses are associated with effective control of HCV replication

Rapport de synthèse : L'infection par le virus de l'hépatite C (VHC) a une évolution sévère chez les patients co-infectés par VIH/VHC, de même que chez les patients transplantés hépatiques. Toutefois, les mécanismes impliqués dans cette évolution restent peu clairs. Dans ce travail, nous étudions le profil fonctionnel des cellules T spécifiques dirigées contre le virus de l'hépatite C chez 86 patients mono-infectés par VHC, 48 patients co-infectés par VIH/VHC et 42 patients ayant bénéficié d'une transplantation hépatique. La production d'IFN-Y et d'IL-2 et la capacité de proliférer des cellules T CD4+ et CD8+ sont évaluées après stimulation par des peptides dérivés du VHC. Chez les patients mono-infectés par le VHC, les cellules T spécifiques au VHC sont polyfonctionnelles du point de vue de la sécrétion de cytokines, avec trois profils de sécrétion pour les cellules T CD4+: sécrétion uniquement de IL-2, sécrétion de IL-2 et IFN-y et sécrétion uniquement de IFN-gamma, et de deux profils pour les cellules T CD8+: sécrétion de IL-2 et IFN-y et sécrétion uniquement de IFN-gamma. En revanche, les cellules T spécifiques au VHC chez les individus coinfectés par VIH/VHC et chez les patients transplantés hépatiques ont un profil de sécrétions de cytokines marqué par l'absence de cellules CD4+ sécrétant uniquement l'Il-2 et l'absence de cellules CD8+ sécrétant à la fois IL-2 et IFN-gamma. De plus, la prolifération de cellules T CD4+ et CD8+ spécifiques au VHC est considérablement réduite chez les patients co-infectés par VIH/VHC, comme chez les transplantés hépatiques. La présence de cellules T effectrices uniquement (définies par l'absence de cellules T CD4+ sécrétants uniquement de l'IL-2 et l'absence de cellules T CD8+ sécrétant à la fois IL-2 et IFN-gamma et altération de la capacité proliférative) est associée avec une charge virale VHC significativement plus élevée et une fibrose hépatique plus sévère. Par conséquent, les présents résultats suggèrent la participation de mécanismes immunitaires dans l'évolution accélérée de l'hépatite C chez les patients co-infectés par VIH-1 et chez les patients greffés hépatiques.

Antibody-indocyanin conjugates for immunophotodetection of human squamous cell carcinoma in nude mice.

We have recently shown that immunophotodetection of human colon carcinomas in nude mice and in patients is possible by using anti-carcinoembryonic antigen monoclonal antibodies (MAb) coupled to fluorescein. The most common clinical application of photodiagnosis has been for the detection of squamous cell carcinomas (SCC) in the upper respiratory tract, but the free dyes used have a poor tumor selectivity. We selected the known MAb E48 directed against SCC and coupled it to a fluorescent dye: indopentamethinecyanin (indocyanin). This dye has an advantage over fluorescein in that it emits a more penetrating fluorescent red signal at 667 nm after excitation with a laser ray of 640 nm. In vitro, an conjugate with an indocyanin:MAb molar ratio of 2, and an additional trace labeling with 125I, showed more than 80% of binding to cells from the SCC line A431. In vivo, when injected i.v. into nude mice bearing xenografts of the same carcinoma line, the MAb E48-(indocyanin)2 conjugate was almost as efficient as the unconjugated MAb E48 in terms of specific tumor localization: 15% of the injected dose per g of tumor at 24 h after injection and a tumor:overall normal tissue ratio of 6-8. There was no selective tumor localization of an irrelevant IgG1-(indocyanin)2 conjugate. Immunophotodetection of the s.c. SCC xenografts on mice given injections of 100 micrograms of MAb E48-(indocyanin), conjugate (representing 1 microgram of indocyanin) was performed at 24 h. Upon laser irradiation, clearly detectable red fluorescence from the indocyanin-MAb conjugate was observed specifically in the SCC xenografts across the mouse skin. In comparison, injection of 100 micrograms of a MAb E48 coupled to 2 micrograms of fluorescein gave a specific green fluorescence signal in the tumor xenografts, which was detectable, however, only after removing the mouse skin. Injection i.v. of a 15 times higher amount of free indocyanin (15 micrograms) gave a diffuse red fluorescence signal all over the mouse body with no definite increase in intensity in the tumor, indicating a lack of tumor selectivity of the free dye. The results demonstrate the possibility of broadening and improving the efficiency of tumor immunophotodiagnosis by coupling to a MAb directed against SCC, a fluorescent dye absorbing and emitting at higher wavelength than fluorescein, and thus having deeper tissue penetration and lower tissue autofluorescence. Such a demonstration opens the way to a new form of clinical immunophotodiagnosis and possibly to the development of a more specific approach to phototherapy of early bronchial carcinomas.

Natural killer cell receptor-repertoire and functions after induction therapy by polyclonal rabbit anti-thymocyte globulin in unsensitized kidney transplant recipients.

Polyclonal rabbit anti-thymocyte globulin (rATG) is widely used in solid organ transplantation (SOT) as induction therapy or to treat corticosteroid-resistant rejection. In vivo, the effect of rATG on natural killer (NK) cells has not been studied. These cells are of particular relevance after SOT because classical immunosuppressive drugs do not inhibit or even can activate NK cells. A cohort of 20 recipients at low immunological risk, that had been receiving rATG as induction therapy, was analyzed for receptor repertoire, cytotoxicity and capacity of NK cells to secrete IFN-γ before kidney transplantation and at different time points thereafter. NK cells expressed fewer killer-cell immunoglobulin-like receptors (KIR), fewer activating receptors NKG2D, but more inhibitory receptor NKG2A compatible with an immature phenotype in the first 6 months post transplantation. Both cytotoxicity of NK cells and the secretion of IFN-γ were preserved over time after transplantation.

The presence of a CD4+ CD25high CD45RO+ CD127high T-cell population correlates with the clinical status of kidney transplant recipients

Background: Recent data have suggested that a population of CD4+ CD25high T cells, phenotypically characterized by the expression of CD45RO and CD127, is significantly expanded in stable liver and kidney transplant recipients and represents alloreactive T cells. We analyzed this putative new alloreactive cellular marker in various groups of kidney transplant recipients. Patients and methods: Flow cytometry was used to analyze the expression of CD25, CD45RO and CD127 on peripheral CD4+ T cells. Of 73 kidney recipients, 59 had a stable graft function under standard immunosuppressive therapy (IS), 5 had biopsy-proven chronic humoral rejection (CHR), 8 were stable under minimal IS and one was an operationally "tolerant" patient who had discontinued IS for more than 3 years. Sixty-six healthy subjects (HS) were studied as controls. Results: Overall, the alloreactive T cell population was found to be significantly increased in the 73 kidney recipients (mean ± SE: 15.03 ± 1.04% of CD4+ CD25high T cells) compared to HS (5.93 ± 0.39%) (p <0.001). In the 5 patients with CHR, this population was highly expanded (31.33 ± 4.16%), whereas it was comparable to HS in the 8 stable recipients receiving minimal IS (6.12 ± 0.86%), in 4 patients who had been switched to sirolimus (4.21 ± 0.53%) as well as in the unique "tolerant" recipient (4.69%). Intermediate levels (15.84 ± 0.93%) were found in the 55 recipients with stable graft function on standard CNI-based IS. Regulatory T cells, defined as CD4+ CD25high FoxP3+ CD127low, were found to be significantly reduced in all recipients except in those with minimal or no IS, and this reduction was particularly striking in recipients with CHR. Conclusion: After kidney transplantation, an alloreactive T cell population was found to be significantly expanded and it correlates with the clinical status of the recipients. Interestingly, in stable patients with minimal (or no) IS as well as in patients on sirolimus, alloreactive T cells were comparable the healthy controls. Measuring circulating CD4+ CD25high CD45RO+ CD127high T cells may become a useful monitoring tool after transplantation.

Harnessing epithelial stem cell potency

Increase in potency of adult stem/progenitor cells holds great expectations for regenerative medicine; reprogramming is achieved by manipulating the genome or indirectly by manipulating the microenvironment. However, the genetic approach, which can result in lineage conversion up to ground pluripotent embryonic state, will certainly face strict regulatory constraints and consequently translation to the clinic may be difficult. Manipulating stem cell fate without altering the genome of adult stem cells is a promising alternative. My laboratory has demonstrated that non hairy squamous epithelia e.g. the cornea, the oral cavity, the oesophagus, the vagina, contain clonogenic stem cells that can respond to skin morphogenetic signals and form epidermis, cycling hair follicles and sebaceous glands. This capacity is maintained in serial transplantation, crosses primary germ line boundaries and is intrinsic to the stem cells, as cells which have never been exposed to cell culture behave in a similar fashion. Even more surprising, the thymus contains a population of clonogenic epithelial cells of endodermal origin that maintain a thymic identity in culture and have the capacity to incorporate into a thymic network, but can acquire the functionality of bona fide multipotent stem cells of the skin when exposed to proper developmental signals. Thymic epithelial cells exposed to a skin microenvironment exhibit a down-regulation or silencing of transcription factors important for thymic function. Hence, it is possible to reveal unsuspected potency and even to robustly reprogram stem cells by solely manipulating the microenvironment.

LRF-mediated Dll4 repression in erythroblasts is necessary for hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) are the most primitive cells in the hematopoietic system and are under tight regulation for self-renewal and differentiation. Notch signals are essential for the emergence of definitive hematopoiesis in mouse embryos and are critical regulators of lymphoid lineage fate determination. However, it remains unclear how Notch regulates the balance between HSC self-renewal and differentiation in the adult bone marrow (BM). Here we report a novel mechanism that prevents HSCs from undergoing premature lymphoid differentiation in BM. Using a series of in vivo mouse models and functional HSC assays, we show that leukemia/lymphoma related factor (LRF) is necessary for HSC maintenance by functioning as an erythroid-specific repressor of Delta-like 4 (Dll4) expression. Lrf deletion in erythroblasts promoted up-regulation of Dll4 in erythroblasts, sensitizing HSCs to T-cell instructive signals in the BM. Our study reveals novel cross-talk between HSCs and erythroblasts, and sheds a new light on the regulatory mechanisms regulating the balance between HSC self-renewal and differentiation.

Mycosis fungoides in a lung transplant recipient with advanced ciclosporin nephropathy: management with mechlorethamine and subsequent renal transplantation.

Posttransplant cutaneous T cell lymphomas are rare and have been reported to have a poor prognosis. We report the case of a follicular mycosis fungoides in a lung transplant recipient who was successfully treated with topical mechlorethamine, prior to subsequent renal transplantation.

Selectively expanding subsets of T cells in mice by injection of interleukin-2/antibody complexes: implications for transplantation tolerance.

The biological activity of interleukin (IL)-2 and other cytokines in vivo can be augmented by binding to certain anti-cytokine monoclonal antibodies (mAb). Here, we review evidence on how IL-2/anti-IL-2 mAb complexes can be used to cause selective stimulation and expansion of certain T-cell subsets. With some anti-IL-2 mAbs, injection of IL-2/mAb complexes leads to expansion of CD8 T effector cells but not CD4 T regulatory cells (Tregs); these complexes exert less adverse side effects than soluble IL-2 and display powerful antitumor activity. Other IL-2/mAb complexes have minimal effects on CD8 T cells but cause marked expansion of Tregs. Preconditioning mice with these complexes leads to permanent acceptance of MHC-disparate pancreatic islets in the absence of immunosuppression.

Bortezomib(Velcade (R))-Thalidomide-Dexamethasone (VTD) Is Superior to Thalidomide-Dexamethasone (TD) In Patients with Multiple Myeloma (MM) Progressing or Relapsing After Autologous Transplantation.

Introduction: The Thalidomide-Dexamethasone (TD) regimen has provided encouraging results in relapsed MM. To improve results, bortezomib (Velcade) has been added to the combination in previous phase II studies, the so called VTD regimen. In January 2006, the European Group for Blood and Marrow Transplantation (EBMT) and the Intergroupe Francophone du Myélome (IFM) initiated a prospective, randomized, parallel-group, open-label phase III, multicenter study, comparing VTD (arm A) with TD (arm B) for MM patients progressing or relapsing after autologous transplantation. Patients and Methods: Inclusion criteria: patients in first progression or relapse after at least one autologous transplantation, including those who had received bortezomib or thalidomide before transplant. Exclusion criteria: subjects with neuropathy above grade 1 or non secretory MM. Primary study end point was time to progression (TTP). Secondary end points included safety, response rate, progression-free survival (PFS) and overall survival (OS). Treatment was scheduled as follows: bortezomib 1.3 mg/m2 was given as an i.v bolus on Days 1, 4, 8 and 11 followed by a 10-Day rest period (days 12 to 21) for 8 cycles (6 months) and then on Days 1, 8, 15, 22 followed by a 20-Day rest period (days 23 to 42) for 4 cycles (6 months). In both arms, thalidomide was scheduled at 200 mg/Day orally for one year and dexamethasone 40 mg/Day orally four days every three weeks for one year. Patients reaching remission could proceed to a new stem cell harvest. However, transplantation, either autologous or allogeneic, could only be performed in patients who completed the planned one year treatment period. Response was assessed by EBMT criteria, with additional category of near complete remission (nCR). Adverse events were graded by the NCI-CTCAE, Version 3.0.The trial was based on a group sequential design, with 4 planned interim analyses and one final analysis that allowed stopping for efficacy as well as futility. The overall alpha and power were set equal to 0.025 and 0.90 respectively. The test for decision making was based on the comparison in terms of the ratio of the cause-specific hazards of relapse/progression, estimated in a Cox model stratified on the number of previous autologous transplantations. Relapse/progression cumulative incidence was estimated using the proper nonparametric estimator, the comparison was done by the Gray test. PFS and OS probabilities were estimated by the Kaplan-Meier curves, the comparison was performed by the Log-Rank test. An interim safety analysis was performed when the first hundred patients had been included. The safety committee recommended to continue the trial. Results: As of 1st July 2010, 269 patients had been enrolled in the study, 139 in France (IFM 2005-04 study), 21 in Italy, 38 in Germany, 19 in Switzerland (a SAKK study), 23 in Belgium, 8 in Austria, 8 in the Czech republic, 11 in Hungary, 1 in the UK and 1 in Israel. One hundred and sixty nine patients were males and 100 females; the median age was 61 yrs (range 29-76). One hundred and thirty six patients were randomized to receive VTD and 133 to receive TD. The current analysis is based on 246 patients (124 in arm A, 122 in arm B) included in the second interim analysis, carried out when 134 events were observed. Following this analysis, the trial was stopped because of significant superiority of VTD over TD. The remaining patients were too premature to contribute to the analysis. The number of previous autologous transplants was one in 63 vs 60 and two or more in 61 vs 62 patients in arm A vs B respectively. The median follow-up was 25 months. The median TTP was 20 months vs 15 months respectively in arm A and B, with cumulative incidence of relapse/progression at 2 years equal to 52% (95% CI: 42%-64%) vs 70% (95% CI: 61%-81%) (p=0.0004, Gray test). The same superiority of arm A was also observed when stratifying on the number of previous autologous transplantations. At 2 years, PFS was 39% (95% CI: 30%-51%) vs 23% (95% CI: 16%-34%) (A vs B, p=0.0006, Log-Rank test). OS in the first two years was comparable in the two groups. Conclusion: VTD resulted in significantly longer TTP and PFS in patients relapsing after ASCT. Analysis of response and safety data are on going and results will be presented at the meeting. Protocol EU-DRACT number: 2005-001628-35.

Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.

According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions.

In vivo mechanisms leading to transplantation tolerance induced by regulatory T cells

Purpose: The mechanisms by which CD4+CD25+Foxp3+ T cells (Tregs) regulate effector T cells in a transplantation setting and their in vivo homeostasis still remain to be clarified. Using a mouse adoptive transfer and skin transplantation model, we analyzed the in vivo expansion, effector function and trafficking of effector T cells and donor-specific Tregs, in response to an allograft. Methods and materials: Antigen-specific Tregs were generated and expanded in vitro by culturing freshly isolated Tregs from BALB/c mice (H2d) with syngeneic dendritic cells pulsed with an allopeptide (here the Kb peptide derived from the MHC class I molecule of allogeneic H2b mice). Fluorescent-labelled CD4+CD25- naive T cells and donor-antigen-specific Tregs were transferred alone or coinjected into syngeneic BALB/c-Nude recipients transplanted with allogeneic C57BL/6xBALB/c donor skin. Results: As opposed to their in vitro hyporesponsiveness, Tregs divided in vivo, migrated and accumulated in the allograft draining lymph nodes (drLN) and within the graft. The co-transfer of Tregs did not modify the early proliferation and homing of CD4+CD25- T cells to secondary lymphoid organs. But, in the presence of Tregs, effector T cells produced significantly less IFN- and IL-2 effector cytokines, while higher amounts of IL-10 were detected in the spleen and drLN of these mice. Furthermore, time-course studies showed that Tregs were recruited into the allograft at a very early stage posttransplantation and prevented infiltration by effector T cells. Conclusion: Overall, our results suggest that suppression of graft rejection involves the early recruitment of donor-specific Tregs at the sites of antigenic challenge and that Tregs mainly regulate the effector arm of T cell alloresponses.

A cell therapy approach for erythropoietin delivery using encapsulated human primary fibroblasts

Summary Cell therapy has emerged as a strategy for the treatment of various human diseases. Cells can be transplanted considering their morphological and functional properties to restore a tissue damage, as represented by blood transfusion, bone marrow or pancreatic islet cells transplantation. With the advent of the gene therapy, cells also were used as biological supports for the production of therapeutic molecules that can act either locally or at distance. This strategy represents the basis of ex vivo gene therapy characterized by the removal of cells from an organism, their genetic modification and their implantation into the same or another individual in a physiologically suitable location. The tissue or biological function damage dictates the type of cells chosen for implantation and the required function of the implanted cells. The general aim of this work was to develop an ex vivo gene therapy approach for the secretion of erythropoietin (Epo) in patients suffering from Epo-responsive anemia, thus extending to humans, studies previously performed with mouse cells transplanted in mice and rats. Considering the potential clinical application, allogeneic primary human cells were chosen for practical and safety reasons. In contrast to autologous cells, the use of allogeneic cells allows to characterize a cell lineage that can be further transplanted in many individuals. Furthermore allogeneic cells avoid the potential risk of zoonosis encountered with xenogeneic cells. Accordingly, the immune reaction against this allogeneic source was prevented by cell macro- encapsulation that prevents cell-to-cell contact with the host immune system and allows to easy retrieve the implanted device. The first step consisted in testing the survival of various human primary cells that were encapsulated and implanted for one month in the subcutaneous tissue of immunocompetent and naturally or therapeutically immunodepressed mice, assuming that xenogeneic applications constitute a stringent and representative screening before human transplantation. A fibroblast lineage from the foreskin of a young donor, DARC 3.1 cells, showed the highest mean survival score. We have then performed studies to optimize the manufacturing procedures of the encapsulation device for successful engraftment. The development of calcifications on the polyvinyl alcohol (PVA) matrix serving as a scaffold for enclosed cells into the hollow fiber devices was reported after one month in vivo. Various parameters, including matrix rinsing solutions, batches of PVA and cell lineages were assessed for their respective role in the development of the phenomenon. We observed that the calcifications could be totally prevented by using ultra-pure sterile water instead of phosphate buffer saline solution in the rinsing procedure of the PVA matrix. Moreover, a higher lactate dehydrogenase activity of the cells was found to decrease calcium depositions due to more acidic microenvironment, inhibiting the calcium precipitation. After the selection of the appropriate cell lineage and the optimization of encapsulation conditions, a retroviral-based approach was applied to DARC 3.1 fibroblasts for the transduction of the human Epo cDNA. Various modifications of the retroviral vector and the infection conditions were performed to obtain clinically relevant levels of human Epo. The insertion of a post-transcriptional regulatory element from the woodchuck hepatitis virus as well as of a Kozak consensus sequence led to a 7.5-fold increase in transgene expression. Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells allowing to reach 200 IU hEpo/10E6 cells /day. These modified cells were encapsulated and implanted in vivo in the same conditions as previously described. All the mouse strains showed a sustained increase in their hematocrit and a high proportion of viable cells were observed after retrieval of the capsules. Finally, in the perspective of human application, a syngeneic model using encapsulated murine myoblasts transplanted in mice was realized to investigate the roles of both the host immune response and the cells metabolic requirements. Various loading densities and anti-inflammatory as well as immunosuppressive drugs were studied. The results showed that an immune process is responsible of cell death in capsules loaded at high cell density. A supporting matrix of PVA was shown to limit the cell density and to avoid early metabolic cell death, preventing therefore the immune reaction. This study has led to the development of encapsulated cells of human origin producing clinically relevant amounts of human EPO. This work resulted also to the optimization of cell encapsulation technical parameters allowing to begin a clinical application in end-stage renal failure patients. Résumé La thérapie cellulaire s'est imposée comme une stratégie de traitement potentiel pour diverses maladies. Si l'on considère leur morphologie et leur fonction, les cellules peuvent être transplantées dans le but de remplacer une perte tissulaire comme c'est le cas pour les transfusions sanguines ou les greffes de moelle osseuse ou de cellules pancréatiques. Avec le développement de la thérapie génique, les cellules sont également devenues des supports biologiques pour la production de molécules thérapeutiques. Cette stratégie représente le fondement de la thérapie génique ex vivo, caractérisée par le prélèvement de cellules d'un organisme, leur modification génétique et leur implantation dans le même individu ou dans un autre organisme. Le choix du type de cellule et la fonction qu'elle doit remplir pour un traitement spécifique dépend du tissu ou de la fonction biologique atteintes. Le but général de ce travail est de développer .une approche par thérapie génique ex vivo de sécrétion d'érythropoïétine (Epo) chez des patients souffrant d'anémie, prolongeant ainsi des travaux réalisés avec des cellules murines implantées chez des souris et des rats. Dans cette perpective, notre choix s'est porté sur des cellules humaines primaires allogéniques. En effet, contrairement aux cellules autologues, une caractérisation unique de cellules allogéniques peut déboucher sur de nombreuses applications. Par ailleurs, l'emploi de cellules allogéniques permet d'éviter les riques de zoonose que l'on peut rencontrer avec des cellules xénogéniques. Afin de protéger les cellules allogéniques soumises à une réaction immunitaire, leur confinement dans des macro-capsules cylindriques avant leur implantation permet d'éviter leur contact avec les cellules immunitaires de l'hôte, et de les retrouver sans difficulté en cas d'intolérance ou d'effet secondaire. Dans un premier temps, nous avons évalué la survie de différentes lignées cellulaires humaines primaires, une fois encapsulées et implantées dans le tissu sous-cutané de souris, soit immunocompétentes, soit immunodéprimées naturellement ou par l'intermédiaire d'un immunosuppresseur. Ce modèle in vivo correspond à des conditions xénogéniques et représente par conséquent un environnement de loin plus hostile pour les cellules qu'une transplantation allogénique. Une lignée fibroblastique issue du prépuce d'un jeune enfant, nommée DARC 3 .1, a montré une remarquable résistance avec un score de survie moyen le plus élevé parmi les lignées testées. Par la suite, nous nous sommes intéressés aux paramètres intervenant dans la réalisation du système d'implantation afin d'optimaliser les conditions pour une meilleure adaptation des cellules à ce nouvel environnement. En effet, en raison de l'apparition, après un mois in vivo, de calcifications au niveau de la matrice de polyvinyl alcohol (PVA) servant de support aux cellules encapsulées, différents paramètres ont été étudiés, tels que les procédures de fabrication, les lots de PVA ou encore les lignées cellulaires encapsulées, afin de mettre en évidence leur rôle respectif dans la survenue de ce processus. Nous avons montré que l'apparition des calcifications peut être totalement prévenue par l'utilisation d'eau pure au lieu de tampon phosphaté lors du rinçage des matrices de PVA. De plus, nous avons observe qu'un taux de lactate déshydrogénase cellulaire élevé était corrélé avec une diminution des dépôts de calcium au sein de la matrice en raison d'un micro-environnement plus acide inhibant la précipitation du calcium. Après sélection de la lignée cellulaire appropriée et de l'optimisation des conditions d'encapsulation, une modification génétique des fibroblastes DARC 3.1 a été réalisée par une approche rétrovirale, permettant l'insertion de l'ADN du gène de l'Epo dans le génome cellulaire. Diverses modifications, tant au niveau génétique qu'au niveau des conditions d'infection, ont été entreprises afin d'obtenir des taux de sécrétion d'Epo cliniquement appropriés. L'insertion dans la séquence d'ADN d'un élément de régulation post¬transcriptionnelle dérivé du virus de l'hépatite du rongeur (« woodchuck ») ainsi que d'une séquence consensus appelée « Kozak » ont abouti à une augmentation de sécrétion d'Epo 7.5 fois plus importante. De même, l'optimisation de la multiplicité d'infection et la sélection plus drastique des cellules hautement productrices ont permis finalement d'obtenir une sécrétion correspondant à 200 IU d'Epo/10E6 cells/jour. Ces cellules génétiquement modifiées ont été encapsulées et implantées in vivo dans les mêmes conditions que celles décrites plus haut. Toutes les souris transplantées ont montré une augmentation significative de leur hématocrite et une proportion importante de cellules présentait une survie conservée au moment de l'explantation des capsules. Finalement, dans la perspective d'une application humaine, un modèle syngénique a été proposé, basé sur l'implantation de myoblastes murins encapsulés dans des souris, afin d'investiguer les rôles respectifs de la réponse immunitaire du receveur et des besoins métaboliques cellulaires sur leur survie à long terme. Les cellules ont été encapsulées à différentes densités et les animaux transplantés se sont vus administrer des injections de molécules anti-inflammatoires ou immunosuppressives. Les résultats ont démontré qu'une réaction immunologique péri-capsulaire était à la base du rejet cellulaire dans le cas de capsules à haute densité cellulaire. Une matrice de PVA peut limiter cette densité et éviter une mort cellulaire précoce due à une insuffisance métabolique et par conséquent prévenir la réaction immunitaire. Ce travail a permis le développement de cellules encapsulées d'origine humaine sécrétant des taux d'Epo humaine adaptés à des traitements cliniques. De pair avec l'optimalisation des paramètres d'encapsulation, ces résultats ont abouti à l'initiation d'une application clinique destinée à des patients en insuffisance rénale terminale.

Role of the REST/RE-1 system in beta-cell life, function and differentiation

SUMMARYDiabetes is characterized by insulin deficiency that results from the destruction of insulin-secreting pancreatic beta-cells (Type 1), or in part from beta-cell death and insulin secretion defects (Type 2). Therefore, understanding the mechanisms of beta cell neogenesis (to generate unlimited supply of beta cells for T1D transplantation] or identifying the specific genes that favors insulin secretion or beta-cell survival is of great importance for the management of diabetes. The transcriptional repressor RE-1 Silencing Transcription Factor (REST) restricts the expression of a large number of genes containing its binding element, called Repressor Element-1 (RE-1), to neurons and beta cells. To do so, REST is ubiquitously expressed but in neurons and beta cells. To identify these essential genes and their functional significance in beta cells, we have generated transgenic mice that express REST specifically in beta cells under the control of the rat insulin promoter (RIP-REST mice). This resulted in the repression of the RE-1- containing genes in beta cells, and we analyzed the consequences.We first showed that RIP-REST mice were glucose-intolerant because of a defective insulin secretion. To explain this defect, we identified that a subset of the REST target genes were necessary for insulin exocytosis, such as Snap25, Synaptotagmin (Syt) IX, Complexin II, and Ica512, and we further demonstrated that among the identified REST targets, Syt IV and VII were also involved in insulin release. We next analyzed a novel RIP-REST mouse line that featured diabetes and we showed that this defect was due to a major loss of beta-cell mass. To explain this phenotype, we identified REST target genes that were involved in beta-cell survival, such as Ibl, Irs2, Ica512 and Connexin36, and revealed that another REST target, Cdk5r2 is also involved in beta-cell protection. In a third part, we finally suggest that REST may be important for pancreatic endocrine differentiation, since transgenic mice expressing constitutive REST in pancreatic multipotent progenitors show impaired formation of Ngn3-expressing endocrine- committed precursors, and impaired formation of differentiated endocrine cells. Mapping the pattern of REST expression in wild type animals indicates that it is expressed in multipotent progenitors to become then excluded from endocrine cells. Preliminary results suggest that a downregulation of REST would result in relieved expression of at least the Mytl target, favoring subsequent acquisition of the endocrine competence by endocrine precursor cells.Thus, we propose that the REST/RE-1 system is an important feature for beta-cell neogenesis, function and survivalRESUMELe diabète se caractérise par une déficience en insuline qui résulte d'une destruction des cellules bêta (β) pancréatiques sécrétant l'insuline [Type 1], ou à un défaut de sécrétion d'insuline qui peut être associé à la mort des cellules β (Type 2). La compréhension des mécanismes de néogenèse des cellules β, ainsi que l'identification de gènes impliqués dans leur survie et dans le contrôle de la sécrétion d'insuline est donc importante pour le traitement du diabète. Le facteur de transcription de type répresseur, RE-1 Silencing Transcription Factor [REST], contribue à la spécificité d'expression dans les neurones et les cellules β, d'un grand nombre de gènes portant son motif de fixation, le Repressor Element-1 (RE-1). Pour cela, REST est exprimé dans toutes les cellules, sauf dans les neurones et les cellules β. Afin d'identifier les gènes cibles de REST ainsi que leur fonction au sein de la cellule β, nous avons généré des souris transgéniques qui expriment REST spécifiquement dans ces cellules, sous la dépendance du promoteur de l'insuline (souris RIP-REST]. Cette expression ectopique de REST a permis de diminuer l'expression des gènes contrôlés par REST, et d'en analyser les conséquences. Nous avons montré que les souris RIP-REST étaient intolérantes au glucose et que ceci était du à un défaut de sécrétion d'insuline. Pour expliquer ce phénotype, nous avons mis en évidence le fait que des gènes cibles de REST codent pour des protéines importantes pour l'exocytose de l'insuline, comme SNAP25, Synaptotagmin (Syt) IX, Complexin II ou ICA512. De plus, nous avons découvert deux nouvelles cibles de REST impliquées dans la sécrétion d'insuline, Syt IV et Syt VII. Par la suite, nous avons démontré qu'une nouvelle lignée de souris RIP-REST étaient atteintes d'un diabète sévère à cause d'une perte massive des cellules β. La disparition de ces cellules a été expliquée par l'identification de gènes cibles de REST impliqués dans la survie des cellules β, comme Ibl, Irs2, Ica512 ou la Connexine36. De plus, nous avons découvert qu'une nouvelle cible, Cdk5r2, était aussi impliquée dans la survie des cellules β. Dans une dernière partie, nous suggérons, grâce à l'analyse de nouvelles souris transgéniques exprimant constitutivement REST dans les cellules progénitrices du pancréas embryonnaire, que REST empêche la formation des précurseurs de cellules endocrines ainsi que la différenciation de ces cellules. L'analyse de l'expression de REST au cours du développement embryonnaire du pancréas indique que la diminution de l'expression de REST conduit en partie, à l'induction d'un de ses gènes cible Mytl, qui favorise la formation de précurseurs endocrines. Nous proposons donc que le système REST/RE-1 est important pour la génération, la fonction et la survie des cellules β.

Expansion and tissue infiltration of an allospecific CD4+CD25+CD45RO+IL-7Ralphahigh cell population in solid organ transplant recipients.

It has been recently shown (Seddiki, N., B. Santner-Nanan, J. Martinson, J. Zaunders, S. Sasson, A. Landay, M. Solomon, W. Selby, S.I. Alexander, R. Nanan, et al. 2006. J. Exp. Med. 203:1693-1700.) that the expression of interleukin (IL) 7 receptor (R) alpha discriminates between two distinct CD4 T cell populations, both characterized by the expression of CD25, i.e. CD4 regulatory T (T reg) cells and activated CD4 T cells. T reg cells express low levels of IL-7Ralpha, whereas activated CD4 T cells are characterized by the expression of IL-7Ralpha(high). We have investigated the distribution of these two CD4 T cell populations in 36 subjects after liver and kidney transplantation and in 45 healthy subjects. According to a previous study (Demirkiran, A., A. Kok, J. Kwekkeboom, H.J. Metselaar, H.W. Tilanus, and L.J. van der Laan. 2005. Transplant. Proc. 37:1194-1196.), we observed that the T reg CD25(+)CD45RO(+)IL-7Ralpha(low) cell population was reduced in transplant recipients (P < 0.00001). Interestingly, the CD4(+)CD25(+)CD45RO(+)IL-7Ralpha(high) cell population was significantly increased in stable transplant recipients compared with healthy subjects (P < 0.00001), and the expansion of this cell population was even greater in patients with documented humoral chronic rejection compared with stable transplant recipients (P < 0.0001). The expanded CD4(+)CD25(+)CD45RO(+)IL-7Ralpha(high) cell population contained allospecific CD4 T cells and secreted effector cytokines such as tumor necrosis factor alpha and interferon gamma, thus potentially contributing to the mechanisms of chronic rejection. More importantly, CD4(+)IL-7Ralpha(+)and CD25(+)IL-7Ralpha(+) cells were part of the T cell population infiltrating the allograft of patients with a documented diagnosis of chronic humoral rejection. These results indicate that the CD4(+)CD25(+)IL-7Ralpha(+) cell population may represent a valuable, sensitive, and specific marker to monitor allospecific CD4 T cell responses both in blood and in tissues after organ transplantation.