Evaluación del uso de exosomas como aloantígenos para la inducción de tolerancia en trasplante

La consecución de tolerancia aloespecífica es de mucha relevancia en trasplante. Las células dendríticas (DC) son las principales responsables de la inducción de la respuesta inmune frente a las moléculas de histocompatibilidad (MHC) del donante, provocando el rechazo del injerto. Sin embargo las DC son también responsables de la inducción de tolerancia. Diversos modelos animales de alotrasplante han mostrado la tolerización del injerto mediante DC diferenciadas in vitro en condiciones tolerogénicas (tDC). En humanos, las fuentes de aloantígenos potencialmente utilizables en terapia son, entre otras, los cuerpos apoptóticos y los exosomas. Éstos expresan antígenos MHC de forma abundante y su composición es relativamente uniforme, lo que supone una ventaja frente a otras fuentes. En este proyecto, se ha evaluado la obtención de exosomas secretados por una línea de linfocitos T y por células dendríticas derivadas de médula ósea. Se ha caracterizado la captura de exosomas derivados de linfocitos T por células dendríticas humanas derivadas de sangre periférica y su presentación a linfocitos T autólogos. Por otra parte, se ha comenzado a desarrollar los experimentos para estudiar la inducción de tolerancia en un modelo de trasplante renal en rata. Se han generado células dendríticas tolerógenicas derivadas de médula ósea (tolDC), en presencia de dexametasona. Las tolDC expresan menos moléculas de histocompatibilidad y de coestimulación e inducen una menor proliferación en reacciones mixtas leucocitaras, comparadas con las células dendríticas maduras. Por último, se han caracterizado los exosomas de plasma humano con el fin de estudiar su posible uso como aloantígenos. El análisis proteómico revela la presencia de proteínas relacionadas con el sistema inmune, la coagulación, la señalización celular y moléculas implicadas en el transporte y metabolismo de nutrientes. El estudio de la captura por diferentes líneas celulares sugiere que deben existir mecanismos específicos para su internalización.

Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T-cell activation in vivo.

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c(high) DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44(+) CD62(-)) CD4(+) and CD8(+) T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC(+) DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0.95% versus 0.47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.

The changing preference of T and B cells for partners as T-dependent antibody responses develop.

Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move to the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class switch-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones B cells migrate to follicles, where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci, but to the follicles; there they proliferate and are subsequently involved in the selection of B cells that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cells found in that site: a substantial proportion of the CD4+ memory T-cell pool may originate from growth in follicles.

Secretory IgA-mediated neutralization of Shigella flexneri prevents intestinal tissue destruction by down-regulating inflammatory circuits.

Shigella, a Gram-negative invasive enteropathogenic bacterium responsible for bacillary dysentery, causes the rupture, invasion, and inflammatory destruction of the human colonic mucosa. We explored the mechanisms of protection mediated by Shigella LPS-specific secretory IgA (SIgA), the major mucosal Ab induced upon natural infection. Bacteria, SIgA, or SIgA-S. flexneri immune complexes were administered into rabbit ligated intestinal loops containing a Peyer's patch. After 8 h, localizations of bacteria, SIgA, and SIgA-S. flexneri immune complexes were examined by immunohistochemistry and confocal microscopy imaging. We found that anti-Shigella LPS SIgA, mainly via immune exclusion, prevented Shigella-induced inflammation responsible for the destruction of the intestinal barrier. Besides this luminal trapping, a small proportion of SIgA-S. flexneri immune complexes were shown to enter the rabbit Peyer's patch and were internalized by dendritic cells of the subepithelial dome region. Local inflammatory status was analyzed by quantitative RT-PCR using newly designed primers for rabbit pro- and anti-inflammatory mediator genes. In Peyer's patches exposed to immune complexes, limited up-regulation of the expression of proinflammatory genes, including TNF-alpha, IL-6, Cox-2, and IFN-gamma, was observed, consistent with preserved morphology. In contrast, in Peyer's patches exposed to Shigella alone, high expression of the same mediators was measured, indicating that neutralizing SIgA dampens the proinflammatory properties of Shigella. These results show that in the form of immune complexes, SIgA guarantees both immune exclusion and neutralization of translocated bacteria, thus preserving the intestinal barrier integrity by preventing bacterial-induced inflammation. These findings add to the multiple facets of the noninflammatory properties of SIgA.

Improvement of the HIV/AIDS vaccine candidate NYVAC-C by deletion of the viral type I IFN inhibitor and/or acquisition of replication competence

Background: Recombinant viruses based on the attenuated vaccinia virus strain NYVAC are promising HIV vaccine candidates as phase I/II clinical trials have shown good safety and immunogenicity profiles. However, this NYVAC strain is non-replicating in most human cell lines and encodes viral inhibitors of the immune system. Methods: With the aim to increase the immune potency of the current NYVAC-C vector (expressing the codon optimized clade C HIV-1 genes encoding gp120 and Gag-Pol-Nef polyprotein), we have generated and characterized three NYVAC-C-based vectors by, 1) deletion of the viral type I IFN inhibitor gene (NYVAC-CdeltaB19R), 2) restoration of virus replication competence in human cells by re-inserting K1L and C7L host range genes (NYVAC-C-KC) and, 3) combination of both strategies (NYVACC- KC-deltaB19R). Results: Insertion of the KC fragment restored the replication competence of the viruses in human cells (HeLa cells and primary dermal fibroblasts and keratinocytes), increased the expression of HIV antigens by more than 3-fold compared to the non-replicating homologs, inhibited apoptosis induced by the parental NYVAC-C and retained attenuation in a newborn mouse model. In adult mice, replication-competent viruses showed a limited capacity to replicate in tissues surrounding the inoculation site (ovaries and lymph nodes). After infection of keratinocytes, PBMCs and dendritic cells these viruses induced differential modulation in specific host cell signal transduction pathways, triggering genes important in immune modulation. Conclusion: We have developed improved NYVAC-C-based vectors with enhanced HIV-1 antigen expression, with the ability to replicate in cultured human cells and partially in some tissues, with an induced expression of cellular genes relevant to immune system activation, and which trigger IFN-dependent and independent signalling pathways, while maintaining a safety phenotype. These new vectors are promising new HIV vaccine candidates. These studies were performed within the Poxvirus Tcell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Protection from radiation-induced colitis requires MHC class II antigen expression by cells of hemopoietic origin.

Ulcerative colitis, an inflammatory bowel disease, is believed to result from a breakdown of dominant tolerance mechanisms that normally control intestinal immunity. Although CD4+ T lymphocyte subpopulations and expression of MHC class II molecules have been shown to play a role in the pathogenesis of the disease, the nature of the responsible mechanisms remains unclear. In this paper we describe a novel mouse model for inflammatory bowel disease, radiation-induced colitis, that occurs with complete penetrance 6-8 wk postinduction. A combination of high dose gamma-irradiation and lack of MHC class II expression on cells of hemopoietic origin results in development of colitis in C57BL/6 mice. Because of its versatility (due to susceptibility of mice of the widely genetically manipulated C57BL/6 background), high reproducibility, and 100% penetrance, radiation-induced colitis will be a useful mouse model for colitis and a significant tool to study dominant immunological tolerance mechanisms. Moreover, our data imply that tolerization to enteric Ags requires MHC class II mediated presentation by APC of hemopoietic origin.

Overlapping, additive and counterregulatory effects of type II and I interferons on myeloid dendritic cell functions.

Dendritic cells (DCs) are central player in immunity by bridging the innate and adaptive arms of the immune system (IS). Interferons (IFNs) are one of the most important factors that regulate both innate and adaptive immunity too. Thus, the understanding of how type II and I IFNs modulate the immune-regulatory properties of DCs is a central issue in immunology. In this paper, we will address this point in the light of the most recent literature, also highlighting the controversial data reported in the field. According to the wide literature available, type II as well as type I IFNs appear, at the same time, to collaborate, to induce additive effects or overlapping functions, as well as to counterregulate each one's effects on DC biology and, in general, the immune response. The knowledge of these effects has important therapeutic implications in the treatment of infectious/autoimmune diseases and cancer and indicates strategies for using IFNs as vaccine adjuvants and in DC-based immune therapeutic approaches.

Expression pattern of sirtuins in innate immune cells and influence of Sirtuin 1/2 inhibition on innate immune responses to Toll-like receptor ligands and to infection

Purpose/Objective: The family of histone deacetylases comprises 18 members in mammals, among which seven sirtuins (SIRT1-7). Sirtuins are NADP-dependent enzymes that have been involved in the control of cell metabolism, proliferation and survival. The expression pattern of sirtuins and their influence on host response to microbial infection remain largely unknown. The aim of the study was to analyze the expression of SIRT1-7 and to address the effects of SIRT1/2 inhibition on innate immune responses in vitro and in vivo.. Materials and methods: in vitro: Bone marrow (BM), BM-derived macrophages (BMDMs) and dendritic cells (BMDCs) and RAW 264.7 and J774.1 macrophage cell lines were stimulated for 0, 2, 6 and 18 h with LPS, Pam3CSK4 and CpG ODN. SIRT1-7 mRNA was quantified by real time-PCR. TNF was measured by ELISA. In vivo: BALB/c mice were challenged with LPS (350 lg i.p.) with or without a SIRT1/2 inhibitor. Blood and organs were collected after 0, 1, 4, 8 and 24 h to quantify SIRT1-7 and TNF. Mortality was assessed daily. Results: Bone marrow, macrophages and DCs express, in order of abundance, SIRT2 > > SIRT1, SIRT3 and SIRT6 > SIRT4, SIRT5 and SIRT7. Microbial products decrease the expression of all sirtuins except SIRT6 in a time dependent manner in BMDMs (0_24 h). SIRT2 is the most expressed sirtuin also in the liver, kidney (together with SIRT3) and spleen. Upon LPS challenge, SIRT1, SIRT3, SIRT4 and SIRT7 mRNA levels decrease in the liver (from 4 h to 24 h), whereas SIRT1-7 mRNA levels decrease within 1 h in both kidney and spleen. Pharmacological inhibition of SIRT1/2 decreases TNF production by macrophages stimulated with LPS, Pam3CSK4 and CpG ODN (n = 6; P < 0.001). In agreement, prophylactic treatment with a SIRT1/2 inhibitor decreases TNF production (n = 8; P = 0.04) and increases survival (n = 13, P = 0.03) of mice challenged with LPS. Conclusions: Sirtuins are expressed in innate immune cells. Inhibition of SIRT1/2 activity decreases cytokine production by macrophages and protects from endotoxemia, suggesting that sirtuin inhibitors may represent novel adjunctive therapy for treating inflammatory disorders such as sepsis.

Induction of apoptosis in A549 pulmonary cells by two Paracoccidioides brasiliensis samples

Paracoccidioidomycosis presents a variety of clinical manifestations and Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The ability of the pathogen to interact with host surface structures is essential to its virulence. The interaction between P. brasiliensis and epithelial cells has been studied, with particular emphasis on the induction of apoptosis. To investigate the expression of different apoptosis-inducing pathways in human A549 cells, we infected these cells with P. brasiliensis Pb18SP (subcultured) and 18R (recently isolated from cell culture and showing a high adhesion pattern) samples in vitro. The expressions of Bcl-2, Bak and caspase 3 were analysed by flow cytometry and DNA fragmentation using the TUNEL technique. Apoptosis of human A549 cells was induced by P. brasiliensis in a sample and time-dependent manner. Using an in vitro model, our data demonstrates that caspase 3, Bak, Bcl-2 and DNA fragmentation mediate P. brasiliensis-induced apoptosis in A549 cells. The overall mechanism is a complex process, which may involve several signal transduction pathways. These findings could partially explain the efficient behaviour of this fungus in promoting tissue infection and/or blood dissemination.

The processing limits the presentation of the melanoma-associated antigen Melan-A in vitro and in vivo

SUMMARY Both proteasomes and additional proteases play an essential role in the generation of most antigenic peptides presented by MHC class I molecules. Therefore, it is of major importance to characterize the mechanisms leading to the production of correct antigenic peptides to improve the design of vaccines. As a model determinant we used the melanoma-associated protein Melan-A, which contains the immunodominant CTL-epitope Melan-A26/27-35/HLA-A*0201 and against which a high frequency of T lymphocytes has been detected in many melanoma patients. In a first part, we have studied the effects of antigen processing on the induction of a specific T cell response in vivo. Our results have shown that the immunoproteasome, expressed in most cells after exposure to Interferon-γ (IFN-γ) and constitutively in some specialized cells such as dendritic cells, does not efficiently process the HLA¬A2-restricted peptide Melan-A26-35. We have produced recombinant lentiviral vectors (rec. 1v) and vaccinia virus (rec. vv) encoding either preprocessed Melan-A26-35(A27L) peptide or full-length Melan-A(A27L). The immunization of HLA-A2/Kb mice with thoses viruses indicates that immunoproteasomes negatively affect the induction of anti-Melan-A T cell responses in animals immunized with vectors coding for the full- length protein. This negative effect was abrogated in HLA-A2/Kb LMP2-/- mice, lacking the immunoproteasomes. Therefore, we can conclude that the expression of immunoproteasomes limits the induction of the anti-Melan-A T cell response. In a second part, we show that the in vitro degradation of a Melan-A26/27-35 precursor by the proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. We demonstrated that the N-terminally extended intermediates were inefficiently trimmed by cytosolic proteases. These results imply that both proteasomes and post-proteasomal peptidases influence the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products. RESUME Le protéasome ainsi que d'autres protéases jouent un rôle essentiel dans l'apprêtement de la plupart des peptides antigéniques présentés par les molécules de MHC classe I. Il est donc particulièrement important de connaître les mécanismes menant à la production du peptide antigénique correct afin de pouvoir mieux définir de futurs vaccins. Nous avons utilisé la protéine associée au mélanome, Melan-A, contenant un épitope immunodominant Melan-A26/27-35/HLA-A*0201 contre lequel une fréquence élevée de lymphocytes T a été detectée dans plusieurs patients atteints de mélanome. Dans une première partie, nous avons étudié les effets de l'apprêtement du peptide antigéniques Melan-A26-35 sur l'induction de cellules T spécifiques dans la souris. Nos résultats ont démontré que l'immunoprotéasome, exprimé dans la plupart des cellules après exposition à de l'IFN-γ et exprimé constitutivement dans certaines cellules spécialisées, telles les cellules dendritiques, n'apprête pas efficacement le peptide antigénique Melan-A26-35 restreint par HLA-A2 in vitro. Nous avons produit des vecteurs lentiviraux recombinants ainsi que des virus vaccinia codant pour le peptide antigénique Melan-A26-35(A27L) et pour la protéine entière Melan-A(A27L). L'immunisation de souris HLA-A2/Kb avec ces virus démontre que l'immunoprotéasome affecte négativement l'induction d'une réponse T contre Melan¬-A dans les souris immunisées avec des virus contenant la séquence de la protéine entière. Cet effet négatif est complètement aboli dans les souris HLA-A2/Kb LMP2-/- qui n'expriment pas l'immunoprotéasome. Deuxièmement, nous avons demontré que la dégradation d'un peptide précurseur contenant Melan-A26/27-35 par le protéasome produit à la fois le peptide antigénique ainsi que des peptides rallongés à leurs extrémités N-terminales. Lorsque ces fragments sont exprimés dans des cellules humaines et exposés à des cellules T cytotoxiques (CTL), celles qui expriment le peptide antigénique final sont reconnus plus efficacement que celles exprimant les peptides rallongés en N-terminus. Nous avons démontré que les peptides rallongés en N-terminus ne sont pas apprêtés efficacement par les peptidases du cytosol. L'inefficacité de l'apprêtement des peptides rallongés dans le cytosol offre un certain avantage pour les peptides directement produits par le protéasome. Ces résultats impliquent donc que le protéasome ainsi que les peptidases post-proteasomales influencent l'accessibilité des peptides antigéniques.

Sustained activation and tumor targeting of NKT cells using a CD1d-anti-HER2-scFv fusion protein induce antitumor effects in mice

Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated. A single injection of the high-affinity CD1d ligand alpha-galactosylceramide (alphaGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when alphaGalCer was loaded on a recombinant soluble CD1d molecule (alphaGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-gamma secretion as well as DC maturation in mice. Most importantly, when alphaGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2-7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free alphaGalCer at this time had no effect. The antitumor activity of the CD1d-anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy

From diabetes to heart disease : studies on dyslipidemia-induced B-cell dysfunction, immunomodulation in heart transplantation, and cardiac stem cell therapy

Diabetes is a growing epidemic with devastating human, social and economic impact. It is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent LDL and HDL particles in insulin-secreting β-cells. Purified human VLDL and LDL particles reduced insulin mRNA levels and β-cell proliferation, and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of β-cells involved caspase-3 cleavage and reduction in levels of the c-Jun N-terminal (JNK) Interacting Protein-1 (IB1/JIP-1). In contrast, the pro-apoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of JNK. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of the protein kinase Akt/PKB. Heart disease is a major cause of morbidity and mortality among patients with diabetes. When heart failure is refractory to medical therapy and cannot be improved by electrical resynchronization, percutaneous angioplasty or coronary graft bypass surgery, heart transplantation remains a "last resort" therapy. Nevertheless, it is limited by the side effects of immunosuppressive drugs and chronic rejection. Localized expression of immunomodulatory genes in the donor organ can create a state of immune privilege within the graft, and was performed in rodent hearts by infecting cells with an adenovirus encoding indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in the catabolism of tryptophane. Other strategies are based on genetic manipulation of dendritic cells (DCs) with immunosuppressive genes and in vitro exposure of DCs to agents that prevent their maturation by inflammatory cytokines. Finally, we used 5-bromo-2'-deoxyuridine, which is incorporated into DNA and diluted with cell division, to identify long-term label retaining cells in the adult rodent heart. The majority of these cells were positive for the stem cell antigen-1 (Sca-1) and negative for the endothelial precursor marker CD31. They formed cardiospheres in vitro and showed differentiation potential into mesenchymal cell lineages. When cultured in cardiomyogenic differentiation medium, they expressed cardiac-specific genes. Taken together, these data provide evidence of slow-cycling stem cells in the rodent heart. Chronic shortage of donor organs opens the way to cardiac stem cell therapy in humans, although the long way from animal experimentation to routine therapy in patients may still take several years. - Du diabète de type 2 à la maladie coronarienne : trois études sur les dysfonctions de la cellule sécrétrice d'insuline induites par les dyslipidémies, l'immunomodulation dans la transplantation cardiaque, et la thérapie par des cellules souches myocardiques. Le diabète de type 2 a pris les dimensions d'une épidémie, avec des conséquences sociales et économiques dont nous n'avons pas encore pris toute la mesure. La maladie s'accompagne souvent d'une dyslipidémie caractérisée par une hypertriglycéridémie, des taux abaissés de cholestérol HDL, et des concentrations de cholestérol LDL à la limite supérieure de ce qui est considéré comme acceptable. L'hypothèse à la base de cette étude est qu'une modification des taux plasmatiques de lipoprotéines pourrait avoir une influence directe sur la cellule β sécrétrice d'insuline en modifiant sa fonction, sa durée de vie et son taux de régénération. Dans un premier temps, nous avons mis en évidence, sur la cellule β, la présence de plusieurs récepteurs impliqués dans la captation des lipoprotéines. Nous avons confirmé la fonctionnalité de ces récepteurs en suivant l'internalisation de LDL et de HDL marqués. En présence de VLDL ou de LDL humains, nous avons observé une diminution de la transcription du gène de l'insuline, une prolifération cellulaire réduite, et une augmentation de l'apoptose, toutes fonctions de la dose et du temps d'exposition. L'apoptose induite par les VLDL passe par une activation de la caspase-3 et une réduction du taux de la protéine IB1/JIP-1 (Islet Brain1/JNK Interacting Protein 1), dont une mutation est associée à une forme monogénique de diabète de type 2. Par opposition, les HDL, ainsi que des peptides inhibiteurs de JNK, sont capables de contrer la cascade pro-apoptotique déclenchée, respectivement, par les LDL et les VLDL. Ces effets protecteurs comprennent l'inhibition du clivage de la caspase-3 et l'activation de la protéine kinase Akt/PKB. En conclusion, les lipoprotéines sont des éléments clés de la survie de la cellule β, et pourraient contribuer au dysfonctionnement observé dans le pancréas endocrine au cours du développement du diabète. La maladie cardiaque, et plus particulièrement la maladie coronarienne, est une cause majeure de morbidité et de mortalité chez les patients atteints de diabète. Plusieurs stratégies sont utilisées quotidiennement pour pallier les atteintes cardiaques: traitements médicamenteux, électromécaniques par resynchronisation électrique, ou communément appelés « interventionnels » lorsqu'ils font appel à l'angioplastie percutanée. La revascularisation du myocarde par des pontages coronariens donne également de très bons résultats dans certaines situations. Il existe toutefois des cas où plus aucune de ces approches n'est suffisante. La transplantation cardiaque est alors la thérapie de choix pour un nombre restreint de patients. La thérapie génique, en permettant l'expression locale de gènes immunomodulateurs dans l'organe greffé, permet de diminuer les réactions de rejet inhérentes à toute transplantation (à l'exception de celles réalisées entre deux jumeaux homozygotes). Nous avons appliqué chez des rongeurs cette stratégie en infectant le coeur greffé avec un adénovirus codant pour l'enzyme indoleamine 2,3-dioxygénase (IDO), une enzyme clé dans le catabolisme du tryptophane. Nous avons procédé de manière identique in vitro en surexprimant IDO dans les cellules dendritiques, dont le rôle est de présenter les antigènes aux lymphocytes Τ du receveur. Des expériences similaires ont été réalisées en traitant les cellules dendritiques avec des substances capables de prévenir, en partie du moins, leur maturation par des agents pro-inflammatoires. Finalement, nous avons exploré une stratégie utilisée couramment en hématologie, mais qui n'en est encore qu'à ses débuts au niveau cardiaque : la thérapie par des cellules souches. En traitant des rongeurs avec un marqueur qui s'incorpore dans l'ADN nucléaire, le 5-bromo- 2'-deoxyuridine, nous avons identifié une population cellulaire se divisant rarement, positive en grande partie pour l'antigène embryonnaire Sca-1 et négative pour le marqueur endothélial CD31. En culture, ces cellules forment des cardiosphères et sont capables de se différencier dans les principaux types tissulaires mésenchymateux. Dans un milieu de differentiation adéquat, ces cellules expriment des gènes cardiomyocytaires. En résumé, ces données confirment la présence chez le rongeur d'une population résidente de précurseurs myocardiques. En addenda, on trouvera deux publications relatives à la cellule β productrice d'insuline. Le premier article démontre le rôle essentiel joué par la complexine dans l'insulino-sécrétion, tandis que le second souligne l'importance de la protéine IB1/JIP-1 dans la protection contre l'apoptose de la cellule β induite par certaines cytokines.

The role of dentritic cells in leishmania infection : use of novel DC lines for the development of monoclonal antibodies

Les cellules dendritiques (DCs) sont des cellules multifonctionnelles qui font le lien entre le sytème immunitaire inné et adaptatif chez les mammifères. Il existe plusieurs sous-types de DCs basés sur leurs fonctions et l'endroit où elles se situent dans le corps. Dans le cadre de cette thèse, nous avons étudié le rôle de ces cellules face à une infection parasitaire. La Leishmania est un parasite causant une maladie appelée Leishmaniose, maladie endémique de l'Afrique, de l'Asie et de certaines régions de l'Amérique du Sud. Certaines espèces causent des lésions cutanées, alors que d'autres causent des lésions dans les muqueuses ou dans les organes internes. Le système immunitaire répond en générant une réponse inflammatoire qui élimine l'infection. Lors d'une réponse non-inflammatoire (de type cytokines, chemokines), cela va amener à une persistance du parasite sur le long terme. Les DC s'activant en présence du parasite dans la peau, vont le transporter vers un ganglion. A cet endroit, se trouvent différents sous-types de DC qui ont la particularité de présenter l'antigène (spécifique à la Leishmaniose) aux lymphocytes T, ce qui va alors amener à une réponse immunitaire puissante contre le parasite. Nous avons comparé différentes espèces de Leishmaniose dans leur façon d'activer les DC et différents modèles de souris ont été utilisé dans ce but-là. Les souris du type C57BL/6 sont connues pour être résistantes à L. major et sensibles à L. mexicana, alors qu'au contraire, les souris Balb/c sont connues pour être sensibles à ces deux espèces. En utilisant des parasites fluorescents transgéniques, nous avons comparé ces deux espèces de parasites (L. major et L. mexicana) en recherchant quelles cellules elles sont capables d'infecter in-vivo dans un modèle murin. Le rôle général des DC dans une infection à L. major a déjà été décrit. Dans notre étude, nous avons étudié le besoin en DC CD8a+ dans les ganglions afin d'engendrer une réponse face à une infection à L. major. Les souris qui n'ont pas ce sous-type de DC sont beaucoup plus sensibles à l'infection : elles ont des marqueurs inflammatoires plus bas et des lésions plus grandes. Nous avons également remarqué que les DC CD8a+ jouent un rôle crucial dans une phase plus avancée de l'infection. Dans notre laboratoire, nous avons la chance d'avoir une source illimitée de DCs de sous-type CD8a+ provenant d'une souris génétiquement modifiée par nos soin. Grâce à cela, nous avons utilisé ces cellules CD8a+ pour immuniser des rats afin de produire des anticorps monoclonaux ayant des propriétés spécifiques comme l'identification de protéines uniques présentes à la surface des DC et qui ensuite, modulent une réponse immunitaire in-vivo. Nous sommes actuellement en phase de caractérisation de plus de 750 hybridomes générés dans notre laboratoire. - Les cellules dendritiques (DCs) constituent le lien entre le système inné et adaptatif de la réponse immunitaire, car elles sont capables de présenter l'antigène, de donner la co- stimulation et de relâcher des cytokines et chimokines. Au cours de cette thèse, nous avons exploré différentes familles de DC lors d'infections parasitaires, telles que la Leishmaniose, parasite intracellulaire qui infecte les mammifères. La plupart des lésions cutanées résistantes sont caractérisées par une réponse pro-inflammatoire générée par l'IL-12. A l'inverse, pour la forme non résistante, la réponse est générée par l'IL-4 et l'IL-10, dans les modèles murins vulnérables. L'infection avec Lmajor a été caractérisée chez la souris C57BL/6 (Thl) et chez la souris Balb/c (Th2). Chez la souris C57BL/6 la lésion guérit, alors que chez la souris Balb/c, la lésion est au contraire non-cicatrisante. Nous avons comparé l'activation causée dans l'ensemble des DC par différentes espéces de Leishmania, et plus spécifiquement dans les DC CD8a+ présentes dans les ganglions lymphatiques et leur rôle dans la vulnérabilité à L. major. Ces cellules sont spécialisées dans la présentation croisée d'antigènes exogènes par le CMH-I et le haut taux de production d'IL-12 après activation. En utilisant des DC dérivées de moelle osseuse, nous avons constaté que L. guyanensis V+ (transportant un retrovirus) était le plus efficace pour l'activation des DC in-vitro comparé à L. major, L. mexicana et L. guyanensis (V-). Toutefois, in-vivo, les souris infectées avec L. major ont vu la taille de leur ganglions lymphatiques drainants augmentée, 3-6 semaines après l'infection dans les deux espèces de souris (les C57BL/6 résistantes et les Balb/c sensibles). En utilisant un parasite fluorescent transgénique, nous avons trouvé que les souris C57BL/6 sensibles à Lmexicana ont un nombre plus important de cellules Β infectées et un plus petit nombre de DC dérivées des monocytes inflammatoires, comparé au souris infectées avec L. major. Les conséquences de ces observations sont encore à l'étude. Des souris déficientes en CD8ct+DC et CD103+ sont plus sensibles à L. major que les souris WT: leurs lésions sont plus grandes et la charge parasitaire est plus importante. Nous avons généré une chimère de moelles osseuse CD11-DTR et Batf3-/- en mélangeant les moelles de ces deux souris, afin de déterminer le temps après infection où le manque de DC's CD8a+ contribue le plus à l'augmentation de la vulnérabilité chez la souris KO. Ces souris produisent plus d'IgG1 et IgE, font une réponse Th2 plus forte et Thl moins forte. Nous avons constaté que les souris déficientes en DC CD8a+ au début de la réponse immunitaire adaptive (trois semaines après injection) maintiennent un haut taux de lésions de grande taille, semblable à celui des souris chez qui les cellules ont été déplétées avant l'injection. Cela indique que les DC CD8a+ sont nécessaires pour l'efficacité de l'immunité dans la phase chronique de l'infection à L. major. Parallèlement à cela, nous avons aussi commencé une génération d'anticorps monoclonaux dirigés contre les DC CD8a+ activés en utilisant des souches établies dans notre laboratoire. En partant d'une librairie de 763 hybridomes, nous avons identifié plusieurs clones dignes d'intérêt avec une capacité fonctionnelle à moduler la prolifération et la sécrétion de cytokines des cellules T, ainsi que les molécules de co-stimulation présentes à la surface des DC activées elle-même. - Dendritic cells (DCs) are the bridge between the innate and the adaptive arms of the immune systems. They are professional antigen presentation cells and have important cytokine/chemokine release functions. In this dissertation we have focussed on the study of the different subsets of DCs in parasitic infection immunity. Leishmania are intra-cellular parasites of many different species that infect mammals. Most cutaneous lesions that are self- healing are characterized with a pro-inflammatory response with IL-12 while high levels of cytokines such as IL-4 and IL-10 characterized in susceptible mouse models. In mice L. major infection has been well characterized in C57BL/6 mice (Thl) that form healing lesions while Balb/c mice (Th2) form non-healing lesions. This thesis is focussed on comparing DC activation at large by different strains of Leishmania and more specifically, dLN resident CD8a+ DCs and their role in L. major susceptibility. This subset is specialized in cross- presentation of exogenous antigens in the MHC-I pathway and produce high levels of EL-12. Using bone marrow derived DCs we found that L. guyanensis V+ (carrying a retro-virus) was the most efficient at activating DCs in-vitro. In-vivo however L. major infected mice had the largest dLNs 3-6 weeks after infection in both genetically resistant C57BL/6 and susceptible Balb/c mice. Using transgenic fluorescent parasites, we found that C57BL/6 mice which are susceptible to L. mexicana had more number of infected Β cells and fewer number of infected inflammatory monocyte derived DCs in contrast to L. major infection. Using mice deficient in CD8a+ DCs, we found that these mice were more susceptible to L. major than their WT counterparts. They made larger lesions, had higher parasite burdens, higher levels of Th2 indicating immunolgloblins as measured by higher serie IgE levels and lower CD4+ IFNy+ cells. A mixed bone marrow chimera system of CDllc-DTR and Batf3~'~ was generated to determine the time point at which the lack of CD8a+ DCs most contributes to the increased susceptibility in KO mice. We found that mice depleted of CD8a+ DCs at the advent of the adaptive response (3 weeks after infection) maintained the significantly higher lesion size similar to mice whose cells were depleted from the onset of infection. This indicates that CD8a+ DCs are required for effective immunity in the chronic phase of L. major infection. We also began the generation of a valuable tool of monoclonal antibodies against activated CD8a+ DCs using our in-house DC line. From a library of 763 hybridomas we have identified several interesting clones with a functional ability to modulate Τ cell proliferation and cytokine secretion as well as down-modulating co-stimulatory molecules on activated DC cells themselves.

Classification and clinical behavior of blastic plasmacytoid dendritic cell neoplasms according to their maturation-associated immunophenotypic profile.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of leukemia/lymphoma, whose diagnosis can be difficult to achieve due to its clinical and biological heterogeneity, as well as its overlapping features with other hematologic malignancies. In this study we investigated whether the association between the maturational stage of tumor cells and the clinico-biological and prognostic features of the disease, based on the analysis of 46 BPDCN cases classified into three maturation-associated subgroups on immunophenotypic grounds. Our results show that blasts from cases with an immature plasmacytoid dendritic cell (pDC) phenotype exhibit an uncommon CD56- phenotype, coexisting with CD34+ non-pDC tumor cells, typically in the absence of extramedullary (e.g. skin) disease at presentation. Conversely, patients with a more mature blast cell phenotype more frequently displayed skin/extramedullary involvement and spread into secondary lymphoid tissues. Despite the dismal outcome, acute lymphoblastic leukemia-type therapy (with central nervous system prophylaxis) and/or allogeneic stem cell transplantation appeared to be the only effective therapies. Overall, our findings indicate that the maturational profile of pDC blasts in BPDCN is highly heterogeneous and translates into a wide clinical spectrum -from acute leukemia to mature lymphoma-like behavior-, which may also lead to variable diagnosis and treatment.

Insulated retroviral vectors towards safe and efficient genetic modification of stem cells

In otherwise successful gene therapy trials for the treatment of SCID patients and others, insertional mutagenesis has resulted in leukemia development. Besides the integration of vectors that including strong enhancers, more recently, SIN-vectors have been shown to partially retain oncogenic potential. The identification of genetic elements which would both prevent such activation effects and shield the transgene from silencing, is a main challenge. Previous attempts met with difficulties in producing the vectors and poor efficacy of the insulators (GIE). The improvement of integrating vectors safety has been investigated using new candidate synthetic GIEs. The latter have been introduced in retroviral and lentiviral vectors. Native LTRs, SIN-LTRs, and SIN-insulated constructs have been designed and compared, using two sets of internal promoter, i.e. strong and housekeeping. We could establish that a specific insulator translates at best into functional activity and boundary effect in both vector types. We could also determine that other genetic elements are key determinants in order to achieve accurate expression and viral titre, from these insulated vectors. A dramatic shift in the expression profile is observed in target cells, with a homogenous pattern including data on both cell-lines and primary HSCs from cord blood. The assessment of potential genotoxicity will be presented, based on the comparison of the integration patterns ingenuity in human target cells sampled over a three months period with both reference LTRs and SIN versus test insulated vectors, using high-throughput pyro-sequencing.