Role of Tyk-2 in Th9 and Th17 cells in allergic asthma.

In a murine model of allergic asthma, we found that Tyk-2((-/-)) asthmatic mice have induced peribronchial collagen deposition, mucosal type mast cells in the lung, IRF4 and hyperproliferative lung Th2 CD4(+) effector T cells over-expressing IL-3, IL-4, IL-5, IL-10 and IL-13. We also observed increased Th9 cells expressing IL-9 and IL-10 as well as T helper cells expressing IL-6, IL-10 and IL-21 with a defect in IL-17A and IL-17F production. This T helper phenotype was accompanied by increased SOCS3 in the lung of Tyk-2 deficient asthmatic mice. Finally, in vivo treatment with rIL-17A inhibited local CD4(+)CD25(+)Foxp3(+) T regulatory cells as well as Th2 cytokines without affecting IL-9 in the lung. These results suggest a role of Tyk-2 in different subsets of T helper cells mediated by SOCS3 regulation that is relevant for the treatment of asthma, cancer and autoimmune diseases.

Dual Blockade of PD-1 and CTLA-4 Combined with Tumor Vaccine Effectively Restores T-Cell Rejection Function in Tumors.

Tumor progression is facilitated by regulatory T cells (Treg) and restricted by effector T cells. In this study, we document parallel regulation of CD8(+) T cells and Foxp3(+) Tregs by programmed death-1 (PD-1, PDCD1). In addition, we identify an additional role of CTL antigen-4 (CTLA-4) inhibitory receptor in further promoting dysfunction of CD8(+) T effector cells in tumor models (CT26 colon carcinoma and ID8-VEGF ovarian carcinoma). Two thirds of CD8(+) tumor-infiltrating lymphocytes (TIL) expressed PD-1, whereas one third to half of CD8(+) TIL coexpressed PD-1 and CTLA-4. Double-positive (PD-1(+)CTLA-4(+)) CD8(+) TIL had characteristics of more severe dysfunction than single-positive (PD-1(+) or CTLA-4(+)) TIL, including an inability to proliferate and secrete effector cytokines. Blockade of both PD-1 and CTLA-4 resulted in reversal of CD8(+) TIL dysfunction and led to tumor rejection in two thirds of mice. Double blockade was associated with increased proliferation of antigen-specific effector CD8(+) and CD4(+) T cells, antigen-specific cytokine release, inhibition of suppressive functions of Tregs, and upregulation of key signaling molecules critical for T-cell function. When used in combination with GVAX vaccination (consisting of granulocyte macrophage colony-stimulating factor-expressing irradiated tumor cells), inhibitory pathway blockade induced rejection of CT26 tumors in 100% of mice and ID8-VEGF tumors in 75% of mice. Our study indicates that PD-1 signaling in tumors is required for both suppressing effector T cells and maintaining tumor Tregs, and that PD-1/PD-L1 pathway (CD274) blockade augments tumor inhibition by increasing effector T-cell activity, thereby attenuating Treg suppression. Cancer Res; 73(12); 3591-603. ©2013 AACR.

The Role of Interleukin-2 in Memory CD8 Cell Differentiation

The current literature on the role of interleukin (IL)-2 in memory CD8(+) T-cell differentiation indicates a significant contribution of IL-2 during primary and also secondary expansion of CD8(+) T cells. IL-2 seems to be responsible for optimal expansion and generation of effector functions following primary antigenic challenge. As the magnitude of T-cell expansion determines the numbers of memory CD8(+) T cells surviving after pathogen elimination, these events influence memory cell generation. Moreover, during the contraction phase of an immune response where most antigen-specific CD8(+) T cells disappear by apoptosis, IL-2 signals are able to rescue CD8(+) T cells from cell death and provide a durable increase in memory CD8(+) T-cell counts. At the memory stage, CD8(+) T-cell frequencies can be boosted by administration of exogenous IL-2. Significantly, only CD8(+) T cells that have received IL-2 signals during initial priming are able to mediate efficient secondary expansion following renewed antigenic challenge. Thus, IL-2 signals during different phases of an immune response are key in optimizing CD8(+) T-cell functions, thereby affecting both primary and secondary responses of these T cells.

CCL17 Promotes Intestinal Inflammation in Mice and Counteracts Regulatory T Cell-Mediated Protection From Colitis.

BACKGROUND & AIMS: Priming of T cells by dendritic cells (DCs) in the intestinal mucosa and associated lymphoid tissues helps maintain mucosal tolerance but also contributes to the development of chronic intestinal inflammation. Chemokines regulate the intestinal immune response and can contribute to pathogenesis of inflammatory bowel diseases. We investigated the role of the chemokine CCL17, which is expressed by conventional DCs in the intestine and is up-regulated during colitis. METHODS: Colitis was induced by administration of dextran sodium sulfate (DSS) to mice or transfer of T cells to lymphopenic mice. Colitis activity was monitored by body weight assessment, histologic scoring, and cytokine profile analysis. The direct effects of CCL17 on DCs and the indirect effects on differentiation of T helper (Th) cells were determined in vitro and ex vivo. RESULTS: Mice that lacked CCL17 (Ccl17(E/E) mice) were protected from induction of severe colitis by DSS or T-cell transfer. Colonic mucosa and mesenteric lymph nodes from Ccl17-deficient mice produced lower levels of proinflammatory cytokines. The population of Foxp3(+) regulatory T cells (Tregs) was expanded in Ccl17(E/E) mice and required for long-term protection from colitis. CCR4 expression by transferred T cells was not required for induction of colitis, but CCR4 expression by the recipients was required. CCL17 promoted Toll-like receptor-induced secretion of interleukin-12 and interleukin-23 by DCs in an autocrine manner, promoted differentiation of Th1 and Th17 cells, and reduced induction of Foxp3(+) Treg cells. CONCLUSIONS: The chemokine CCL17 is required for induction of intestinal inflammation in mice. CCL17 has an autocrine effect on DCs that promotes production of inflammatory cytokines and activation of Th1 and Th17 cells and reduces expansion of Treg cells.

Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4(+) T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4(+) T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+) T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+) T cells and forkhead box P3 (FoxP3)(+) regulatory T (Treg) cells. IFN-γ produced mainly by CD4(+) T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.

Fatty acid amide hydrolase deficiency enhances intraplaque neutrophil recruitment in atherosclerotic mice.

OBJECTIVE: Endocannabinoid levels are elevated in human and mouse atherosclerosis, but their causal role is not well understood. Therefore, we studied the involvement of fatty acid amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in atherosclerotic plaque vulnerability. METHODS AND RESULTS: We assessed atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)FAAH(-/-) mice. Before and after 5, 10, and 15 weeks on high-cholesterol diet, we analyzed weight, serum cholesterol, and endocannabinoid levels, and atherosclerotic lesions in thoracoabdominal aortas and aortic sinuses. Serum levels of FAAH substrates anandamide, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were 1.4- to 2-fold higher in case of FAAH deficiency. ApoE(-/-)FAAH(-/-) mice had smaller plaques with significantly lower content of smooth muscle cells, increased matrix metalloproteinase-9 expression, and neutrophil content. Circulating and bone marrow neutrophil counts were comparable between both genotypes, whereas CXC ligand1 levels were locally elevated in aortas of FAAH-deficient mice. We observed enhanced recruitment of neutrophils, but not monocytes, to large arteries of ApoE(-/-) mice treated with FAAH inhibitor URB597. Spleens of ApoE(-/-)FAAH(-/-) mice had reduced CD4+FoxP3+regulatory T-cell content, and in vitro stimulation of splenocytes revealed significantly elevated interferon-γ and tumor necrosis factor-α production in case of FAAH deficiency. CONCLUSIONS: Increased anandamide and related FAAH substrate levels are associated with the development of smaller atherosclerotic plaques with high neutrophil content, accompanied by an increased proinflammatory immune response.

A novel mucosal orthotopic murine model of human papillomavirus-associated genital cancers.

Cervical cancer results from infection with high-risk type human papillomaviruses (HPV). Therapeutic vaccines aiming at controlling existing genital HPV infections and associated lesions are usually tested in mice with HPV-expressing tumor cells subcutaneously implanted into their flank. However, effective vaccine-induced regression of these ectopic tumors strongly contrasts with the poor clinical results of these vaccines produced in patients with HPV-associated genital neoplasia. To assess HPV therapeutic vaccines in a more relevant setting, we have, here, established an orthotopic mouse model where tumors in the genital mucosa (GM) develop after an intravaginal instillation of HPV16 E6/E7-expressing tumor cells transduced with a luciferase-encoding lentiviral vector for in vivo imaging of tumor growth. Tumor take was 80-90% after nonoxynol-9 induced damage of the epithelium. Tumors remained localized in the genital tract, and histological analysis showed that most tumors grew within the squamous epithelium of the vaginal wall. Those tumors induced (i) E7-specific CD8 T cells restricted to the GM and draining lymph nodes, in agreement with their mucosal location and (ii) high Foxp3+ CD4+ infiltrates, similarly to those found in natural non-regressing HPV lesions. This novel genital HPV-tumor model by requiring GM homing of vaccine-induced immune responses able to overcome local immuno-suppression may be more representative of the situation occurring in patients upon therapeutic vaccination.

A role for altered TLR gene expression in association with increased expression of CD200R in the induction of mucosal tissue CD4(+) Treg in aged mice following gavage with a liver extract along with intramuscular monophosphoryl lipid A (MPLA) injection.

Previous studies showed a fetal sheep liver extract (FSLE), in association with LPS, injected into aged (>20 months) mice reversed the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFN-gamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. Aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+)Treg and so-called Tr3 (CD4(+)TGFbeta(+)). Their number/function is restored to levels seen in control (8-week-old) mice by FSLE. We have reported at length on the ability of a novel pair of immunoregulatory molecules, members of the TREM family, namely CD200:CD200R, to control development of dendritic cells (DCs) which themselves regulate production of Foxp3(+) Treg. The latter express a distinct subset of TLRs which control their function. We report that a feature of the altered Treg expression following combined treatment with FSLE and monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS) is the altered gene expression both of distinct subsets of TLRs and of CD200Rs. We speculate that this may represent one of the mechanisms by which FSLE and MPLA alter immunity in aged mice.

Autoimmunity-Associated LYP-W620 Does Not Impair Thymic Negative Selection of Autoreactive T Cells.

A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a "gain-of-function" of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4(+)Foxp3(+) Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4(+) T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.

CTLA-4 controls the thymic development of both conventional and regulatory T cells through modulation of the TCR repertoire.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is of pivotal importance for self-tolerance, with deficiency or unfavorable polymorphisms leading to autoimmune disease. Tolerance to self-antigens is achieved through thymic deletion of highly autoreactive conventional T (Tconv) cells and generation of FoxP3(+) regulatory T (Treg) cells. The main costimulatory molecule, CD28, augments the negative selection of Tconv cells and promotes the generation of FoxP3(+) Treg cells. The role of its antagonistic homolog CTLA-4, however, remains a topic of debate. To address this topic, we investigated the thymic development of T cells in the presence and absence of CTLA-4 in a T-cell receptor (TCR) transgenic mouse model specific for the myelin basic protein peptide Ac1-9. We reveal that CTLA-4 is expressed in the corticomedullary region of the thymus. Its absence alters the response of CD4(+)CD8(-) thymocytes to self-antigen recognition, which affects the quantity of the Treg cells generated and broadens the repertoire of peripheral Tconv cells. T-cell repertoire alteration after deletion of CTLA-4 results from changes in TCR Vα and Jα segment selection as well as CDR3α composition in Tconv and Treg cells. CTLA-4, therefore, regulates the early development of self-reactive T cells in the thymus and plays a key role in central tolerance.

A microRNA profile of human CD8.

BackgroundRecently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8+CD25+ Treg cells and the impact of microRNAs on molecules associated with immune regulation.MethodsWe purified human natural CD8+ Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA `signature¿ for CD8+CD25+FOXP3+CTLA-4+ natural Treg cells. We used the `TargetScan¿ and `miRBase¿ bioinformatics programs to identify potential target sites for these microRNAs in the 3¿-UTR of important Treg cell-associated genes.ResultsThe human CD8+CD25+ natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo.ConclusionsWe are examining the biological relevance of this `signature¿ by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

Influence of CD4+/CD25+ regulatory T cells on atherogenesis in patients with end-stage kidney disease.

Atherosclerosis, which is influenced by both traditional and nontraditional cardiovascular risk factors and has been characterized as an inflammatory process, is considered to be the main cause of the elevated cardiovascular risk associated with chronic kidney disease. The inflammatory component of atherosclerosis can be separated into an innate immune response involving monocytes and macrophages that respond to the excessive uptake of lipoproteins and an adaptive immune response that involves antigen-specific T cells. Concurrent with the influx of immune cells to the site of atherosclerotic lesion, the role of the adaptive immune response gradually increases. One of those cells are represented by the CD4+/CD25+ Tregs, which play indispensable roles in the maintenance of natural self-tolerance and negative control of pathological, as well as physiological, immune responses. Altered self-antigens such as oxidized LDLs may induce the development of CD4+/CD25+ Tregs with atheroprotective properties. However, atherosclerosis may be promoted by an imbalance between regulatory and pathogenic immunity that may be represented by the low expression of the forkhead box transcription factor (Foxp3) in CD4+/CD25+ Tregs. Such a defect may break immunologic tolerance and alter both specific and bystander immune suppression, leading to exacerbation of plaque development. Patients with end-stage kidney disease (ESKD) display a cellular immune dysfunction and accelerated atherosclerosis. Uremic solutes that accumulate during ESKD may be involved in these processes. In patients with ESKD and especially in those that are chronically hemodialyzed, oxidative stress induced by oxidized LDLs may increase CD4+/CD25+ Treg sensitivity to Fas-mediated apoptosis as a consequence of specific dysregulation of IL-2 expression. This review will focus on the current state of knowledge regarding the influence of CD4+/CD25+ Tregs on atherogenesis in patients with ESKD, and the potential effect of statins on the circulating number and the functional properties of these cells.

Tissue-based immune monitoring I: tumor core needle biopsies allow in-depth interrogation of the tumor microenvironment.

We sought to assess the feasibility and reproducibility of performing tissue-based immune characterization of the tumor microenvironment using CT-compatible needle biopsy material. Three independent biopsies were obtained intraoperatively from one metastatic epithelial ovarian cancer lesion of 7 consecutive patients undergoing surgical cytoreduction using a 16-gauge core biopsy needle. Core specimens were snap-frozen and subjected to immunohistochemistry (IHC) against human CD3, CD4, CD8, and FoxP3. A portion of the cores was used to isolate RNA for 1) real-time quantitative (q)PCR for CD3, CD4, CD8, FoxP3, IL-10 and TGF-beta, 2) multiplexed PCR-based T cell receptor (TCR) CDR3 Vβ region spectratyping, and 3) gene expression profiling. Pearson's correlations were examined for immunohistochemistry and PCR gene expression, as well as for gene expression array data obtained from different tumor biopsies. Needle biopsy yielded sufficient tissue for all assays in all patients. IHC was highly reproducible and informative. Significant correlations were seen between the frequency of CD3+, CD8+ and FoxP3+ T cells by IHC with CD3ε, CD8A, and FoxP3 gene expression, respectively, by qPCR (r=0.61, 0.86, and 0.89; all p< 0.05). CDR3 spectratyping was feasible and highly reproducible in each tumor, and indicated a restricted repertoire for specific TCR Vβ chains in tumor-infiltrating T cells. Microarray gene expression revealed strong correlation between different biopsies collected from the same tumor. Our results demonstrate a feasible and reproducible method of immune monitoring using CT-compatible needle biopsies from tumor tissue, thereby paving the way for sophisticated translational studies during tumor biological therapy.

Differential effects of immunosuppressive drugs on effector and regulatory T cells.

Purpose: Current experimental data suggest that CD4+CD25+Foxp3+regulatory T cells (Tregs) based immunotherapy would be of greatinterest to promote donor-specific immune tolerance in transplantation(Tx). Whether and how adoptive transfer of Tregs could be bestcombined with current immunosuppressive regimens in clinicalsettings remains to be defined. Using an experimental Tx model,we had previously shown that the transfer of antigen-specific Tregspromoted long-term skin allograft acceptance in lymphopenic mice,in the absence of any immunosuppressive drug. However, allograftsurvival was only slightly prolonged when Tregs were transferredalone into non-lymphopenic mice, suggesting that in more stringentconditions such as in clinical settings adjuvant therapies may beneeded to effectively control alloreactive T cells (Teff).Methods and Materials: Here we have investigated the effects ofvarious immunosuppressive drugs on the survival, proliferation andeffector function of Teff and Tregs in response to alloantigens in in vitroassays and in our in vivo Tx model.Results: Teff proliferation was inhibited in a dose-dependant mannerby rapamycin and cyclosporine A, while anti-CD154 only marginallyaffected Teff proliferation and survival in vitro. Rapamycin promotedapoptosis of Teff as compared to Tregs that were more resistant underthe same culture conditions. In vivo, the transfer of donor-specificTregs could be advantageously combined with rapamycin andanti-CD154 to significantly prolong MHC-mismatched skin allograftsurvival in non-lymphopenic recipients.Conclusion: Taken together, our data indicate thatimmunosuppressive drugs differentially target T-cell subsets and couldpromote Tregs expansion and/or function while controlling the Teff pool.

Tolerogenic dendritic cells and their role in induction of immune tolerance

Les cellules dendritiques sont des cellules du système immunitaire qui permettent d'instruire les lymphocytes T, autres cellules de ce système, pour mettre en place une réponse immunitaire adaptée afin de combattre et vaincre une infection. Ces cellules dendritiques vont reconnaître des motifs spécifiquement exprimés par des pathogènes par l'intermédiaire de récepteurs exprimés à leur surface. En détectant ces molécules, elles vont s'activer et subir diverses modifications pour pouvoir activer les lymphocytes T. Elles vont alors interagir avec les lymphocytes Τ et transférer les informations nécessaires pour que ces cellules s'activent à leur tour et produisent différentes protéines de façon à éliminer le pathogène. En fonction du type de pathogène, les informations transférées entre les cellules dendritiques et les lymphocytes seront différentes de manière à produire la réponse immunitaire la mieux adaptée pour supprimer l'élément infectieux. Dans le corps, les cellules dendritiques circulent continuellement afin de détecter les éléments étrangers. Quand elles reconnaissent une protéine étrangère, elles la phagocytent, c'est-à-dire qu'elles la mangent afin de pouvoir la présenter aux lymphocytes T. Mais quand elles phagocytent un élément étranger, elles peuvent également prendre des éléments du soi, comme par exemple quand elles phagocytent une cellule infectée par un virus. Les cellules dendritiques doivent alors être capables de différentier les molécules du soi et du non-soi de façon à ne pas induire une réponse en présentant un antigène du soi aux lymphocytes T. D'autant plus que lors de leur développement, les lymphocytes Τ qui sont capables de reconnaître le soi sont éliminés mais ce système n'est pas parfait et donc certains lymphocytes Τ auto-reactifs peuvent se trouver dans le corps. Il existe ainsi d'autres mécanismes en périphérie du site de développement pour inhiber ces lymphocytes Τ auto-reactifs. Ce sont les mécanismes de tolérance. Quand les lymphocytes Τ induisent une réponse aux antigènes du soi, cela résulte à des maladies auto-immunes. Dans mon projet de recherche, nous avons travaillé avec des lignées de cellules dendritiques, c'est-à-dire des cellules dendritiques semblables à celles que l'on peut trouver in vivo mais qui sont immortalisées, elles peuvent donc être cultiver et manipuler in vitro. Nous avons génétiquement modifiées ces lignées cellulaires pour qu'elles expriment des molécules immunosuppressives afin d'étudier comment induire une tolérance immunitaire, c'est-à-dire si l'expression de ces molécules permet d'éviter de générer une réponse immunitaire. Pour cela, nous avons utilisé des modèles murins de tumeurs et de maladies auto-immunes. Nous avons démontré que ces lignées de cellules dendritiques peuvent être un grand outil de recherche pour étudier les bénéfices de différentes molécules immuno-modulatrices afin d'induire une tolérance immunitaire à différents antigènes. - Les cellules dendritiques sont responsables de l'induction des réponses immunitaires adaptatives. Suite à une infection microbienne, les cellules dendritiques s'activent, elles induisent l'expression de molécules de costimulation à leur surface, sécrètent des cytokines et induisent la différentiation des cellules Τ effectrices et mémoires. De plus, les cellules dendritiques ont un rôle important dans l'induction et la maintenance de la tolérance immunitaire au niveau du thymus et en périphérie, en induisant l'anergie, la délétion ou la conversion des cellules Τ naïves en cellules régulatrices. Dans notre groupe, une nouvelle lignée de cellules dendritiques appelée MuTu a été crée par la culture de cellules dendritiques tumorales isolées à partir d'une rate d'une souris transgénique, dans laquelle l'expression de l'oncogène SV40 et du GFP sont sous le contrôle du promoteur CD1 le, et sont ainsi spécifiquement exprimés dans les cellules dendritiques. Ces nouvelles lignées appartiennent au sous-type des cellules dendritiques conventionnelles exprimant CD8a. Elles ont conservé leur capacité d'augmenter l'expression des marqueurs de costimulation à leur surface ainsi que le production de cytokines en réponse à des ligands des récepteurs Toll, ainsi que leur capacité à présenter des antigènes associés aux molécules du complexe majeur d'histocompatibilité (CMH) de classe I ou II pour activer la prolifération et la différentiation des lymphocytes T. En utilisant un système de transduction de lentivirus de seconde génération, ces nouvelles lignées de cellules dendritiques ont été génétiquement modifiées pour sur-exprimer des molécules immunosuppressives (IL-10, TGFP latent, TGFp actif, Activin A, Arginase 1, IDO, B7DC et CTLA4). Ces lignées permettent d'étudier de manière reproductible le rôle de ces molécules potentiellement tolérogènes sur les réponses immunitaires in vitro et in vivo. Ces lignées potentiellement tolérogènes ont été testées, tout d'abord, in vitro, pour leur capacité à inhiber l'activation des cellules dendritiques, à bloquer la prolifération des cellules Τ ou à modifier leur polarisation. Nos résultats démontrent qu'en réponse à une stimulation, la sur-expression des molécules costimulatrices et la sécrétion de molécules pro- inflammatoires est réduite quand les cellules dendritiques sur-expriment l'IL-10. La sur¬expression de TGFp sous sa forme active induit le développement de cellules régulatrices CD4+ CD25+ Foxp3+ et bloque la réponse CD8 cytotoxique tandis que la sur-expression de CTLA4 à la surface des cellules dendritiques inhibe une réponse Thl et induit des lymphocytes Τ anergiques. Ces lignées ont également été utilisées pour étudier l'induction de tolérance in vivo. Tout d'abord, nous avons étudié l'induction de tolérance dans un modèle de développement de tumeurs. En effet, quand les lignées tumorales sont transférées dans les lignées de souris C57BL/6, elles sont reconnues comme du non-soi du à l'expression de l'oncogène SV40 et du GFP et sont éliminées. Ce mécanisme d'élimination a été étudié en utilisant une lignée de cellules dendritiques modifiée pour exprimer la luciférase et qui a permis de suivre le développement des tumeurs par de l'imagerie in vivo dans des animaux vivants. Ces lignées de cellules dendritiques MuTu sont éliminées dans la souris C57BL/6 par les lymphocytes CD8 et l'action cytotoxique de la perforine. Après plusieurs injections, les cellules dendritiques sur-exprimant CTLA4 ou l'actif TGFp peuvent casser cette réponse immunitaire inhérente aux antigènes de la lignée et induire le développement de la tumeur dans la souris C57BL/6. Le développement tumoral a pu être suivi en mesurant la bioluminescence émise par des cellules dendritiques modifiées pour exprimer à la fois l'actif TGFp et la luciférase. Ces tumeurs ont pu se développer grâce à la mise en place d'un microenvironnement suppressif pour échapper à l'immunité en recrutant des cellules myéloïde suppressives, des lymphocytes CD4 régulateurs et en induisant l'expression d'une molécule inhibitrice PD-1 à la surface des lymphocytes CD8 infiltrant la tumeur. Dans un deuxième temps, ces lignées tolérogènes ont également été testées dans un modèle murin de maladies auto-immunes, appelé l'encéphalomyélite auto-immune expérimental (EAE), qui est un modèle pour la sclérose en plaques. L'EAE a été induite dans la souris par le transfert de cellules de ganglions prélevées d'une souris donneuse préalablement immunisée avec une protéine du système nerveux central, la glycoprotéine myéline oligodendrocyte (MOG) émulsifiée dans de l'adjuvant complet de Freund. La vaccination des souris donneuses et receveuses avec les cellules sur-exprimant l'actif TGFP préalablement chargées avec la protéine MOG bloque l'induction de l'EAE. Nous sommes actuellement en train de définir les mécanismes qui permettent de protéger la souris du développement de la maladie auto-immune. Dans cette étude, nous avons ainsi démontré la possibilité d'induire la tolérance in vivo et in vitro à différents antigènes en utilisant nos nouvelles lignées de cellules dendritiques et en les modifiant pour exprimer des molécules immunosuppressives. En conséquence, ces nouvelles lignées de cellules dendritiques représentent un outil pour explorer les bénéfices de différentes molécules ayant des propriétés immuno-modulatrices pour manipuler le système immunitaire vers un phénotype tolérogène. - Dendritic cells (DC) are widely recognized as potent inducers of the adaptive immune responses. Importantly, after microbial infections, DC become activated, induce co- stimulation, secrete cytokines and induce effector and memory Τ cells. DC furthermore play an important role in inducing and maintaining central and peripheral tolerance by inducing anergy, deletion or commitment of antigen-specific naïve Τ cells into regulatory Τ cells. In our group, stable MuTu DC lines were generated by culture of splenic DC tumors from transgenic mice expressing the SV40 large Τ oncogene and the GFP under DC-specific CDllc promoter. These transformed DC belong to the CD8a+ conventional DC subtype and have fully conserved their capacity to upregulate co-stimulatory markers and produce cytokines after activation with Toll Like Receptors-ligands, and to present Major Histocompatibility class-I or MHCII-restricted antigens to activate Τ cell expansion and differentiation. Using a second- generation lentiviral transduction system, these newly developed MuTu DC lines were genetically modified to overexpress immunosuppressive molecules (IL-10, latent TGFp, active TGFp, Activin A, Arginase 1, IDO, B7DC and CTLA4). This allows to reproducibly investigate the role of these potentially tolerogenic molecules on in vitro and in vivo immune responses. These potentially tolerogenic DC were tested in vitro for their ability to inhibit DC activation, to prevent Τ cell proliferation and to modify Τ cell polarization. Our results show that the upregulation of costimulatory molecules and the secretion of pro-inflammatory cytokines were reduced upon stimulation of DC overexpressing IL-10. The overexpression of active TGFP induced the development of CD4+ CD25+ Foxp3+ regulatory Τ cells and inhibited the cytotoxic CD8 Τ cell response as shown by using the OT-II Τ cell system whereas the surface expression of CTLA-4 on DC prevented the Thl response and prompted an anergic antigen-specific Τ cell response. These MuTu DC lines were also used in vivo in order to study the induction of tolerance. First we addressed the induction of tolerance in a model of tumorogenesis. The adoptively transferred tumor cell lines were cleared in C57BL/6 mice due to the foreign expression of SV40 LargeT and GFP. The mechanism of clearance of MuTu DC line into C57BL/6 mice was investigated by using luciferase-expressing DC line. These DC line allowed to follow, by in vivo imaging, the tumor development in living animals and determined that MuTu DC lines were eliminated in a perforin-mediated CD8 Τ cell dependent and CD4 Τ cell independent response. After multiple injections, DC overexpressing CTLA4 or active TGFp could break the immune response to these inherent antigens and induced DC tumorogenesis in wild type mice. The tumor outgrowth in C57BL/6 mice was nicely observed by double-transduced DC lines to express both luciferase and active TGFp. actTGFp-DC tumor was shown to recruit myeloid-derived suppressor cells, induce CD4+ CD25+ Foxp3+ regulatory Τ cells and induce the expression of the inhibitory receptor PD-1 on tumor- infiltrating CD8+ Τ cells in order to escape tumor immunity. Tolerogenic DC lines were also tested for the induction of tolerance in a murine model of autoimmune disease, the experimental autoimmune encephalitis (EAE) model for human multiple sclerosis. EAE was induced in C57BL/6 mice by the adoptive transfer of lymph node cells isolated from donor mice previously immunized by a protein specific to the central nervous system, the myelin oligodendrocyte glycoprotein (MOG) emulsified in the complete freund adjuvant. The vaccination of donor and recipient mice with MOG-pulsed actTGFP-DC line prevented EAE induction. We are still investigating how the active TGFP protect mice from EAE development. We generated tolerogenic DC lines inducing tolerance in vitro and in vivo. Thereby these MuTu DC lines represent a great tool to explore the benefits of various immuno-modulatory molecules to manipulate the immune system toward a tolerogenic phenotype.