Germ cell-specific targeting of DICER or DGCR8 reveals a novel role for endo-siRNAs in the progression of mammalian spermatogenesis and male fertility.

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process.

Nuclear receptor PPARS function in stem cell differentiation and bone physiology

In adult, bone remodeling is a permanent process, reaching an annual turnover of about 10% of the skeleton. Bone remodeling requires the sequential and coordinated actions of the hematopoietic origin osteoclasts, to remove bone and the mesenchymal origin osteoblasts to replace it. An increased level of bone resorption is the primary cause of age-related bone loss often resulting in osteopenia, and is the major cause of osteoporosis.¦Peroxisome proliferator-activated receptors (PPARs), which are expressed in three isotypes, PPARa, PPARp and PPARy, are ligand-activated transcription factors that control many cellular and metabolic processes, more particularly linked to lipid metabolism. In bone, previous works has shown that PPARy inhibits osteogenesis by favoring adipogenesis from common mesenchymal progenitors. In addition, the pro-osteoclastogenesis activity of PPARy results in an increased bone resorption. Accordingly, treatment with PPARy agonist such as the anti-diabetic drug TZD causes bone loss and accumulation of marrow adiposity in mice as well as in postmenopausal women. The aim of the present thesis work was to elucidate the PPARs functions in bone physiology.¦The initial characterization of the PPARP" bone phenotype mainly revealed a decreased BMD. In vitro studies exploring the potency of mesenchymal stem cells to differentiate in osteoblast showed no differences depending on the genotype. However, we could demonstrate an effect of PPARp in partially inhibiting osteoclastogenesis. These results are further sustained by a study made in collaboration with the group of Dr Kronke, which showed an impressive protection against ovariectomy-generated bone loss when the females are treated with a PPARp agonist.¦Observations in PPARy null mice are more complex. The lab has recently been able to generate mice carrying a total deletion of PPARy. Intriguingly, the exploration of the bone phenotype of these mice revealed paradoxical findings. Whereas short bones such as vertebrae exhibit an elevated BMD as expected, long bones (tibia and femur) are clearly osteoporotic. According to their activity when set in culture, osteoblast differentiation normally occurs. Indeed the phenotype can be mainly attributed to a high density of osteoclasts in the cortical bone of PPARy null mice, associated to large bone resorption areas.¦Our explorations suggest a mechanism that involves regulatory processes linking osteoclastogenesis to adipogenesis, the latter being totally absent in PPARy null mice. Indeed, the lack of adipose tissue creates a favorable niche for osteoclastogenesis since conditioned medium made from differentiated adipocyte 3T3L1 inhibited osteoclastogenesis from both PPARy-/- and WT cells. Thus, adipokines deficiency in PPARy-/- mice contributes to de- repress osteoclastogenesis. Using specific blocking antibody, we further identified adiponectin as the major player among dozens of adipokines. Using flow cytometry assay, we explored the levels at which the osteoclastic commitment was perturbed in the bone marrow of PPARy-/- mice. Intriguingly, we observe a general decrease for hematopoietic stem cell and lineage progenitors but increased proportion of osteoclast progenitor in PPARy-/- bone marrow. The general decrease of HSC in the bone marrow is however largely compensated by an important extra-medullary hematopoeisis, taking place in the liver and in the spleen.¦These specific characteristics emphasize the key role of PPARy on a cross road of osteogenesis, adipogenesis and hematopoiesis/osteoclastogenesis. They underline the complexity of the bone marrow niche, and demonstrate the inter-dependance of different cell types in defining bone homeostasis, that may be overseen when experimental design single out pure cell populations.¦Chez l'adulte, même après la fin de la croissance, le renouvellement des os se poursuit et porte sur environ 10% de l'ensemble du squelette adulte, par année. Ce renouvellement implique à la fois des mécanismes séquentiels et coordonnés des ostéoclastes d'origine hématopoïetique, qui dégradent l'os, et des ostéoblastes d'origine mésenchymale, qui permettent la régénération de l'os. La perte en densité osseuse due à l'âge entraîne un fort niveau de résorption, conduisant souvent à une ostéopénie, elle-même cause de l'ostéoporose.¦Les trois isotypes PPAR (Peroxisome proliferator-activated receptor, PPARa, PPARp, et PPARy) sont des récepteurs nucléaires qui contrôlent de nombreux mécanismes cellulaires et métaboliques, plus particulièrement liés au métabolisme lipidique. Au niveau osseux, des travaux précédents ont montré que PPARy inhibe l'ostéoblastogenèse en favorisant la formation d'adipocytes à partir de la cellule progénitrice commune. De plus, l'activité pro- ostéoclastogénique de PPARy induit une résorption osseuse accrue. Condormément à ces observations, les patients diabétiques traités par les thiazolidinediones qui agissent sur PPARy, ont un risque accrue d'ostéoporose liée à une perte osseuse accrue et un accroissement de l'adiposité au niveau de la moelle osseuse. Dans ce contexte, l'objectif de mon travail de thèse a été d'élucider le rôle des PPAR dans la physiologie osseuse, en s'appuyant sur le phénotype des souris porteuses de mutation pour PPAR.¦La caractérisation initiale des os des souris porteuses d'une délétion de ΡΡΑΕφ a principalement révélé une diminution de la densité minérale osseuse (DMO). Alors que l'ostéogenèse n'est pas significativement altérée chez ces souris, l'ostéoclastogenèse est elle augmentée, suggérant un rôle modérateur de ce processus par ΡΡΑΕΙβ. Ces résultats sont par ailleurs soutenus par une étude menée par le groupe du Dr Krônke en collaboration avec notre groupe, et qui monte une protection très importante des souris traitées par un activateur de PPARP contre l'ostéoporose provoquée par l'ovariectomie.¦Les observations concernant PPARy donnent des résultats plus complexes. Le laboratoire a en effet été capable récemment de générer des souris portant une délétion totale de PPARy. Alors que les os courts chez ces souris présentent une augmentation de la DMO, comme attendu, les os longs sont clairement ostéoporotiques. Ce phénotype corrèle avec une densité élevée d'ostéoclastes dans l'os cortical de ces os longs. Deux processus semblent contribuer à ce phénotype. En premier lieu, nous démontrons qu'un milieu conditionné provenant de cultures de cellules 3T3-L1 différenciées en adipocytes contiennent une forte activité inhibitrice d'osteoclastogenesis. L'utilisation d'anticorps neutralisant permet d'identifier l'adiponectine comme l'un des facteurs principaux de cette inhibition. Les souris PPARy étant totalement dépourvues d'adipocytes et donc de tissu adipeux, la sécrétion locale d'adiponectine dans la moelle osseuse est donc également absente, entraînant une désinhibition de l'ostéoclastogenèse. En second lieu, des analyses par FACS révèle une proportion accrue des cellules progénitrices d'ostéoclastes dans la moelle osseuse. Cela s'accompagne par une diminution globale des cellules souches hématopoïétiques, qui est cependant largement compensée par une importante hématopoëise extra-médullaire, dans le foie comme dans la rate.¦L'ensemble de notre travail montre toute l'importance de PPARy au carrefour de l'ostéogenèse, adipogenèse, et hématopoëise/osteoclastogenèse. Il souligne la complexité de la niche que représente la moelle osseuse et démontre l'inter-dépendance des différents types cellulaires définissant l'homéostasie osseuse, complexité qui peut facilement être masqué lorsque le travail expérimental se concentre sur le comportement d'un type cellulaire donné.

Hypoinsulinaemia, glucose intolerance and diminished beta-cell size in S6K1-deficient mice.

Insulin controls glucose homeostasis by regulating glucose use in peripheral tissues, and its own production and secretion in pancreatic beta cells. These responses are largely mediated downstream of the insulin receptor substrates, IRS-1 and IRS-2 (refs 4-8), through distinct signalling pathways. Although a number of effectors of these pathways have been identified, their roles in mediating glucose homeostasis are poorly defined. Here we show that mice deficient for S6 kinase 1, an effector of the phosphatidylinositide-3-OH kinase signalling pathway, are hypoinsulinaemic and glucose intolerant. Whereas insulin resistance is not observed in isolated muscle, such mice exhibit a sharp reduction in glucose-induced insulin secretion and in pancreatic insulin content. This is not due to a lesion in glucose sensing or insulin production, but to a reduction in pancreatic endocrine mass, which is accounted for by a selective decrease in beta-cell size. The observed phenotype closely parallels those of preclinical type 2 diabetes mellitus, in which malnutrition-induced hypoinsulinaemia predisposes individuals to glucose intolerance.

Expression of genes encoding cytotoxic cell-associated serine proteases in thymocytes.

A family of homologous serine esterases designated granzyme A-H and the pore-forming protein perforin are present in cytoplasmic granules of mature peripheral cytolytic T lymphocytes and natural killer cells. In vivo, the majority of cytotoxic T cells containing these granule-associated proteins are of the CD4-CD8+ phenotype. It is generally assumed that these cells are derived from immature CD4-CD8- thymocytes. However, the precise intrathymic differentiation steps leading to functionally mature cytotoxic T cells are unclear. Thus we decided to analyze the expression of genes in the thymus which are preferentially expressed in mature cytotoxic cells, i.e. granzyme A, granzyme B, and perforin. In situ hybridization on tissue sections revealed the expression of genes coding for granzyme A and granzyme B in the thymus. No evidence was found, however, for thymocytes expressing the perforin gene. Granzyme A and granzyme B mRNA positive cells in the thymus are almost exclusively CD4-CD8- thymocytes, particularly of the CD3- IL2R- phenotype.

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo.

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.

Phenotypic heterogeneity of human cardiac stem cell clones and cardiosphere-forming cells

Although cardiac stem cells have been isolated based on stem cell surface markers, no single marker is stem cell-specific. Clonogenicity is a defining functional property of stemness. We therefore analyzed cardiac cell clones derived from human hearts.Methods: Clonogenic cells were derived from adult human atrial samples. Cells were either cultured in the absence of an initial marker selection or, in separate experiments, they were initially selected for c-kit (CD117), CD31 or CD164 by magnetic immunobeads, or for high aldehyde dehydrogenase activity (ALDH) by FACS. High ALDH activity has been linked to stem/progenitor cells in several tissues. Surface marker analysis was performed by flow cytometry. Cultured cells were also exposed to different factors that modulate cell differentiation, including Dikkopf-1, Noggin, and Wnt-5.Results: Clonogenic cells mainly showed fibroblast-like morphology, ability to grow for more than 30 passages in vitro, and a heterogeneous marker profile even in clones derived from the same cardiac sample. The predominant phenotype was positive for CD13, CD29, CD31, CD44, CD54, CD105 and CD146, but negative for CD10, CD11b, CD14, CD15, CD34, CD38, CD45, CD56, CD106, CD117, CD123, CD133, CD135 and CD271, primarily consistent with endothelial/vascular progenitor cells. However, a minority of clones showed a different profile characterized by expression of CD90, CD106 and CD318, but not CD31 and CD146, consistent with mesenchymal stem/progenitor cells. When initial cell selection was performed, both phenotypes were observed, similarly to unselected cells, irrespective of the selection marker used. Of note, CD117+ sorted cell clones were CD117-negative in culture. Regardless of the immunophenotype, several clones were able to form spheric cell aggregates (cardiospheres), a distinct stem cell property. Dikkopf-1 induced marked CD15 and CD106 upregulation, consistent with stromal differentiation; this effect was prevented by Noggin.Conclusions: The adult human heart contains clonogenic stem/progenitor cells that can be expanded for many passages and form cardiospheres. The surface marker profile of these cells is heterogeneous, consistent with a majority of clones being comprised of endothelial or vascular progenitor cells and a minority of clones consisting of mesenchymal stem/progenitor cells. Dikkopf-1 and Noggin showed opposing effects on stromal differentiation of human cardiac cell clones.

Comparison of human and rhesus macaque T-cell responses elicited by boosting with NYVAC encoding human immunodeficiency virus type 1 clade C immunogens.

Rhesus macaques (Macaca mulatta) have played a valuable role in the development of human immunodeficiency virus (HIV) vaccine candidates prior to human clinical trials. However, changes and/or improvements in immunogen quality in the good manufacturing practice (GMP) process or changes in adjuvants, schedule, route, dose, or readouts have compromised the direct comparison of T-cell responses between species. Here we report a comparative study in which T-cell responses from humans and macaques to HIV type 1 antigens (Gag, Pol, Nef, and Env) were induced by the same vaccine batches prepared under GMP and administered according to the same schedules in the absence and presence of priming. Priming with DNA (humans and macaques) or alphavirus (macaques) and boosting with NYVAC induced robust and broad antigen-specific responses, with highly similar Env-specific gamma interferon (IFN-gamma) enzyme-linked immunospot assay responses in rhesus monkeys and human volunteers. Persistent cytokine responses of antigen-specific CD4(+) and CD8(+) T cells of the central memory as well as the effector memory phenotype, capable of simultaneously eliciting multiple cytokines (IFN-gamma, interleukin 2, and tumor necrosis factor alpha), were induced. Responses were highly similar in humans and primates, confirming earlier data indicating that priming is essential for inducing robust NYVAC-boosted IFN-gamma T-cell responses. While significant similarities were observed in Env-specific responses in both species, differences were also observed with respect to responses to other HIV antigens. Future studies with other vaccines using identical lots, immunization schedules, and readouts will establish a broader data set of species similarities and differences with which increased confidence in predicting human responses may be achieved.

In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease.

In contrast to intact BALB/c mice, BALB/c mice rendered deficient in Vbeta4+ CD4+ T cells develop a Th1 response to infection with Leishmania major and are resistant. Vbeta4-deficient BALB/c mice are unable to generate the early IL-4 transcription occurring in Vbeta4 Valpha8 CD4+ T cells of BALB/c mice within 1 day of infection. Here we demonstrate that treatment of Vbeta4-deficient BALB/c mice with IL-4 during the first 64 h after infection instructs Th2 cell development and susceptibility to infection. The demonstrated inability of IL-4 to reverse the resistant phenotype of BALB/c mice treated with anti-CD4 mAb the day before infection suggest that these effects of IL-4 require its interaction with CD4+ T cells. In contrast to draining lymph node cells from BALB/c mice, cells from Vbeta4-deficient BALB/c mice remain responsive to IL-12 following infection. Strikingly, administration of IL-4 to Vbeta4-deficient BALB/c mice renders their lymph node cells unresponsive to IL-12 by down-regulating IL-12R beta2-chain expression. This study directly demonstrates that in BALB/c mice IL-4 is necessary and sufficient to initiate the molecular events steering Th2 cell maturation and susceptibility to L. major.

Macrophage migration inhibitory factor is critically involved in basal and fluoxetine-stimulated adult hippocampal cell proliferation and in anxiety, depression, and memory-related behaviors.

Intensive research is devoted to unravel the neurobiological mechanisms mediating adult hippocampal neurogenesis, its regulation by antidepressants, and its behavioral consequences. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is expressed in the CNS, where its function is unknown. Here, we show, for the first time, the relevance of MIF expression for adult hippocampal neurogenesis. We identify MIF expression in neurogenic cells (in stem cells, cells undergoing proliferation, and in newly proliferated cells undergoing maturation) in the subgranular zone of the rodent dentate gyrus. A causal function for MIF in cell proliferation was shown using genetic (MIF gene deletion) and pharmacological (treatment with the MIF antagonist Iso-1) approaches. Behaviorally, genetic deletion of MIF resulted in increased anxiety- and depression-like behaviors, as well as of impaired hippocampus-dependent memory. Together, our studies provide evidence supporting a pivotal function for MIF in both basal and antidepressant-stimulated adult hippocampal cell proliferation. Moreover, loss of MIF results in a behavioral phenotype that, to a large extent, corresponds with alterations predicted to arise from reduced hippocampal neurogenesis. These findings underscore MIF as a potentially relevant molecular target for the development of treatments linked to deficits in neurogenesis, as well as to problems related to anxiety, depression, and cognition.

The Th2 lymphoproliferation developing in LatY136F mutant mice triggers polyclonal B cell activation and systemic autoimmunity.

Lat(Y136F) knock-in mice harbor a point mutation in Tyr(136) of the linker for activation of T cells and show accumulation of Th2 effector cells and IgG1 and IgE hypergammaglobulinemia. B cell activation is not a direct effect of the mutation on B cells since in the absence of T cells, mutant B cells do not show an activated phenotype. After adoptive transfer of linker for activation of T cell mutant T cells into wild-type, T cell-deficient recipients, recipient B cells become activated. We show in vivo and in vitro that the Lat(Y136F) mutation promotes T cell-dependent B cell activation leading to germinal center, memory, and plasma cell formation even in an MHC class II-independent manner. All the plasma and memory B cell populations found in physiological T cell-dependent B cell responses are found. Characterization of the abundant plasmablasts found in secondary lymphoid organs of Lat(Y136F) mice revealed the presence of a previously uncharacterized CD93-expressing subpopulation, whose presence was confirmed in wild-type mice after immunization. In Lat(Y136F) mice, B cell activation was polyclonal and not Ag-driven because the increase in serum IgG1 and IgE concentrations involved Abs and autoantibodies with different specificities equally. Although the noncomplement-fixing IgG1 and IgE are the only isotypes significantly increased in Lat(Y136F) serum, we observed early-onset systemic autoimmunity with nephritis showing IgE autoantibody deposits and severe proteinuria. These results show that Th2 cells developing in Lat(Y136F) mice can trigger polyclonal B cell activation and thereby lead to systemic autoimmune disease.

Transcription factor PROX1 induces colon cancer progression by promoting the transition from benign to highly dysplastic phenotype.

The Drosophila transcription factor Prospero functions as a tumor suppressor, and it has been suggested that the human counterpart of Prospero, PROX1, acts similarly in human cancers. However, we show here that PROX1 promotes dysplasia in colonic adenomas and colorectal cancer progression. PROX1 expression marks the transition from benign colon adenoma to carcinoma in situ, and its loss inhibits growth of human colorectal tumor xenografts and intestinal adenomas in Apc(min/+) mice, while its transgenic overexpression promotes colorectal tumorigenesis. Furthermore, in intestinal tumors PROX1 is a direct and dose-dependent target of the beta-catenin/TCF signaling pathway, responsible for the neoplastic transformation. Our data underscore the complexity of cancer pathogenesis and implicate PROX1 in malignant tumor progression through the regulation of cell polarity and adhesion.

Human CD8 T-cell responses to Epstein-Barr virus and Cytomegalovirus in healthy donors and melanoma patients

Summary The mechanisms regulating the protective immune T-cell responses generated against the persistent Epstein-Barr virus (EBV) and Cytomegaloviru_s (CNIV) remain poorly understood. We analyzed the dynamics of cellular differentiation and T-cell receptor (TCR) clonotype selection of EBV- and CMV-specific T-cells in healthy adults and melanoma patients. While these responses could be subdivided into four T lymphocyte populations, théir proportions varied between EBV and CMV specific responses. Phenotypic and TCR clonotypic analyses supported a linear model of differentiation from the early-differentiated (EM/CD28pos) subset to the late-differentiatdc (EMRA/CD28neg) subset. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28neg subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, as some clonotypes were selected with differentiation, while others were not. Latedifferentiated CMV-specific clonotypes were mostly characterized by TCRs with lower dependency on CD8 co-receptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of four years. This work was extended to the study of EBV-specific CD8 T-cell responses in melanoma patients undergoing transient lymphodepletion, followed by adoptive cell transfer (ACT) and immune reconstitution for thè treatment of their tumors. Following treatment regimen, we first observed an increase in the proportion of virus-specific T-cells in 3 out of 5 patients, accompanied by a more differentiated phenotype (EMRA/CD28neg), compared to specific cells of healthy individuals. Yet, similarly to healthy donors, clonotype selection and composition of virus-specific T-cells varied along the pathway of cellular differentiation, with some clonotypes being selected with differentiation, while others were not. Intriguingly, no novel clonotypes emerged following transient immuno-suppression and homeostatic proliferation, finding which was subsequently explained by the absence of EBV reactivation. The distribution of each clonotype within early- and late-differentiated T-cell subsets in 4 out 5 patients was highly stable over time, with those clonotypes initially found before the start of treatment that were again present at specific differentiation stages after transient lymphodepletion and ACT. These findings uncover novel features of the highly sophisticated control of steady state protective T-cell immune responses against persistent herpesviruses in healthy adults. Furthermore they reveal the striking stability of these responses in terms of clonotype selection and composition with T-cell differentiation even in situations where the immune system has been. challenged. Résumé : Les mécanismes qui régulent les réponses immunitaires de type protectrices, générées contre les virus chroniquement persistants tels que l'Epstein-Barr (EBV) ou le Cytomegalo (CMV) restent largement inconnus. Nous avons analysé la différenciation des lymphocytes T spécifiques pour ces virus, ainsi que la composition des clonotypes T (par leur récepteur T) chez les donneurs sains. Les réponses immunes peuvent être classifiées en quatre souspopulations majeures de lymphocytes T, cependant, leur proportion varie entre les réponses spécifiques contre EBV ou CMV. Ces analyses soutiennent le modèle linéaire de différenciation, à partir de la population non différenciée (EM/CD28pos) vers la population plus différenciée (ENIIZA/CD28neg). De plus, nos données sur la composition clonale de ces cellules T spécifiques ont révélé des répertoires TCR restreints, pour la réponse anti-CMV, et relativement diversifiés contre EBV. Tous les clonotypes spécifiques de ces virus identifiés dans la sous-population différenciée EMRA/CD28neg, ont également été retrouvés dans la population de cellules "mémoires". Toutefois, de fortes différences ont été observées dans les schémas de domination de ces sous-populations, en effet, certains clonotypes étaient sélectionnés avec la différenciation, alors que d'autres ne l'étaient pas. Nous avons également démontré que ces clonotypes différenciés et spécifiques pour le CMV sont caractérisés par des TCRs à faible dépendance en regard de la coopération du corécepteur CD8. Néanmoins, tous les clonotypes affichent une avidité fonctionnelle similaire, suggérant un rôle compensatoire du CD8, dans le cas des clonotypes avec une faible avidité du TCR En définitive, la composition et la sélection des clonotypes spécifiques pour chaque virus et pour chaque sous-population suit un schéma de différenciation hautement conservé au cours du temps, avec la présence de ces mêmes clonotypes au même stade de différenciation sur une période de quatre ans. Ce travail a été étendu à l'étude des réponses T CD8+ spécifiques pour le virus EBV chez les patients atteints de mélanome et recevant dans le cadre du traitement de leurs tumeurs une lymphodéplétion transitoire, suivie d'un transfert adoptif de cellules et d'une reconstitution immunitaire. Au cours de cette thérapie, nous avons en premier lieu observé pour 3 des 5 patients une augmentation de la proportion de cellules T spécifiques pour le virus, accompagné d'un phénotype plus différencié (EMRA/CD28neg), et ceci comparativement à des cellules spécifiques d'individus sains. Pourtant, comme nous l'avons observé chez les donneurs sains, la sélection et la composition des clonotypes T spécifiques varient tout au long de la différenciation cellulaire, avec certains clonotypes sélectionnés et d'autres qui ne le sont pas. Étonnamment, aucun nouveau clonotype n'a émergé après l'immuno-suppression transitoire et la prolifération homéostatique. Cette observation trouve son explication par une absence de réactivation du virus EBV chez ces patients, et ce malgré leur traitement. De plus, la distribution de chaque clonotype parmi ces sous-populations non-différenciées et différenciées reste stable au cours du traitement. Ainsi, les mêmes clonotypes initialement identifiés avant le début du traitement sont présents aux mêmes stades de différenciation après la lymphodéplétion et la prolifération homéostatique. Ces résultats ont permis d'identifier de nouveaux mécanismes impliqués dans la régulation hautement «sophistiquée » des réponses immunitaires T contre les virus persistants EBV et CMV chez les donneurs sains. En particulier, ils révèlent la grande stabilité de ces réponses en termes de sélection et de composition des clonotypes avec la différenciation cellulaire, et ce dans les situations chroniques, ainsi que dans les situations dans lesquelles le système immunitaire a été profondément perturbé.

Ex vivo detectable human CD8 T-cell responses to cancer-testis antigens.

Clinical trials have shown that strong tumor antigen-specific CD8 T-cell responses are difficult to induce but can be achieved for T-cells specific for melanoma differentiation antigens, upon repetitive vaccination with stable emulsions prepared with synthetic peptides and incomplete Freund's adjuvant. Here, we show in four melanoma patients that ex vivo detectable T-cells and thus strong T-cell responses can also be induced against the more universal cancer-testis antigens NY-ESO-1 and Mage-A10. Interestingly, all patients had ex vivo detectable T-cell responses against multiple antigens after serial vaccinations with three peptides emulsified in incomplete Freund's adjuvant. Antigen-specific T-cells displayed an activated phenotype and secreted IFNgamma. The robust immune responses provide a solid basis for further development of human T-cell vaccination.

The role of the c-myc gene in pigment cell development and homeostasis

Résumé La dérégulation de c-Myc est un événement fréquent de la transformation cellulaire. Une régulation positive de cette oncoprotéine a été démontrée dans divers mélanomes cutanés primaires et métastatiques et est associée à un pronostic défavorable (Grover et al., 1996; Zhuang et al., 2008). c-Myc est considéré comme une molécule centrale impliquée dans plusieurs processus de l'homéostasie cellulaire. En raison de sa contribution importante dans la progression tumorale, la fonction de c-Myc a été étudiée intensément. Cependant nous connaissons peu le rôle de ce facteur de transcription dans l'embryogenèse et dans la spécification tissulaire. Un déficit total de c-Myc pendant l'embryogenèse conduit à la mort embryonnaire avant 10.5 jours de gestation. Cette mort est causée par de multiples imperfections du développement touchant la taille de l'embryon, le coeur, le péricarde, le tube neural et les cellules sanguines (Davis et al., 1993; Trumpp et al., 2001). Récemment, il a été montré que la plupart de ces anomalies sont secondaires et résultent d'une insuffisance du placenta dans les embryons c-myc-/- (Dubois et al., 2008). Sachant que c-Myc est important dans la maintenance des lignées de la crête neurale (Wei et al., 2007), nous nous sommes intéressés au rôle de c-Myc dans le développement des cellules pigmentaires et à leur homéostasie après la naissance. Un allèle floxé de c-myc (Trumpp et al., 2001) a été utilisé pour supprimer ce gène spécifiquement dans la lignée mélanocytaire à l'aide d'une souris transgénique Tyr::Cre (Delmas et al., 2003). L'ablation des deux allèles de c-myc dans les mélanocytes des souris c-myccKO conduit au phénotype de grisonnement des poils, observé directement après la naissance et associé à une diminution du nombre de mélanocytes dans le bulbe des follicules pileux. Les cellules pigmentaires restantes expriment les marqueurs mélanogéniques (Tyr, TRP-1, Dct and MITF) et semblent être fonctionnelles puisqu'elles peuvent produire et transférer la mélanine. De plus, la capacité de prolifération des mélanocytes déficients en c-Myc dans le bulbe des follicules pileux ne semble pas être affectée chez les nouveaux-nés. Les cellules souches mélanocytaires sont présentes, mais en nombre réduit, dans le bulge des follicules pileux à la fin de la morphogenèse chez les souris c-myccKO âgées de huit jours. Ces cellules sont maintenues sans changement durant le premier cycle pileux (vérifié à l'âge de trente jours), ce qui sous-entend que la fonction de c-Myc n'est pas nécessaire pour ce processus. Ceci explique pourquoi, en supposant que des cellules souches mélanocytaires fonctionnelles sont présentes dans la peau, nous n'observons pas de dilution de couleur de la robe liée à l'âge. Cependant, la présence de ces cellules souches mélanocytaires dans la peau c-myccKO ne suffit pas à assurer une quantité normale de mélanocytes différenciés dans le bulbe des follicules pileux. Cette population de cellules pigmentaires matures est sévèrement affectée par la suppression de c-Myc, ce qui contribue amplement au phénotype de grisonnement des poils. De plus, c-Myc paraît être important pour le développement des mélanocytes. Ainsi, le nombre de mélanoblastes diminue dans les embryons c-myccKO à partir du douzième jour de gestation. A treize jours de gestation, au stade où les mélanoblastes pénètrent dans l'épiderme et prolifèrent, les mélanoblastes déficients en c-Myc ne s'adaptent pas aux signaux de prolifération et se retrouvent en nombre réduit dans l'épiderme. Finalement, nous nous sommes intéressés, au rôle de N-Myc, un homologue proche de c-Myc, dans la lignée mélanocytaire. Nos expériences ont montré que. N-Myc était superflu pour le développement et l'homéostasie des mélanocytes, une seule copie du gène c-myc étant suffisante pour maintenir une pigmentation normale de la robe des souris c-mycc-myccKO/+~N_ myccKO/KO. Cependant, le rôle essentiel de N-Myc dans la maintenance des cellules mélanocytaires précurseurs apparaît lorsque c-Myc est absent, puisque la suppression simultanée des deux Myc résulte en une perte complète de la coloration de la robe. Ceci implique la présence d'un mécanisme compensatoire entre c- et N-Myc dans la lignée mélanocytaire, avec un rôle prédominant de c-Myc. Summary Deregulation of c-Myc is known to be a common event in cellular transformation. Upregulation of this oncoprotein was shown in a variety of primary and metastatic cutaneous melanomas and has been associated with a poor prognosis (Grover et al., 1996; Zhuang et al., 2008). c-myc is seen as a central molecule involved in many aspects of cellular homeostasis. c-Myc function has been intensively studied mostly because of its significant contribution to tumour progression. However little is known on the role of this transcription factor in embryogenesis and tissue specification. Complete loss of c-Myc during embryogenesis results in embryonic death before E10.5 due to multiple developmental defects including embryonic size, heart, pericardium, neural tube and blood cells (Davis et al., 1993; Trumpp et al., 2001). Recently it was discovered that most of these abnormalities are secondary and results of placental insufficiency in c-Myc-/- embryos (Dubois et al., 2008). Here, we focused on the role of c-Myc in pigment cell development and homeostasis after birth, knowing that c-Myc is important in the maintenance of neural crest lineages (Wei et al., 2007). A floxed allele of c-Myc (Trumpp et al., 2001) was used to specifically delete this gene in the melanocyte lineage using Tyr::Cre transgenic mice (Delmas et al., 2003). Removal of both c-Myc alleles in melanocytes of c-MyccKO mouse led to the grey hair phenotype which is seen directly after birth and was associated with a decrease in the melanocyte number in the bulb of the hair follicle. The remaining population of pigment cells express melanogenic markers (Tyr, TRP-1, Dct and MITF) and seem functionally normal since they can produce and transfer melanin. Furthermore proliferation capacity of c-Myc deficient melanocytes in the bulb of hair follicle seems not to be affected in newborn animals. Melanocyte stem cells (MSCs) are present but reduced in numbers in the bulge of the hair follicle at the end of morphogenesis in 8 days old c-MyccKO mice. These cells are maintained through the first hair cycle (as verified at P30) without any further changes, suggesting that c-Myc function is not required for this process. This explains why we did not detect any agerelated coat color dilution, assuming a presence of functional MSCs in the skin. Importantly, presence of MSCs in c-MyccKO skin was not sufficient for assuring a normal number of differentiated melanocytes in the bulb of the hair follicle. This population of mature pigmented cells is severely affected upon c-myc deletion thus largely contributing to the grey hair phenotype. Moreover, c-Myc appears to be important for melanocyte development. Thus, melanoblast number is affected in c-MyccKO embryos day 12 of gestation onwards. At E13.5, when melanoblasts enter the epidermis and proliferate, c-myc deficient melanoblasts failed to adapt to proliferation signals and are therefore reduced in number in the epidermis. Finally, we addressed the role of N-Myc, a closest homologue of c-Myc, in the melanocyte lineage. In these experiments, N-Myc was dispensable for melanocyte development and homeostasis, and even one copy of the c-myc gene was sufficient to maintain normal coat color pigmentation in c-mycc-mycCKO/+ ,N-myccKO/KO mice. However the crucial role of N-Myc in maintenance of melanocyte precursor cells became apparent when c-myc is eliminated since simultaneous deletion of both Myc results in complete loss of coat color pigmentation. This suggests compensatory mechanisms between c- and N-Myc with a predominant role of c-Myc in melanocyte lineage.

Role of lymph node fibroblasts in T cell priming

SummarySecondary lymphoid organs, such as lymph nodes or spleen, are the only places in our body where primary adaptive immune responses are efficiently elicited. These organs have distinct Β and Τ cell rich zones and Τ lymphocytes constantly migrate from the bloodstream into Τ zones to scan dendritic cells (DCs) for antigens they present. Specialized fibroblasts, the Τ zone reticular cells (HR.Cs), span the Τ zone in the form a three-dimensional network. lK.Cs guide incoming Τ cells in their migration, both chemically, by the secretion of the chemokines CCL19 and CCL21, and physically, by construction of a road system to which also DCs adhere. In this way TRCs are thought to facilitate encounters of Τ cells with antigen-bearing DCs and thereby accelerate the selection of rare antigen-specific Τ cells. The resulting Τ cell activation, proliferation and differentiation all take place within the TRC network. However, the influence of TRCs on Τ cell activation has so fer not been elucidated with the possible reasons being that TRCs represent a relative rare cell population and that mice devoid of TRCs have not been described.To circumvent these technical limitations, we established TRC clones and lines to have an abundant source to functionally characterize TRCs. Both the clones and lines show a fibroblastic phenotype, express a surface marker profile comparable to ex vivo TRCs and produce extracellular matrix molecules. However, expression of Ccl19, Ccl21 and ZL-7 is lost and could not be restored by cytokine stimulation. When these TRC clones or lines were cultured in a three-dimensional cell culture system, their morphology changed and resembled that of in vivo TRCs as they formed networks. By adding Τ cells and antigen-loaded DCs to these cultures we successfully reconstructed lymphoid Τ zones that allowed antigen-specific Τ cell activation.To characterize the role of TRCs in Τ cell priming, TRCs were co-cultured with antigen-specific Τ cells in the presence antigen-loaded DCs. Surprisingly, the presence of TRC lines and ex vivo TRCs inhibited rather than enhanced CD8+ Τ cell activation, proliferation and effector cell differentiation. TRCs shared this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. TRCs were identified as a strong source of nitric oxide (NO) thereby directly dampening Τ cell expansion as well as reducing the Τ cell priming capacity of DCs. The expression of inducible NO synthase (iNOS) was up- regulated in a subset of TRCs by both DC-signals as well as interferon-γ produced by primed CD8+ Τ cells. Importantly, iNOS expression was induced during viral infection in vivo in both lymph node TRCs and DCs. Consistent with a role for NO as a negative regulator, the primary Τ cell response was exaggerated in iNOS-/- mice. Our findings highlight that in addition to their established positive roles in Τ cell responses TRCs and DCs cooperate in a negative feedback loop to attenuate Τ cell expansion during acute inflammation.RésuméLes organes lymphoïdes secondaires, comme les ganglions lymphoïdes ou la rate, sont les seuls sites dans notre corps où la réponse primaire des lymphocytes Β et Τ est initiée efficacement. Ces organes ont des zones différentes, riches en cellules Β ou T. Des lymphocytes Τ circulent constamment du sang vers les zones T, où ils échantillonent la surface des cellules dendritiques (DCs) pour identifier les antigènes qu'ils présentent. Des fibroblastes spécialisés - nommés Τ zone reticular cells (TRCs)' forment un réseau tridimensionnel dans la zone T. Les TRCs guident la migration des cellules Τ par deux moyens: chimiquement, par la sécrétion des chimiokines CCL19 et CCL21 et physiquement, par la construction d'un réseau routier en trois dimensions, auquel adhèrent aussi des DCs. Dans ce? cas, on pense que la présence des TRCs facilite les rencontres entre les cellules Τ et les DCs chargées de l'antigène et accélère la sélection des rares cellules Τ spécifiques. Ensuite, l'activation de cellules T, ainsi que la prolifération et la différenciation se produisent toutes à l'intérieur du réseau des TRCs. L'influence des TRCs sur l'activation des cellules T n'est que très peu caractérisée, en partie parce que les TRCs représentent une population rare et que les souris déficientes dans les TRCs n'ont pas encore été découvertes.Pour contourner ces limitations techniques, nous avons établi des clones et des lignées cellulaires de TRC pour obtenir une source indéfinie de ces cellules permettant leur caractérisation fonctionnelle. Les clones et lignées établis ont un phénotype de fibroblaste, ils expriment des molécules de surface similaires aux TRCs ex vivo et produisent de la matrice extracellulaire. Mais l'expression de Ccl19, Ccl21 et 11-7 est perdue et ne peut pas être rétablie par stimulation avec différentes cytokines. Les clones TRC ou les lignées cultivées en un système tridimensionnel de culture cellulaire, montrent une morphologie changée, qui ressemble à celle de TRC ex vivo inclus la construction de réseaux tridimensionnels.Pour caractériser le rôle des TRC dans l'activation des cellules T, nous avons cultivé des TRCs avec des cellules T spécifiques et des DCs chargées avec l'antigène. Etonnamment, la présence des TRC (lignées et ex vivo) inhibait plutôt qu'elle améliorait l'activation, la prolifération et la différenciation des lymphocytes T CDS+. Les TRCs partageaient cette fonction avec des fibr-oblastes des organes non lymphoïdes et des cellules souches du type mésenchymateux. Dans ces conditions, les TRCs sont une source importante d'oxyde nitrique (NO) et par ce fait limitent directement l'expansion des cellules T et réduisent aussi la capacité des DCs à activer les cellules T. L'expression de l'enzyme NO synthase inductible (ïNOS) est régulée à la hausse par des signaux dérivés des DCs et par l'interféron-γ produit par des cellules T de type CD8+ activées. Plus important, l'expression d'iNOS est induite pendant une infection virale in vivo, dans les TRCs et dans les DCs. Par conséquent, la réponse primaire de cellules T est exagérée dans des souris iNOS-/-. Nos résultats mettent en évidence qu'en plus de leur rôle positif bien établi dans la réponse immunitaire, les TRCs et les DCs coopèrent dans une boucle de rétroaction négative pour atténuer l'expansion des cellules T pendant l'inflammation aigiie pour protéger l'intégrité et la fonctionnalité des organes lymphoïdes secondaires.