Lifelong dynamics of human CD4+CD25+ regulatory T cells: insights from in vivo data and mathematical modeling.

Despite their limited proliferation capacity, regulatory T cells (T(regs)) constitute a population maintained over the entire lifetime of a human organism. The means by which T(regs) sustain a stable pool in vivo are controversial. Using a mathematical model, we address this issue by evaluating several biological scenarios of the origins and the proliferation capacity of two subsets of T(regs): precursor CD4(+)CD25(+)CD45RO(-) and mature CD4(+)CD25(+)CD45RO(+) cells. The lifelong dynamics of T(regs) are described by a set of ordinary differential equations, driven by a stochastic process representing the major immune reactions involving these cells. The model dynamics are validated using data from human donors of different ages. Analysis of the data led to the identification of two properties of the dynamics: (1) the equilibrium in the CD4(+)CD25(+)FoxP3(+)T(regs) population is maintained over both precursor and mature T(regs) pools together, and (2) the ratio between precursor and mature T(regs) is inverted in the early years of adulthood. Then, using the model, we identified three biologically relevant scenarios that have the above properties: (1) the unique source of mature T(regs) is the antigen-driven differentiation of precursors that acquire the mature profile in the periphery and the proliferation of T(regs) is essential for the development and the maintenance of the pool; there exist other sources of mature T(regs), such as (2) a homeostatic density-dependent regulation or (3) thymus- or effector-derived T(regs), and in both cases, antigen-induced proliferation is not necessary for the development of a stable pool of T(regs). This is the first time that a mathematical model built to describe the in vivo dynamics of regulatory T cells is validated using human data. The application of this model provides an invaluable tool in estimating the amount of regulatory T cells as a function of time in the blood of patients that received a solid organ transplant or are suffering from an autoimmune disease.

Productive HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane permeabilization leading to a caspase-independent cell death.

We have explored in vitro the mechanism by which human immunodeficiency virus, type 1 (HIV-1) induces cell death of primary CD4+ T cells in conditions of productive infection. Although HIV-1 infection primed phytohemagglutinin-activated CD4+ T cells for death induced by anti-CD95 antibody, T cell death was not prevented by a CD95-Fc decoy receptor, nor by decoy receptors of other members of the TNFR family (TNFR1/R2, TRAILR1/R2/OPG, TRAMP) or by various blocking antibodies, suggesting that triggering of death receptors by their cognate ligands is not involved in HIV-induced CD4 T cell death. HIV-1 induced CD4 T cell shrinkage, cell surface exposure of phosphatidylserine, loss of mitochondrial membrane potential (Deltapsim), and mitochondrial release of cytochrome c and apoptosis-inducing factor. A typical apoptotic phenotype (nuclear chromatin condensation and fragmentation) only occurred in around half of the dying cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a broad spectrum caspase inhibitor, prevented nuclear chromatin condensation and fragmentation in HIV-infected CD4+ T cells and in a cell-free system (in which nuclei were incubated with cytoplasmic extracts from the HIV-infected CD4+ T cells). Nevertheless, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not prevent mitochondrial membrane potential loss and cell death, suggesting that caspases are dispensable for HIV-mediated cell death. Our findings suggest a major role of the mitochondria in the process of CD4 T cell death induced by HIV, in which targeting of Bax to the mitochondria may be involved.

Quelques mécanismes moléculaires impliqués dans la pathogenèse du diabète de type II et leur importance thérapeutique potentielle [Various molecular mechanisms involved in the pathogenesis of type II diabetes and their potential therapeutic importance]

The pancreatic beta cell presents functional abnormalities in the early stages of development of non-insulin dependent diabetes mellitus (NIDDM). The disappearance of the first phase of insulin secretion induced by a glucose load is a early marker of NIDDM. This abnormality could be secondary to the low expression of the pancreatic glucose transporter GLUT2. Together with the glucokinase enzyme, GLUT2 is responsible for proper beta cell sensing of the extracellular glucose levels. In NIDDM, the GLUT2 mRNA levels are low, a fact which suggests a transcriptional defect of the GLUT2 gene. The first phase of glucose-induced insulin secretion by the beta pancreatic cell can be partly restored by the administration of a peptide discovered by a molecular approach, the glucagon-like peptide 1 (GLP-1). The gene encoding for the glucagon is expressed in a cell-specific manner in the A cells of the pancreatic islet and the L cells of the intestinal tract. The maturation process of the propeptide encoded by the glucagon gene is different in the two cells: the glucagon is the main hormone produced by the A cells whereas the glucagon-like peptide 1 (GLP-1) is the major peptide synthesized by the L cells of the intestine. GLP-1 is an incretin hormone and is at present the most potent insulinotropic peptide. The first results of the administration of GLP-1 to normal volunteers and diabetic patients are promising and may be a new therapeutic approach to treating diabetic patients.

Incrétines, sécrétion d'insuline et diabète

Nutrient ingestion triggers a complex hormonal response aimed at stimulating glucose utilization in liver, muscle and adipose tissue to minimize the raise in blood glucose levels. Insulin secretion by pancreatic beta cells plays a major role in this response. Although the beta cell secretory response is mainly controlled by blood glucose levels, gut hormones secreted in response to food intake have an important role in potentiating glucose-stimulated insulin secretion. These gluco-incretin hormones are GLP-1 (glucagon-like peptide-1) and GIP (gluco-dependent insulinotropic polypeptide). Their action on pancreatic beta cells depends on binding to specific G-coupled receptors linked to activation of the adenylyl cyclase pathway. In addition to their effect on insulin secretion both hormones also stimulate insulin production at the transcriptional and translational level and positively regulate beta cell mass. Because the glucose-dependent insulinotropic action of GLP-1 is preserved in type 2 diabetic patients, this peptide is now developed as a novel therapeutic drug for this disease.

The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter.

Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associated SLC6A8. Here, we identified a second creatine transporter (CRT2) known as monocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration in MCT12 (p.G407S) found in a patient with age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis-Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 µm. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels. We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventive power of creatine supplementation against the most prominent age-related vision impaired condition.

Complex genetics in idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) is an important human disease model. Investigations of the genetics of IHH have facilitated insights into critical pathways regulating sexual maturation and fertility. IHH has been traditionally considered a monogenic disorder. This model holds that a single gene defect is responsible for the disease in each patient. In the case of IHH, 30% of cases are explained by mutations in one of eleven genes. In recent years, several lines of evidence have challenged the monogenic paradigm in IHH. First, disease-associated mutations display striking incomplete penetrance and variable expressivity within and across IHH families. Second, each locus is responsible for only a small percentage of cases. Third, more than one disease-associated mutation seems to be segregating in some families with IHH, and their combined or separate presence in individuals accounts for the variability in disease severity. Finally, IHH is not strictly a congenital and life-long disorder; occasionally it manifests itself during adulthood (adult-onset IHH); in other cases, the disease is not permanent, as evidenced by normal activity of the hypothalamic-pituitary-gonadal axis after discontinuation of treatment in adulthood (IHH reversal). Together, these observations suggest that IHH is not strictly a monogenic mendelian disease, as previously thought. Rather, it is emerging as a digenic, and potentially oligogenic disease, in which hormonal and/or environmental factors may critically influence genetic predisposition and clinical course. Future investigations of IHH should characterize the extent of the involvement of multiple genes in disease pathogenesis, and elucidate the contributions of epigenetic factors.

DL4-mediated Notch signaling is required for the development of fetal αβ and γδ T cells.

T-cell development depends upon interactions between thymocytes and thymic epithelial cells (TECs). The engagement of delta-like 4 (DL4) on TECs by Notch1 expressed by blood-borne BM-derived precursors is essential for T-cell commitment in the adult thymus. In contrast to the adult, the earliest T-cell progenitors in the embryo originate in the fetal liver and migrate to the nonvascularized fetal thymus via chemokine signals. Within the fetal thymus, some T-cell precursors undergo programmed TCRγ and TCRδ rearrangement and selection, giving rise to unique γδ T cells. Despite these fundamental differences between fetal and adult T-cell lymphopoiesis, we show here that DL4-mediated Notch signaling is essential for the development of both αβ and γδ T-cell lineages in the embryo. Deletion of the DL4 gene in fetal TECs results in an early block in αβ T-cell development and a dramatic reduction of all γδ T-cell subsets in the fetal thymus. In contrast to the adult, no dramatic deviation of T-cell precursors to alternative fates was observed in the fetal thymus in the absence of Notch signaling. Taken together, our data reveal a common requirement for DL4-mediated Notch signaling in fetal and adult thymopoiesis.

Direct presentation of a melanocyte-associated antigen in peripheral lymph nodes induces cytotoxic CD8+ T cells.

Encounter of self-antigens in the periphery by mature T cells induces tolerance in the steady-state. Hence, it is not understood why the same peripheral antigens are also promiscuously expressed in the thymus to mediate central tolerance. Here, we analyzed CD8(+) T-cell tolerance to such an antigen constituted by ovalbumin under the control of the tyrosinase promoter. As expected, endogenous CD8(+) T-cell responses were altered in the periphery of transgenic mice, resulting from promiscuous expression of the self-antigen in mature medullary epithelial cells and deletion of high-affinity T cells in the thymus. In adoptive T-cell transfer experiments, we observed constitutive presentation of the self-antigen in peripheral lymph nodes. Notably, this self-antigen presentation induced persisting cytotoxic cells from high-affinity CD8(+) T-cell precursors. Lymph node resident melanoblasts expressing tyrosinase directly presented the self-antigen to CD8(+) T cells, independently of bone marrow-derived antigen-presenting cells. This peripheral priming was independent of the subcellular localization of the self-antigen, indicating that this mechanism may apply to other melanocyte-associated antigens. Hence, central tolerance by promiscuous expression of peripheral antigens is a mandatory, rather than a superfluous, mechanism to counteract the peripheral priming, at least for self-antigens that can be directly presented in lymph nodes. The peripheral priming by lymph node melanoblasts identified here may constitute an advantage for immunotherapies based on adoptive T-cell transfer.

Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment.

Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor-mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1- and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifically inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the first steps of intrathymic T cell development.

Genes for normal sleep and sleep disorders.

Sleep and wakefulness are complex behaviors that are influenced by many genetic and environmental factors, which are beginning to be discovered. The contribution of genetic components to sleep disorders is also increasingly recognized as important. Point mutations in the prion protein, period 2, and the prepro-hypocretin/orexin gene have been found as the cause of a few sleep disorders but the possibility that other gene defects may contribute to the pathophysiology of major sleep disorders is worth in-depth investigations. However, single gene disorders are rare and most common disorders are complex in terms of their genetic susceptibility, environmental effects, gene-gene, and gene-environment interactions. We review here the current progress in the genetics of normal and pathological sleep.

Lymph node stromal cells in homeostasis and immune response

Summary : Antigen-specific T lymphocytes constantly patrol the body to search for invading pathogens. Given the large external and internal body surfaces that need to be surveyed, a sophisticated strategy is necessary to facilitate encounters between T cells and pathogens. Dendritic cells present at all body surfaces are specialized in capturing pathogens and bringing them to T zones of secondary lymphoid organs, such as the lymph nodes and the spleen. Here, dendritic cells present antigenic fragments and activate the rare antigen-specific T lymphocytes. This induction of an immune response is facilitated in multiple ways by a dense network of poorly characterized stromal cells, termed fibroblastic reticular cells (FRCs). They constitutively produce the chemokines CCL21 and CCL19, which attract naïve T cells and dendritic cells into the T zone. Further, they provide an adhesion scaffold for dendritic cells and a migration scaffold for naïve T cells, allowing efficient screening of dendritic cell by thousands of T cells. FRCs also form a system of microchannels (conduits) that allows rapid transport of antigen or cytokines from the subcapsular sinus to the T zone. We characterized lymph node FRCS by flow cytometry, immunofluorescence microscopy, real time PCR and functional assays and could show that FRCs are a unique type of myofibroblasts which produce the T cell survival factor IL-7. This function was shown to be critically involved in regulating the size of the peripheral T cell pool and further demonstrates the importance of FRCs in maintaining immunocompetence. As we observed that some dendritic cells also express the receptor for IL-7, we expected a similar function of IL-7 in their survival. Surprisingly, we found no role for IL-7 in their survival but in their development. Analysis of hematopoietic precursors suggested that part of the dendritic cell pool develops out of an IL-7 dependent precursor, which maybe shared with lymphocytes. During the induction of an immune response, lymph node homeostasis is drastically altered when the lymph node expands several-fold in size to accommodate many more lymphocytes. Here, we describe that this expansion of the T zone is accompanied by the activation and proliferation of FRCs thereby preserving T zone architecture and function. This expansion of the FRC network is regulated by antigen-independent and -dependent events. It demonstrates the incredible plasticity of this organ allowing clonal expansion of antigen-specific lymphocytes. Résumé : Les lymphocytes T, spécifiques pour un antigène particulier, patrouillent constamment le corps à la recherche de l'invasion de pathogène. A cause des grandes surfaces externes et internes du corps, une stratégie sophistiquée est nécessaire afin de faciliter les rencontres entre les cellules T et les agents pathogènes. Les cellules dendritiques présentes dans toutes les surfaces du corps sont spécialisées dans la capture des agents pathogènes et dans le transport vers les zones T des organes lymphoïdes secondaires, comme les ganglions lymphatiques et la rate. Dans ces organes, les cellules dendritiques présentent les fragments antigéniques et activent les lymphocytes T rares. L'induction de cette réponse immunitaire est facilitée de différentes manières par un réseau dense de cellules strornales mal caractérisé, appelées 'fibroblastic reticular tells' (FRCs). FRCs produisent constitutivement les chimiokines CCL21 et CCL19, qui attirent les lymphocytes T naïfs et les cellules dendritiques vers la zone T. En outre, elles donnent une base d'adhérence pour les cellules dendritiques et elles attirent les cellules T naïves vers les cellules dendritiques. Les FRCs forment des petits canaux (ou conduits) qui permettent le transport rapide d'antigènes solubles ou de cytokines vers la zone T. Nous avons caractérisé les FRCs par cytométrie en flux, immunofluorescence et par PCR en temps réel et nous avons démontré que les FRCs sont un type unique de rnyofibroblastes qui produisent un facteur de survie des cellules T, l'Interleukine-7. Il a été démontré que cette fonction est cruciale afin d'augmenter la taille et la diversité du répertoire de cellules T, et ainsi, maintenir l'immunocompétence. Comme nous avons observé que certaines cellules dendritiques expriment également le récepteur de l'IL-7, nous avons testé une fonction similaire dans leur survie. Étonnamment, nous n'avons pas trouvé de rôle pour l'IL-7 dans leur survie, mais dans leur développement. L'analyse des précurseurs hématopoïétiques a suggéré qu'une fraction des cellules dendritiques se développe à partir des précurseurs dépendants de l'IL-7, qui sont probablement partagés avec les lymphocytes. Au cours de l'induction d'une réponse immunitaire, l'homéostasie du ganglion lymphatique est considérablement modifiée. En effet, sa taille augmente considérablement afin d'accueillir un plus grand nombre de lymphocytes. Nous décrivons ici que cet élargissement de la zone T est accompagné par l'activation et 1a prolifération des FRCs, préservant l'architecture et la fonction de la zone T. Cette expansion du réseau des FRCs est régie par des évènements à la fois dépendants et indépendants de l'antigène. Cela montre l'incroyable plasticité de cet organe qui permet l'expansion clonale des lymphocytes T spécifiques.

T cell fate specification and alphabeta/gammadelta lineage commitment.

The development of T cells from pluripotent stem cells involves a coordinated series of lineage-commitment steps. Common lymphoid precursors in the fetal liver or adult bone marrow must first choose between a T, B or NK cell fate. Committed T cell precursors in the thymus then differentiate into cells committed to the alphabeta or gammadelta lineages. Recent advances have been made in our understanding of the mechanisms underlying T cell fate specification and alphabeta/gammadelta lineage divergence.

A critical lineage-nonspecific role for pTalpha in mediating allelic and isotypic exclusion in TCRbeta-transgenic mice.

Although it is well established that early expression of TCRbeta transgenes in the thymus leads to efficient inhibition of both endogenous TCRbeta and TCRgamma rearrangement (also known as allelic and "isotypic" exclusion, respectively) the role of pTalpha in these processes remains controversial. Here, we have systematically re-evaluated this issue using three independent strains of TCRbeta-transgenic mice that differ widely in transgene expression levels, and a sensitive intracellular staining assay that detects endogenous TCRVbeta expression in individual immature thymocytes. In the absence of pTalpha, both allelic and isotypic exclusion were reversed in all three TCRbeta-transgenic strains, clearly demonstrating a general requirement for pre-TCR signaling in the inhibition of endogenous TCRbeta and TCRgamma rearrangement. Both allelic and isotypic exclusion were pTalpha dose dependent when transgenic TCRbeta levels were subphysiological. Moreover, pTalpha-dependent allelic and isotypic exclusion occurred in both alphabeta and gammadelta T cell lineages, indicating that pre-TCR signaling can potentially be functional in gammadelta precursors. Finally, levels of endogenous RAG1 and RAG2 were not down-regulated in TCRbeta-transgenic immature thymocytes undergoing allelic or isotypic exclusion. Collectively, our data reveal a critical but lineage-nonspecific role for pTalpha in mediating both allelic and isotypic exclusion in TCRbeta-transgenic mice.

Spatial relationship between the atmospheric circulation and the precipitation measured in the western Swiss Alps by means of the analogue method

An adaptation technique based on the synoptic atmospheric circulation to forecast local precipitation, namely the analogue method, has been implemented for the western Swiss Alps. During the calibration procedure, relevance maps were established for the geopotential height data. These maps highlight the locations were the synoptic circulation was found of interest for the precipitation forecasting at two rain gauge stations (Binn and Les Marécottes) that are located both in the alpine Rhône catchment, at a distance of about 100 km from each other. These two stations are sensitive to different atmospheric circulations. We have observed that the most relevant data for the analogue method can be found where specific atmospheric circulation patterns appear concomitantly with heavy precipitation events. Those skilled regions are coherent with the atmospheric flows illustrated, for example, by means of the back trajectories of air masses. Indeed, the circulation recurrently diverges from the climatology during days with strong precipitation on the southern part of the alpine Rhône catchment. We have found that for over 152 days with precipitation amount above 50 mm at the Binn station, only 3 did not show a trajectory of a southerly flow, meaning that such a circulation was present for 98% of the events. Time evolution of the relevance maps confirms that the atmospheric circulation variables have significantly better forecasting skills close to the precipitation period, and that it seems pointless for the analogue method to consider circulation information days before a precipitation event as a primary predictor. Even though the occurrence of some critical circulation patterns leading to heavy precipitation events can be detected by precursors at remote locations and 1 week ahead (Grazzini, 2007; Martius et al., 2008), time extrapolation by the analogue method seems to be rather poor. This would suggest, in accordance with previous studies (Obled et al., 2002; Bontron and Obled, 2005), that time extrapolation should be done by the Global Circulation Model, which can process atmospheric variables that can be used by the adaptation method.

Vancomycin-intermediate Staphylococcus aureus selected during vancomycin therapy of experimental endocarditis are not detected by culture-based diagnostic procedures and persist after treatment arrest.

OBJECTIVES: Laboratory detection of vancomycin-intermediate Staphylococcus aureus (VISA) and their heterogeneous VISA (hVISA) precursors is difficult. Thus, it is possible that vancomycin failures against supposedly vancomycin-susceptible S. aureus are due to undiagnosed VISA or hVISA. We tested this hypothesis in experimental endocarditis.¦METHODS: Rats with aortic valve infection due to the vancomycin-susceptible (MIC 2 mg/L), methicillin-resistant S. aureus M1V2 were treated for 2 days with doses of vancomycin that mimicked the pharmacokinetics seen in humans following intravenous administration of 1 g of the drug every 12 h. Half of the treated animals were killed 8 h after treatment arrest and half 3 days thereafter. Population analyses were done directly on vegetation homogenates or after one subculture in drug-free medium to mimic standard diagnostic procedures.¦RESULTS: Vancomycin cured 14 of 26 animals (54%; P<0.05 versus controls) after 2 days of treatment. When vegetation homogenates were plated directly on vancomycin-containing plates, 6 of 13 rats killed 8 h after treatment arrest had positive cultures, 1 of which harboured hVISA. Likewise, 6 of 13 rats killed 3 days thereafter had positive valve cultures, 5 of which harboured hVISA. However, one subculture of vegetations in drug-free broth was enough to revert all the hVISA phenotypes to the susceptible pattern of the parent. Thus, vancomycin selected for hVISA during therapy of experimental endocarditis due to vancomycin-susceptible S. aureus. These hVISA were associated with vancomycin failure. The hVISA phenotype persisted in vivo, even after vancomycin arrest, but was missed in vitro after a single passage of the vegetation homogenate on drug-free medium.¦CONCLUSIONS: hVISA might escape detection in clinical samples if they are subcultured before susceptibility tests.