The thymus is a complex cellular structure made up of several interdependent cell types and is the primary site for T cell development. A population of fetal thymic epithelial cells (TEC), marked by MTS20 and MTS24, when grafted in vivo can generate a functional thymus containing all thymic epithelial cells and is capab,e of supporting T cell differentiation. Further analysis using in vivo grafting experiments have determined the endoderm as the sole origin for all major thymic epithelial subsets. These findings suggest the possibility that a bipotent thmic epithelial progenitor cell (TEPC) gives rise to both cortical and medullary epithelial compartments. The first ai of this study was to address whether bipotent mouse TEPC give rise to both medullary and cortical epithelial cell populations and to begin to establish a model of TEC differentiation through ontogeny. Its second aim was to start to define condidtions for maintaining functionally undifferentiated RTEPC in vitro. Finally, as little is knowth about the genetic regulation of human thymic development and TEC differentiation, I hve used comparative analysis to investigate the similaarities in expression of key regulations of thymus development between human and mouse, which will aid in the translation of mouse thmic research to human. the main findings of this thesis are i) that a bipotent thymic epithelial progenitor cell population that contribute to both medullary and cortical epithelial cell compartments exists in vivo, but is at low frequency even by E12.5. ii) That a unipotent progenitor population committed to a cortical epithelial cell fate is also present as early as E11.5. iii) That E11.5 TEC can be propagated in vitro in semi defined conditions, but appear to revert ro an early endodermal phenotype on prolonged culture and iv) that the genetic program regulating thymus organogenesis appears to be conserved between mouse and human. In addition I have defined the exact time of haematopoietic cell entry into the human thymus, and the time of entry of mesenchymal cells and the mesenchymal distribution pattern throughout human thymic ontogeny. I also establish the time of onset of differentiation and maturation of hTEC, and using known mouse TEPC markets, show the existence of a population of Claudin4+Ueal+ hTEC at week 8, presumed equivalent to the precursor cells for the AIRE1+ subpopulation of medullary TEC in the mouse.

Appropriate early microbial colonisation of the gut dramatically affects the incidences of infectious, inflammatory and autoimmune diseases, a concept embraced by the ‘Hygiene Hypothesis’. Hence, factors which influence microbial colonisation and succession during early life have important implications for both human and animal health. This thesis addressed some of the basic principles of the ‘Hygiene Hypothesis’ by investigating how early-life environment impacts on microbial diversity of the adult gut, and how this in turn affects development of the mucosal immune system. The developing pig was used as an experimental model to study host-microbe interactions in early life. The mucosa-adherent microbiota was characterised using both 16S rRNA gene libraries and DGGE and the corresponding host response determined using Affymetrix microarray technology. Initial colonisation of animals derived from two rearing systems with distinct levels of hygiene (outdoor ‘organic/rural’ and indoor ‘hygienic/urban’) was characterised by a highly diverse microbiota that was similar between treatments. These early microbial colonisers were unrelated to those found colonising the adult gut. Establishment of the adult microbiota required continual environmental exposure which led to microbial succession and stabilisation at the adult life-stage. A natural microbiota dominated by Firmicutes reduced both microbial diversity and the number of pathogenic micro-organisms within the gut ecosystem. It also facilitated enhanced innate immune responses. The impact of long-term antibiotic use on the mucosal microbiota was also investigated. This resulted in a sustained disturbance of the gut microbiota which contained phylotypes of a pathogenic phenotype and correlated with altered host immune responses. The work presented supports the concept of the ‘Hygiene Hypothesis’ and has identified windows of opportunity during early life when appropriate manipulation of the mucosal microbiota may enhance immune function and natural disease resistance of the host. Prophylactic administration of broad-spectrum antibiotics in early life is not beneficial to the host.

Pas de résumé / Background: Mendelian mutations causing monogenic enteropathies are identified in an increasing number of genes and are responsible for either chronic inflammatory diseases (frequently called VEO-IBD for very early-onset inflammatory bowel diseases) or for congenital diarrheal disorders (CDD). Management of many patients with monogenic enteropathies requires difficult therapeutic decisions and heavy treatments, such as hematopoietic stem cell transplantation for VEO-IBD patients, or total parenteral nutrition and intestinal transplantation for CDD patients. Early molecular diagnosis is crucial to define the most pertinent treatment and increase life expectancy. During my thesis, I introduced in the laboratory big data management tool (e. g. online dedicated database) and applied next-generation sequencing tools (whole exome sequencing (WES) and targeted gene panel sequencing (TGPS)) to a cohort of patients suffering from monogenic enteropathies in order to characterize them phenotypically and genetically. Methods: My thesis was divided in 4 steps. In Step 1, patients (n=216 in January 2016, n=260 in August 2016) recruited through a French research protocol (Immunobiota, 12 centers) and European network (GENIUS, 33 centers) were phenotypically characterized through an online dedicated database. Following precise phenotyping, molecular diagnoses were obtained by Sanger sequencing of candidate genes suggested by functional tests in Step 2. Step 3 was the adaptation of WES for our cohort of patients (59 patients were sequenced in trio and 11 sequenced by themselves or in duo) and lastly, in Step 4, TGSP was designed and applied to our cohort (173 patients without a molecular diagnosis). Findings: The cohort gathered 57 patients including 22 with a molecular diagnosis in January 2012, and 216 patients including 70 with a diagnosis in January 2016, corresponding to a global diagnosis rate of 1/3. Approximately 50 new patients are recruited each year, with blood samples taken from each patient, both parents and siblings. During this period, 11 diagnoses were obtained by a phenotype-based approach, with identification of mutations notably in IL-10R (4 patients) and XIAP (4 patients). Eleven patients obtained a genetic diagnosis by WES including two siblings with a MALT1 deficiency responsible for an IPEX-like syndrome. Because of the increasing number of genes involved in monogenic enteropathies, we developed, in collaboration with Genomics, Bioinformatics and Translational Genetics platforms from the Institut IMAGINE, a custom-made TGPS gathering 68 genes responsible for either VEO-IBD or CDD. The sequencing of all negative patients (n=173) on this panel allowed to identify 28 new diagnoses (among which 8 were made in patients included before 2012). Interpretation: This work lead to the identification of the genetic diagnosis in 1/3 patients. The close investigations of phenotype-genotype correlations highlighted frequent overlaps among monogenic enteropathies. Following completion of this work, we suggest to use TGPS as a first-line genetic test in addition to a precise phenotyping of the patient. Depending on the results, TGPS will either reach an early molecular diagnosis crucial to optimize treatments in a cost-effective manner, or allow to perform further genetic analysis notably by WES.

Au cours du développement d’un cancer, la tumeur se forme dans un environnement adapté à sa croissance appelé microenvironnement tumoral. Celui-ci est constitué d’un réseau complexe qui orchestre la progression tumorale et dans lequel sont présents différents types cellulaires, des facteurs solubles ainsi que des composants de la matrice extracellulaire. Parmi les cellules du microenvironnement tumoral, on trouve les cellules du système immunitaire. Certaines ont la capacité de reconnaître et d’éliminer directement les cellules cancéreuses, tandis que d’autres promeuvent la croissance tumorale et la dissémination métastatique. Les lymphocytes T γδ ont longtemps été considérés uniquement comme des acteurs anti-tumoraux, agissant grâce à leur fonction cytotoxique et à leur sécrétion précoce d’interféron (IFN)-γ. Dans de plus en plus de travaux récents, l’idée que les lymphocytes T γδ soient, également, des acteurs pro-tumoraux est mise en évidence. Leurs fonctions pro-tumorales sont principalement attribuées à leur production d’interleukine (IL)-17, favorisant l’angiogenèse et le recrutement de cellules myéloïdes suppressives (MDSCs). Le mécanisme induisant la polarisation des T γδ vers la production d’IL-17 plutôt que d’IFN-γ restait à préciser. Nous avons identifié la synthétase de l’oxyde nitrique de type 2 (NOS2) comme candidat potentiel étant donné son action exercée sur la production d’IL-17 par les lymphocytes T αβ CD4+ (Th17). Nous montrons que les cellules T γδ expriment NOS2 dans les tumeurs primaires de mélanome chez les patients et également chez la souris, dans un modèle de développement spontané de mélanome. Nous avons identifié que l’expression de NOS2 endogène était un inducteur clé de la sécrétion d’IL-17 par les T γδ dans le mélanome, ce qui a pour conséquence de recruter les MDSCs dans la tumeur primaire et d’induire la dissémination métastatique. En parallèle, les T γδ exprimant NOS2 produisent moins d’IFN-γ et présentent, in vitro, des capacités de lyse des cellules tumorales réduites en comparaison avec les T γδ déficients pour NOS2. Nous émettons l’hypothèse que le microenvironnement tumoral puisse fournir les facteurs nécessaires à l’activation des T γδ (comme l’IL-1β, l’IL-6 et ou la reconnaissance d’un antigène par le TCR) pour permettre l’expression endogène de NOS2 par les cellules T γδ. En plus de favoriser la polarisation des T γδ en effecteurs pro-tumoraux, nous montrons que NOS2 favorise leur expansion par un mécanisme dépendant de l’IL-2. En effet, les T γδ déficients pour NOS2 montrent des capacités de prolifération et un métabolisme glycolytique réduits qui sont restaurés par l’ajout d’IL-2 exogène. / Consequences of the expression of nitric oxide synthetase type 2 (NOS2) on the functions of γδ T lymphocytes during the development of melanoma

The early B cell response to antigen is analyzed, probing the mechanisms of divergent differentiation into plasmablasts or germinal centre (GC) B cells. Heterozygous QMxB6 mice, in which 5% of B cells are specific for the hapten 4-hydroxy-3-nitrophenol (NP), were immunized with NP-Ficoll. Varying the antigen dose altered the proportion of B cells that entered the extra follicular or GC responses and the amount of class switch recombination (CSR). The expression of phenotypic markers and switched antibody protein in parallel with gene array analysis and single cell real time RT-PCR in responding B cells was used to identify GC and plasmablast precursors and when and where CSR occurred. The results suggest CSR occurs in B blasts before GC B cells or plasmablasts emerge in both thymus dependent and thymus independent type II responses. The protein IRF4, which is essential for CSR and plasmablast differentiation, is expressed in all responding cells immediately after immunization and selectively upregulated to high levels in plasmablasts. A hierarchy of gene expression was identified as B cells differentiate into plasmablasts whereby high IRF4 mRNA expression precedes CD138 protein, which in turn precedes Blimp1 expression

Germinal centres (GCs) are the sites where V-gene hypermutation and B cell selection are taking place. Testing specificity and affinity of GC B cell receptor by interaction with antigen on follicular dendritic cells (FDCs) may be an important selection process to select high affinity B cell clone. As antigen on FDC is present in the form of antigen-antibody immune complex, GC B cells are expected to have to compete with antibody to get access antigen. Initially this antibody will be of low affinity. However, during the course of an immune response, this affinity may increase. We have tested this competitive selection model by following the replacement of antibodies in the GC over the course of an immune response. The speed of this replacement is dependent on affinity. Antibody added during an ongoing GC reaction can replace antibody in the GC, but only, if it is of high enough affinity. Presence of high or low affinity antibodies on FDC influences centrocyte selection, leading to variations in apoptosis within the GC, serum affinity, and plasma cell output. Parallel in silico experiments support the idea that a dynamic GC selection threshold, dependent on the affinity of GC output cells increases affinity maturation, because it enhances selection efficiency over a longer period during the course of a GC reaction. A dynamic selection threshold may explain the termination of the GC reaction, when affinity of new B cell variants is not sufficient to overcome the affinity of antibodies produced outside the GC. IRF4 is essential for the plasma cell differentiation and Ig class switch. IRF4 mRNA and protein rapidly upregulate within one hour after naive B cells get stimulation with NP-Ficoll in QM×C57BL/6 mice, and then activated B blasts expressing intermediate level of IRF4 either go into the red pulp to form the early extrafollicular response by upregulating high level of IRF4, or travel into the follicle to differentiate into GC founding cells. IRF4 completely shuts down when cells becomes proliferating centrocytes. But IRF4 expresses again in centorcytes, which have been committed to differentiate into the plasmablasts. Its high level expression shows the GC emigrants. Here IRF4 is selected as the marker for the early plasmablats appearance on the GC-T zone interface at the beginning of the GC reaction. And further experiments by using cytokines such as IL-6, IL-10, IL-21, costimulatory signals OX40, CD30 deficient mice show that these signals can affect the development of these early IRF4\(^+\) plasmablasts on the GC-T zone interface in the TD antigen response.

The role of the alternative NF-\(\kappa\)B pathway in the development of secondary lymphoid organs (SLOs) has been well described. Tertiary lymphoid organs (TLOs) include intestinal cryptopatches (CPs) and isolated lymphoid follicles (ILFs). This thesis investigates the role of the alternative NF-\(\kappa\)B pathway in the development of colonic ILFs, using the p100\(\Delta\) mouse model, where the alternative pathway is constitutively active. We present compelling data that p100\(\Delta\) mice develop significantly more lymphoid aggregates in the colon, compared with WT littermates, and provide several lines of evidence showing that these aggregates are analogous to ILFs. Additionally, we demonstrate that in the p100\(\Delta\) a significant increase in the numbers of B and T cells, and DCs in the colon and show alterations in the subsets of colonic T cells and DCs. We also present evidence that constitutively active NF-\(\kappa\)B2 p100 in LT\(\alpha\) deficient mice induces recovery of B and T cell segregation in the spleen. Most strikingly, we show recovery of iLNs and mLN development in one of five \(p100\)\(\Delta\)\(LT\)\(\alpha^{-/-}\) mice generated. These findings demonstrate that the alternative NF-\(\kappa\)B pathway plays an important role in not only SLO development and splenic organisation, but also in the development of TLOs, specifically colonic ILFs.

The autoimmune regulator (AIRE), a key player in negative selection of developing thymocytes, acts as a transcriptional regulator, inducing the expression of tissue restricted antigens (TRA) within medullary thymic epithelial cells in a process known as promiscuous gene expression (PGE). Here we demonstrate how AIRE influences PGE through a direct impact on post-translational modifications of core histones which are associated with the regulation of transcription. Through native chromatin immunoprecipitation on thymic epithelial cells transfected with AIRE, we show how in vitro, TRA are enriched in active acetylation and methylation of core histones, yet retain silencing modifications. Furthermore, across a cluster of AIRE-regulated genes, histone modifications were deposited across the entire domain, dependent upon the expression profile of each gene, suggesting a role for domain-wide epigenetic regulation by AIRE. Extension of these studies in vivo, utilising the recently developed carrier ChIP technique, allowed examination of the epigenetic status of TRA throughout the thymic developmental pathway. We report how poised TRA are marked with combinations of active and silent modifications early in thymic development and that the chromatin signatures re-organise as the cells differentiate. The epigenetic patterning differs on a gene by-gene basis, however their significance is implied upon disruption to normal development as the predictive pattern of modifications is lost.

Fibroblasts are the most abundant cell type of the stroma, producing extracellular matrix components which provide mechanical strength to tissues. Recent work has shown that fibroblasts are not simply passive structural cells but active participants in the immune response. In this study fibroblast heterogeneity within the murine lymphoid system was investigated by analysing gene and protein expression. Fibroblasts were isolated from a range of lymphoid and peripheral sites. Our findings show that fibroblasts grown from different sites show heterogeneous gene and protein expression and are functionally different in their response to lymphotoxin α treatment in vitro and in their capacity to recruit host leucocytes in vivo. A novel in vivo functional assay has been developed using a collagen sponge as a three-dimensional structure in the kidney capsule transfer model, to assess the ability of fibroblasts to form and maintain lymphoid structures. We conclude that murine fibroblasts isolated from lymphoid tissues, display site specific features. They are phenotypically distinct with site specific gene and protein expression profiles. This suggests that murine lymphoid fibroblasts have positional memory and help impart anatomical identity. An important implication of these findings is the effect this diversity may have in conveying site specificity to immune responses.

No description available.