The genetic mechanisms underlying the specification of thymic epithelial (TE) lineage cells are poorly understood. Foxn1 is an early specific marker of thymic epithelial cells (TECs) in the third pharyngeal pouch (3PP) and is required for development of all mature TE lineage cells but does not specify the TE lineage. The upstream regulators of Foxn1 are currently unknown, however evidence points to a potential role for Pax1 and Pax9. While the thymus phenotypes of the Pax1-/- and Pax9-/- mutant mice have been investigated in detail and TECs in these mice are known to express Foxn1, the possibility of functional redundancy exists and the compound mutants of these genes have not been studied. Therefore, the aim of this thesis was to test the hypothesis that Pax1 and Pax9 are required for TE lineage specification and regulation of Foxn1 expression. This hypothesis was addressed by analysis of thymus development and TEC function in Pax1/Pax9 compound mutant mice. The data presented in this thesis indicates that prenatally, Pax1 and Pax9 cooperatively regulate thymus organogenesis, such that the size, structure and location of the thymus is affected in a Pax1/Pax9 gene dosage-dependent manner, and the Pax1unex/unexPax9lacZ/lacZ embryo is functionally athymic. Furthermore, they establish that the thymic rudiment of Pax1unex/unexPax9lacZ/lacZ embryos does not express Foxn1, establishing that Pax1 and Pax9 are required together for the initiation of Foxn1 and suggesting they are required to specify the TEC lineage. Postnatally, enlarged blood vessels observed in the Pax1unex/unex thymus suggested a role for Pax1 in vascularisation of the thymus. In addition, the effect of loss of one or more Pax1/Pax9 alleles on the expression of Foxn1 and other genes known to regulate TEC development or function was assessed. These data demonstrate that Pax1 and Pax9 co-operate to regulate Foxn1 in a dosage-dependent manner. Furthermore, Pax1 and Pax9 appear to negatively regulate both Hoxa3 and Vegfa, providing a possible explanation for the enlarged blood vessels in the postnatal Pax1unex/unex thymus. Finally, an inducible and reversible recombinase-mediated cassette exchange system that will allow the knockdown of Pax1 and Pax9 at defined time points during development has been established, that has the potential to test the function of these genes during thymus organogenesis and in the postnatal thymus.

Thesis--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 60-90).

Thymic epithelial cells (TECs) are indispensable for the development of T cells in the thymus. Two subtypes of TECs exist in the thymus, medullary mTECs and cortical cTECs. Both mTECs and cTECs originate from endodermal thymic epithelial progenitor cells (TEPCs) in the embryo, but how the differentiation of TEPCs is regulated is not well understood. The aims of this thesis were to establish the role of Notch signalling in TEPC differentiation, and how it interacts with known regulators such as FOXN1 and the NFκB pathway. Gene expression data showed that Notch is active in TEPCs and exhibits a correlation with the mTEC lineage. Loss of Notch function led to a significant reduction in the number of mTECs in the thymus, and this can be attributed to aberrant mTEC specification. Furthermore, the duration of Notch activity in determining mTEC number appears limited to the early phase of organogenesis, and precedes RANK/NFκB mediated mTEC proliferation. Gain of Notch function resulted in a considerable shift to a primitive, TEPC-like phenotype, and subsequently a latent increase in mTEC frequency. Finally, transcriptomic and functional analyses pointed to a cross-repressive mechanism between Notch and FOXN1 in TEPCs. Taken together, these results identified Notch as a novel regulator of mTEC specification, likely through maintaining the potency of fetal TEPCs, a prerequisite for mTEC lineage commitment.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18160号 / 医博第3880号 / 新制||医||1003(附属図書館) / 31018 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 生田 宏一, 教授 宮地 良樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13160号 / 論医博第2147号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 杉田 昌彦, 教授 椛島 健治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

Les tumeurs épithéliales thymiques (TET) humaines sont rares (250 - 300 cas/an en France). On distingue les thymomes de type A, AB ou B d'évolution lente avec une survie actuarielle > 95% à 5 ans pour les stades précoces et les carcinomes thymiques d'évolution plus sévère avec une survie actuarielle à 5 ans < 20% pour les stades IV. La pierre angulaire du traitement des TET est l'exérèse chirurgicale complète, facteur pronostique le plus significatif identifié à ce jour. Les récidives des TET, essentiellement pleurales pour les thymomes et générales pour les carcinomes thymiques, sont de prise en charge complexe. Les avancées thérapeutiques sont limitées notamment par l'absence de modèles d'étude de la cellule épithéliale thymique tumorale. Dans le cadre d'une prise en charge multidisciplinaire des récidives pleurales métastatiques, nous avons développé la pleurectomie de cytoréduction associée à une chimio hyperthermie (cisplatine/ mitomycine ; 42°C) intra thoracique (CHIT) pour la prise en charge des métastases pleurales de thymome. Chez des patients sélectionnés (n=19), la médiane de survie sans récidive était de 53 mois et les survies actuarielles à 1 an et 5 ans étaient respectivement de 93% et 86%. Cette technique chirurgicale innovante a permis de développer une alternative à la morbide pleuro pneumonectomie. L'efficacité de la CHIT pose des questions sur le rôle de l'hyperthermie et sur le type de chimiothérapie à associer. Avec pour objectif d'améliorer la prise en charge des patients, la connaissance de la biologie tumorale thymique et l'identification de potentielles cibles thérapeutiques sont des voies de recherche importantes pour améliorer la survie des patients. Nous avons développé des cultures de cellules épithéliales thymiques dérivées in vitro de 12 TET (11 thymomes A, AB ou B et un carcinome thymique), caractérisées par leur potentiel prolifératif et leur expression de cytokératine. La voie PI3K / Akt / mTOR joue un rôle clé dans de nombreux cancers ; plusieurs études de phases I / II ont rapporté un effet positif des inhibiteurs de mTOR pour le contrôle de l'évolution du thymome chez les patients. Nous avons mis en évidence l'expression et l'activation des effecteurs mTOR, Akt et P70S6K dans les thymomes et dans les cellules épithéliales thymiques dérivées in vitro. Nous avons montré l'efficacité de la rapamycine, inhibiteur de mTOR, à réduire la prolifération cellulaire (30%) sans induire de mort cellulaire. Nos résultats suggèrent que l'activation de la voie Akt / mTOR participe à la prolifération cellulaire associée à la croissance tumorale. Nous avons établi un nouvel outil permettant l'étude de la dérégulation cellulaire au cours des thymomes. Dans un contexte de tumeurs rares, ces cellules permettront d'aborder des études mécanistiques in vitro et de tester l'efficacité de drogues anti tumorales / Human thymic epithelial tumors (TETs) are rare (250 – 300 cases/ year in France). We distinguish thymomas (A, AB and B subtypes) with indolent evolution (5 years actuarial survival in early stages >95%) and more aggressive thymic carcinomas (5 years actuarial survival <20% in stage IV). Surgical complete resection when feasible is the corner stone of a multimodal therapy and the most significant factor on survival. Relapse of TETs principally in pleura for thymomas (75%) and general for thymic carcinomas are difficult to treat. Therapeutics advances are limited given the lack of studies models of tumoral thymic epithelial cell. In a multidisciplinary approach for the treatment of metastatic pleural relapse of thymomas we developed an innovative surgical technique: cytoreductive pleurectomy associated with hyper thermic intra pleural chemotherapy (Cysplatin/ Mitomycin; 42°C) (ITCH). In selected patient (n=19), ITCH provides an efficient alternative to the morbid pleuro pneumonectomy. The median of free disease survival was 53 months, one year and five years actuarial survival were respectively 93% and 86%. However, the effectiveness of ITCH procedure questions on the played role ok hyperthermia, on the choice of chemotherapy association. With the aim to improve TETs therapies, the knowledge of TETs biology to identify potential target therapies is currently challenging. We developed an in vitro study model of tumoral thymic epithelial cells derived from 12 TETs (11 A, AB and B thymomas and one thymic carcinoma) characterized by their proliferative abilities and the cytokeratin expression. The PIK3 / Akt / mTOR pathway is implicated in numerous cancers. Several phase I, II studies advocate the potential role of mTOR inhibitors in the control of the metastatic disease. We highlighted the expression and the activation of mTOR, Akt and P70S6K effectors in TETs and in thymic epithelial cells in vitro derived. We showed the efficacy of rapamycin (mTOR inhibitor) in the inhibition (-30%) of in vitro cell proliferation without cell death induction. Our results suggest the implication of the PIK3 / Akt / mTOR pathway in the tumoral cell growth. We established a new tool to study cell dysregulation in TETs. In the context of rare tumors, these cells could allow in vitro mechanistic studies and test the efficacy of new anti tumoral therapies

T lymphocytes are an essential component of the adaptive immune response and are highly versatile in function. Each T cell has a unique T cell receptor that can recognize an antigenic peptide in the context of the major his to compatibility complex (MHC) encoded molecules, thus offering a high degree of specificity to the immune response. T cells play a central role in the development of an effective host immune response and the quantitative and qualitative regulation of the T cell response is critical. T cells develop in the thymus, an important primary immune organ, where immature thymocytes undergo differentiation and maturation. Through the process of thymic differentiation, immature cluster of differentiation (CD)4-CD8- thymocytes progress to a CD4+CD8+ stage and are subjected to positive and negative selection to give rise to MHC restricted, single positive CD4+ or CD8+ naive T cells that emigrate from the thymus and populate the peripheral lymphocyte pool. Thymic atrophy is well known to occur naturally during the process of aging with thymocyte depletion and reduced thymic output. Along with age associated changes leading to atrophy, the thymus is exquisitely sensitive to starvation and several stresses. In addition, thymic atrophy is a characteristic feature during several viral, bacterial and parasitic infections. Egress of immature thymocytes, loss of thymic populations due to sensitivity to glucocorticoids and cytokine modulation, etc. have been variously proposed to be involved in this process. However there is limited understanding on the numerous mechanisms involved and the crosstalk between these diverse pathways. In this study, a model for thymic atrophy during acute Salmonella enterica serovar Typhimurium (S. typhimurium) infection was developed. S. typhimurium is a Gram negative bacterium that resides and grows in intracellular compartments within host cells. It causes gastroenteritis in humans but leads to typhoid like disease in mice, similar to that caused by S. typhi in humans. Initially, it was established that acute infection of C57BL/6 mice with 108 CFU S. typhimurium, via the oral, i.e. the physiological, route of infection leads to extensive depletion (8-10 fold) of thymocytes in an infection-dependent manner. Infected mice had higher CFU burden in the Peyer’s patches, spleen, liver, and mesenteric lymph node (MLN) as compared to the thymus. The thymic atrophy was dependent upon the infection caused by live S. typhimurium since oral feeding of mice even with higher doses (1010 CFU) of heat-killed bacteria did not lead to thymic atrophy. The susceptible populations in the thymus were identified by staining for expression of CD4 and CD8 on cell surface using specific monoclonal antibodies tagged to fluorophores, e.g. Fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively. The double labelled samples were analyzed by flow cytometry. Interestingly, significant death of CD4+CD8+, the major population of thymocytes, but not single positive thymocytes or peripheral lymphocytes (MLN and spleen cells), was observed at later stages during infection. To gain greater understanding of the processes involved, the mechanisms leading to thymic atrophy were investigated. To this purpose, small molecule inhibitors and mice lacking key molecules important for the immune response were utilized. Also, various assays to assess death of thymocytes, including analysis of death markers such as Annexin V based detection of membrane flipping and caspase activation were performed. I. The extrinsic death pathway involving Fas/FasL interactions is a major death pathway. Therefore, the expression and functional role of the components of the pathway in this model of thymocyte death was investigated. It was observed that thymocytes from infected mice expressed more Fas and Fas ligand (FasL) on their surface than cells from uninfected mice. To address the role of the death receptor, Fas, infection studies were performed with lpr mice that lack functional Fas expression. The depletion of CD4+CD8+ thymocytes in lpr mice was comparable to that in C57BL/6 mice indicating that it was independent of the Fas pathway. However, extensive loss of mitochondrial membrane potential was observed upon analysis with mitochondrial potential specific dyes MitoTracker Red and DiOC6. Most likely, the intrinsic death pathway involving mitochondrial depolarization is involved in this model of thymic atrophy. II. Since thymocytes are known to be sensitive to glucocorticoids both in vitro and in vivo, the involvement of the same in this model of thymic atrophy was assessed. The amounts of cortisol, a glucocorticoid, as detected by ELISA, were elevated during infection. To investigate the functional implication of the increase in cortisol, studies were performed using RU486, a glucocorticoid receptor antagonist. RU486 did not modulate cortisol amounts and treatment of mice with RU486 did not affect CFU burden or survival of mice. However there was a moderate rescue in the number of viable CD4+CD8+ thymocytes, with only a 3-4 fold drop as compared to the 8-10 fold drop in vehicle treated infected mice. III. As glucocorticoids appeared to play a partial role in this model, it was reasonable to assume that other pathways were also involved in the thymic atrophy. The quantitative and qualitative modulation of the cytokine milieu has a profound effect upon the thymus. In fact, inflammatory cytokines, Tnfα and Ifnγ, increased upon infection. In order to study the role of Ifnγ mediated inflammatory responses in this model, infection studies with Ifnγ-/- mice were performed. Ifnγ-/- mice had higher CFU and lower survival; however the drop in thymocyte numbers was 3-4 fold as compared to the 8-10 fold drop in the infected C57BL/6 mice, again indicating a partial involvement of the Ifnγ mediated pathways. In order to study the interactions, if any, between the two pathways mentioned above, corticosteroid signaling was blocked in the Ifnγ-/- mice with RU486. Upon infection, the number of CD4+CD8+ thymocytes was significantly higher in Ifnγ-/- mice treated with RU486 (~1.5 fold drop in viable thymocyte numbers) along with lower caspase 3 activity and mitochondrial damage. Importantly, cortisol amounts in infected Ifnγ-/- mice were comparable to those in infected C57BL/6 mice and the administration of RU486 did not modulate Tnfα and Ifnγ cytokine amounts in sera. Thus, the glucocorticoid and Ifnγ mediated pathways are parallel but synergize in an additive manner to induce death of CD4+CD8+ thymocytes during S. typhimurium nfection. IV. Although thymic atrophy is known to occur, a detailed characterization of cell surface changes in thymocyte populations has not been performed. To investigate this aspect, thymocytes and MLN cells from uninfected and infected animals were stained for cell surface expression of CD3, CD4, CD5, CD8, CD24, CD25, CD44, CD69, MHC I and MHC II. This analysis was initially performed by studying the changes in expression of these molecules within the total thymocyte and MLN populations. Although there was no change in the expression of CD25 and MHC II in the total thymocyte population upon infection, CD24 expression reduced, whereas, the expression of CD3, CD5, CD44, CD69 and MHC I increased. Notably, changes in the frequency of expression of CD3, CD69 and MHC I were observed before the development of extensive thymic atrophy. The depletion of majority of the CD4+CD8+ thymocytes enriches the mature CD4+ or CD8+ thymocyte population This was corroborated with the observation that, upon in vitro stimulation with PMA and Ionomycin (pharmacological agents used to activate T cells) the residual thymocytes from infected mice produced more IL2 compared to thymocytes from uninfected mice. Subsequently, cells were stained with anti-CD4-FITC, anti-CD8-PE and a third biotinylated antibody, which was detected by a streptavidin-APC conjugate, against one of the remaining six markers. This three colour analysis made it possible to determine the changes in the expression of the third marker in each of the CD4-CD8-, CD4+CD8+, CD4+ and CD8+ populations upon infection. Distinct differences were observed in the phenotypes of uninfected and infected CD4+CD8+ thymocytes and the latter were CD3high, CD5high, CD24low, CD69high and MHC Ihigh indicating that the surviving population had a possibly more mature phenotype. Also, the changes in the phenotypes of the thymocyte populations were dependent upon the extent of thymic atrophy as indicated by time course and CFU studies with C57BL/6 and BALB/c mice respectively. Finally, the roles of glucocorticoids, Ifnγ and Nos2 in modulation of expression of these markers during infection were addressed. Interestingly, the expression of CD3, CD24 and MHC class I significantly correlated with increase in the number of surviving thymocytes upon inhibition of glucocorticoids signaling and in Ifnγ-/- mice. The implications of these changes in the thymocyte surface phenotype during thymic atrophy are discussed. V. Finally, the roles of downstream signalling molecules in S. typhimurium induced thymic atrophy were studied. Although the MAP kinase family members, Erk, Jnk and p38 have been implicated to play a role in the positive and/or negative selection of thymocytes during development, their role in infection induced thymocyte depletion has not been studied. Interestingly, the amounts of Jnk and pJnk, but not p38, increased in thymocytes upon infection. Importantly, pJnk amounts increased predominantly in CD3-/low thymocytes during infection. Furthermore, inhibition of Jnk signalling, using a specific inhibitor SP600125, lead to an increase in survival of CD4+CD8+ thymocytes during infection due to multiple reasons: lowering of cortisol, Tnfα and Ifnγ amounts, and better maintenance of thymic architecture. Thus, inhibition of Jnk mediated signaling protected CD4+CD8+ and CD3-/low thymocytes from death during S. typhimurium infection. Overall, the main conclusions of this study are as follows: First, extensive analysis of the surface phenotype of cells during thymic atrophy throws light on the sensitive and resistant thymocyte populations, thus offering a potential predictive marker profile. Second, glucocorticoids, Ifnγ and, importantly, Jnk mediated signaling play functional roles in the death of immature CD4+CD8+ thymocytes during S. typhimurium infection. The mechanistic details uncovered in this study may be important in designing effective strategies for reducing thymic atrophy during other infections. In fact, enhancement of thymic output may lead to greater numbers and diversity of thymic T cell emigrants in the periphery which is likely to enhance host responses during infections.

T cells, which constitute an essential arm in the adaptive immunity, complete their development in the thymus through a process called thymopoiesis. However, thymic involution can be induced by a couple of factors, which impairs T cell functions and is slow to recover. Therefore, understanding how thymopoiesis is regulated may lead effort to accelerate thymic recovery and improve immune functions in thymocyte-depleted patients. In this project, we identified scavenger receptor BI (SR-BI), a high density lipoprotein (HDL) receptor, as a novel modulator in thymopoiesis. In mice, absence of SR-BI causes a significant reduction in thymus size after puberty and a remarkable decrease in thymic output. Consequently, SR-BI-null mice show a narrowed naïve T cell pool in the periphery and blunted T cell responses, indicating that the impaired thymopoiesis due to SR-BI deficiency leads to compromised T cell homeostasis and functions. The impaired thymopoiesis of SR-BI-null mice is featured by a significant reduction in the percentage of earliest T progenitors (ETPs) but unchanged percentages of other thymocyte subtypes, suggesting that SR-BI deficiency causes a reduction in progenitor thymic entry. Further investigations reveal that SR-BI deficiency impairs thymopoiesis through affecting bone marrow progenitor thymic homing without influencing the lymphoid progenitor development in bone marrow. Importantly, SR-BI-null mice exhibit delayed thymic recovery after sublethal irradiation, indicating that SR-BI is also required for thymic regeneration. Using bone marrow transplantation models, we elucidate that it is non-hematopoietic rather than hematopoietic SR-BI deficiency that results in the defects in thymopoiesis. However, SR-BI deficiency-induced hypercholesterolemia is not responsible for the impaired thymopoiesis. Using adrenal transplantation models, we found that absence of adrenal SR-BI is responsible for the impaired thymopoiesis, as shown by that adrenalectomized mice transplanted with SR-BI-null adrenal gland display reduced thymus size, decreased percentage of ETPs and delayed thymic regeneration compared with those transplanted with wild-type adrenal. Altogether, results from this study elucidate a previously unrecognized role of SR-BI in thymopoiesis. We reveal that SR-BI expressed in adrenal gland is critical in maintaining normal T cell development and enhancing thymic regeneration, providing novel links between adrenal functions and T cell development.