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In this study, we have analyzed the steady-state kinetics of B lymphocytes in mouse and rat bone marrow using previously published experimental data. Over many years, Prof D.G. Osmond and his colleagues have built up a scheme of B cell development in mouse bone marrow based on the sequential expression of markers associated with the B lineage. The earliest precursor B cells comprise three populations of proliferating pro-B cells, i.e. early, intermediate, and late pro-B cells. The subsequent populations comprise pre-B cells that give rise to nondividing B lymphocytes expressing surface IgM. / In our analysis, we have checked the available published data for consistency with the proliferation of precursor B cells and their death via apoptosis at certain stages of cell development. We made an extensive summary of the existing data on the various B cell precursors and organized it into a comprehensible framework. We built a mathematical model for the proliferation and differentiation of mammalian B lymphocytes in laboratory mice and rats and estimated all of the parameters to explain the existing steady state data. In this context, mathematical modeling acts as a useful tool to analyze hypotheses and experimental results concerning the steady state numbers of B lymphocytes.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.

Les cyclines D jouent un rôle essentiel dans les mécanismes du cycle cellulaire. Cette famille de protéines est composée de trois membres (D1,D2,D3) qui partagent un domaine très homologue de la « cyclin box » (codée par les exons 1-3). Ce domaine est responsable de leur activité redondante dans la phosphorylation de la protéine du rétinoblastome lors de l'association avec les kinases cycline-dépendantes CDK4/6. Parmi les trois cyclines, la cycline Dl, bien que faiblement exprimée dans les lymphocytes, est la cycline la plus impliquée dans les cancers lymphoïdes ou elle aurait une fonction de facteur de transcription indépendante de Cdk. Etant donné qu'après stimulation antigénique, les lymphocytes T et B ont une capacité remarquable de division, essentielle à la génération d’une réponse immunitaire efficace, nous avons porté un intérêt particulier au rôle des cyclines D dans la lymphopoïèse. Pour étudier le rôle de la cycline Dl dans la différenciation des lymphocytes, nous avons utilisé des souris déficientes pour les exons 1,2,3 de la « cycline box » Dl mais conservé les exons 4 et 5. Étonnamment, ces souris présentaient des phénotypes très différents que nous avons subdivisés en quatre groupes. Dans le groupe I, les souris avaient un thymus réduit car la différenciation de la lignée lymphoide est bloquée à un stade très précoce, avec un faible nombre de cellules progénitrices (CLP) dans la moelle osseuse. Dans le thymus, les progéniteurs des thymocytes (ETP) étaient pratiquement absents et les précurseurs CD4CD8CD3 (TN) immatures essentiellement constitués par des cellules CD44*CD25 (TN1) et CD44*CD25+ (TN2) les plus immatures. De plus, les CD4*CD8* (DP) qui donnent naissance aux thymocytes matures CD4+ et CD8+ étaient présents en très faible quantité. Dans la moelle osseuse, on observe un blocage majeur dans la différenciation de la lignée B au stade pré-proB. Dans les ganglions, la forte réduction du nombre de lymphocytes T observée était liée au faible nombre d'ETP et à l’absence du récepteur aux chimiokines CCR7. Dans le groupe II, les souris présentaient une diminution moins sévère des ETP et une atrophie modérée du thymus. La différenciation était bloquée à un stade ultérieur, soit dans la transition des étapes TN3 à TN4. Dans la moelle osseuse, les lymphocytes B ont subi un blocage partiel au stade pré-proB et une réduction des cellules pré-B. Le nombre de CLP est également réduit, mais dans une moindre mesure que dans les souris du groupe I. dans les groupes III et IV, les souris ont une répartition normale des thymocytes mais présentaient une augmentation du compartiment ETP. Alors que les souris du groupe III contenaient un nombre normal de thymocytes, les souris du group IV présentaient une hyperplasie thymique. Par ailleurs, en comparaison avec des souris normales, bien que la différenciation des lymphocytes B soit normale, on observe dans les deux groupes une augmentation des CLP et des progéniteurs hématopoïétlque (LSK). L’implication de la cycline Dl dans la transition de G1 à S nous a conduit à analyser les divisions cellulaires in vivo. De manière surprenante, les souris du groupe I étaient fortement dépourvues de cellules en cycle dans tous les compartiments lymphoïdes, ce qui peut expliquer les blocages de la différenciation lymphoide. Par contre, dans les trois autres groupes, on observe une augmentation du nombre de divisions cellulaires. Ces résultats différents peuvent être dû à l'expression ou l'absence d'une protéine Dl tronquée qui contient cependant les exons 4-5. Alors que ces ARNm tronqués ne sont pas détectables dans les souris de groupe I, on observe des niveaux élevés d'expression dans les autres groupes. De plus, nous avons observé une corrélation entre l'absence d’expression des exons 4-5 et la très faible expression des gènes CCND2 et CCND3, ce qui attribue à cette protéine tronquée un rôle prépondérant dans la régulation des cyclines D et permet d’expliquer l'aplasie profonde et… / D Cyclins play an essential role connecting exogenous stimulation to the intrinsic cell cycle machinery. This family of proteins is composed of three members sharing a highly homologous domain, the cyclin box (coded by exons 1-3), which is responsible for their redundant role in the phosphorylation of the retinoblastoma protein upon association with cydin-dependent kinases Cdk4/6. Both mature T and B-cells have a remarkable division capability after antigen stimulation, essential to the generation of efficient immune responses, raising the interest of D Cyclins in lymphopoiesis. Cyclin Dl, although weakly expressed by lymphocytes, is the D Cyclin most commonly implicated in lymphoid cancers and as having a Cdk-independent transcriptional role. To study the role of Cyclin Dl, we used mice deficient for the Dl cyclin box but sparing exons 4-5. Surprisingly, individual mice have very different phenotypes that we subdivided into four arbitrary groups. Group I mice show the most precocious block in lymphoid lineage differentiation, illustrated by a low cellularity of common lymphoid progenitor cells (CLP). The thymi showed very few CD4*CD8*, double positive (DP) cells, while the CD4 CD8TCR, triple negative (TN) populations were found to be mostly constituted by the early CD44*CD25' (TNI) and few CD44*CD25* (TN2). TNl's early thymocyte progenitors (ETP) were virtually absent. At the B-cell lineage level in the bone marrow (BM) there was a major block in pre-proB differentiation. The number of peripheral T-cells was severely reduced, mainly in LN, since group I T-cells lack CCR7 expression. Group II mice presented moderate thymus atrophy. The block on TN differentiation occurs at a later stage, i.e., in the TN3 to TN4 transition, and the TNI population was characterized by a less severe depletion of the ETP. Group II mice showed a partial pre-proB block and a reduction in pre-B-cells. CLPs were also reduced but to a lesser extent than in group I mice. Group ill and group IV mice appear to have a normal thymocyte population distribution but showed an increase on ETP compartment. Group IV mice displayed thymic hyperplasia while group III mice possessed normal thymus cellularity. B-cell differentiation on both groups appeared to be normal but BM precursors had an increase in both CLP and early haematopoietic progenitor's (LSK) levels as compared with wild type mice. Cyclin Dl involvement in G1 to S transition led us to analyse in vivo division rates. Strikingly, group I mice were virtually devoid of cycling cellsin all lymphoid compartments, explaining why lymphoid lineage cells do not differentiate in these mice. In contrast, in all other groups we observed an increased BrdU incorporation. These contradicting phenotypes correlated with the expression or absence of a truncated Dl protein coded by exons 4-5. The presence of the cyclin Dl truncated mRNA was not found in group I mice but high levels of expression are consistently observed in the remaining groups. In the absence of the Dl truncated protein only trace values of Cyclins D2 and D3 were found, highlighting the role of this protein as a master D cyclin regulator, which further supports the profound aplasia and arrest in lymphoid lineage division on cells that predominantly express Cyclin D2. These results suggest that, while the function of the Dl cyclin box is redundant, the regulatory domain coded by exons 4-5 is fundamental for lymphopoiesis. Full Dl protein was also eliminated by RNA interference both in vitro and in vivo. These experiments reproduced the phenotype of group I mice. We have developed a lentiviral vector with a truncated Dl (exons 4-5) and conditional knockout (KO) mice by floxing exons 4-5 of cyclin Dl. These tools will allow us to show Cyclin Dl Cdk-independent role as a transcription regulator in lymphopoiesis and to attribute this function to exons 4-5. Understanding how exons 4-5 regulate different transcription factors might be a key in…

Mutation, whether spontaneous or induced, is the premier tool for understanding gene function. One approach is to create mutations in a specific gene, and then use the resulting cell or organism to search for a phenotype. An alternative is to create mutations at random, and focus first on the identification of phenotypes. The mutation that underlies a phenotype can then be tracked down, forming the foundation of testable hypotheses. Using random chemical mutagenesis in mice, I have identified 20 heritable phenotypes affecting either the innate or adaptive branches of immunity. The genetic basis of 18 of these phenotypes was solved, caused by mutations in at least 16 unique genes. Five of these genes were not previously known to be involved in immunity, and a detailed analysis of four of them is provided in this thesis. These include genes encoding the following proteins: the inactive rhomboid protease iRhom2, which is specifically required for the secretion of tumour necrosis factor alpha; the hypothetical phospholipid flippase ATP11C, required for B cell development in the adult bone marrow; the folliculin-interacting protein FNIP1, also required for B cell development; and the zinc finger transcription factor ZBTB1, essential for the development of all lymphocyte lineages. These findings uncover new entry points for the understanding of mammalian immunity, and highlight the value of mouse forward genetics in the understanding of mammalian phenomena in general.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25205号 / 医科博第161号 / 新制||医科||11(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 伊藤 貴浩, 教授 齋藤 潤 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Les vols spatiaux sont source de nombreux stress conduisant à un affaiblissement du système immunitaire. L’efficacité de ce système repose notamment sur la diversité des répertoires de récepteurs aux antigènes présents à la surface des lymphocytes B (BCR) et T (TCR) permettant de reconnaitre un grand nombre d’antigènes différents. Au cours de cette thèse, j’ai étudié la diversité des récepteurs à l’antigène dans trois modèles animaux différents : l’amphibien Pleurodeles waltl, le modèle murin de suspension anti‐orthostatique (micropesanteur simulée) et le modèle murin CUMS (pour « Chronic Unpredictable Mild Socio‐environmental stressors ») qui fait appel à des stress sociaux et environnementaux chroniques similaires à ceux rencontrés lors des vols spatiaux. L’étude des répertoires de chaînes lourdes d’anticorps IgM et IgY de P. waltl a montré que ceux‐ci présentent une diversité importante, faisant de cet animal un bon modèle pour étudier les effets d’un vol spatial sur le système immunitaire humoral. Nous avons aussi montré que l’exposition à la suspension anti‐orthostatique durant 21 jours diminue la lymphopoïèse B mais n’affecte que modérément la diversité du répertoire de chaînes lourdes d’IgM. Ces résultats ont été comparés à ceux obtenus avec des souris âgées afin de savoir si la suspension anti‐orthostatique induit un vieillissement accéléré du système immunitaire. Même si nous avons noté des similitudes intéressantes entre ces deux groupes de souris, nous avons constaté que l’effet du vieillissement sur le répertoire d’IgM est plus important que celui de la suspension anti‐orthostatique, suggérant que le temps d’exposition au modèle anti‐orthostatique devrait être augmenté pour accentuer ses effets sur le répertoire d’anticorps. Quant au modèle CUMS, nous avons montré que son application durant la gestation n’impacte pas la lymphopoïèse T chez les souriceaux nouveau‐nés mais affecte 25% de leur répertoire de chaînes lourdes β du TCR. Ces résultats suggèrent que des stress socio‐environnementaux chroniques, même de faibles intensités, pourraient modifier les capacités de reconnaissance antigénique de l’hôte / Spaceflight is a source of various stresses leading to the weakening of the immune system. The efficiency of this system relies, notably, on the diversity of antigen receptor repertoires present on B (BCR) and T (TCR) cells, allowing the recognition of a vast array of antigens. During this thesis, I studied the diversity of antigen receptors in three different animal models: the amphibian Pleurodeles waltl, the murine anti‐orthostatic suspension model (simulated microgravity) and the CUMS (for "Chronic Unpredictable Mild Socio‐environmental stressors") murine model involving exposure to chronic social and environmental stressors similar to those encountered during spaceflights. Analyses of P. waltl IgM and IgY heavy chain repertoires have shown that they are highly diverse, making this species a nice animal model for studying the effects of spaceflight on the humoral immune system. We have also shown that 21 days of anti‐orthostatic suspension decrease murine B lymphopoiesis and moderately affect IgM heavy chain repertoire diversity. These results were compared with those obtained with old mice to determine if anti‐orthostatic suspension induces an accelerated aging of the immune system. Although we noted interesting similarities between these two groups of mice, we found that the effect of aging on IgM repertoire is stronger than that of the anti‐orthostatic suspension, suggesting that anti‐orthostatic duration should be extended to increase the effects of this model on antibody repertoire. Finally, regarding the CUMS model, we have shown that, when applied during gestation it does not affect T lymphopoiesis in newborn mice but affects 25% of their TCRβ heavy chain repertoire. These results suggest that low‐intensity chronic socio‐environmental stressors may alter antigen recognition capabilities of the host

T lymphocytes comprise one arm of the adaptive immune system and are critical for immunity to neoplasia and infection. A full understanding of their development has important implications for the treatment of autoimmunity, immunodeficiency, and leukemias arising from T cell developmental intermediates. The Notch signaling pathway is already known to be absolutely required for T cell commitment and development, but its collaboration with other factors is poorly understood. Unfortunately, deficiency in many of the genes critical to hematopoiesis, including Notch, causes early embryonic lethality by disrupting multiple developmental processes. This complicates the study of such genes by in vivo models or ex vivo hematopoietic progenitors. To circumvent these difficulties, this thesis describes the use of in vitro-differentiated embryonic stem cell-derived T progenitors to examine the roles of two GATA family members during early T cell development. GATA-2, while not required for T cell development, is shown to act downstream of Notch signals to inhibit myelopoiesis. These findings both characterize a novel role for GATA-2, and demonstrate that T progenitor maturation and exclusion of non-T cell fates are distinct and separable events. GATA-3, in contrast to GATA-2, is absolutely required for T lymphopoiesis. However, the current literature does not distinguish between a requirement for GATA-3 in homing to the thymic environment, committing to the T cell fate, or surviving such a commitment event. This thesis demonstrates that GATA-3 is dispensable for commitment itself, but required to permit survival and proliferation after commitment. Taken together, the results presented in this thesis employ a novel model system to characterize the interactions of two important collaborators with Notch signals during T cell development, and further dissect the stages through which early T cell development is enacted.

T lymphocytes comprise one arm of the adaptive immune system and are critical for immunity to neoplasia and infection. A full understanding of their development has important implications for the treatment of autoimmunity, immunodeficiency, and leukemias arising from T cell developmental intermediates. The Notch signaling pathway is already known to be absolutely required for T cell commitment and development, but its collaboration with other factors is poorly understood. Unfortunately, deficiency in many of the genes critical to hematopoiesis, including Notch, causes early embryonic lethality by disrupting multiple developmental processes. This complicates the study of such genes by in vivo models or ex vivo hematopoietic progenitors. To circumvent these difficulties, this thesis describes the use of in vitro-differentiated embryonic stem cell-derived T progenitors to examine the roles of two GATA family members during early T cell development. GATA-2, while not required for T cell development, is shown to act downstream of Notch signals to inhibit myelopoiesis. These findings both characterize a novel role for GATA-2, and demonstrate that T progenitor maturation and exclusion of non-T cell fates are distinct and separable events. GATA-3, in contrast to GATA-2, is absolutely required for T lymphopoiesis. However, the current literature does not distinguish between a requirement for GATA-3 in homing to the thymic environment, committing to the T cell fate, or surviving such a commitment event. This thesis demonstrates that GATA-3 is dispensable for commitment itself, but required to permit survival and proliferation after commitment. Taken together, the results presented in this thesis employ a novel model system to characterize the interactions of two important collaborators with Notch signals during T cell development, and further dissect the stages through which early T cell development is enacted.

D Cyclins play an essential role connecting exogenous stimulation to the intrinsic cell cycle machinery. This family of proteins is composed of three members sharing a highly homologous domain, the cyclin box (coded by exons 1-3), which is responsible for their redundant role in the phosphorylation of the retinoblastoma protein upon association with cydin-dependent kinases Cdk4/6. Both mature T and B-cells have a remarkable division capability after antigen stimulation, essential to the generation of efficient immune responses, raising the interest of D Cyclins in lymphopoiesis. Cyclin Dl, although weakly expressed by lymphocytes, is the D Cyclin most commonly implicated in lymphoid cancers and as having a Cdk-independent transcriptional role. To study the role of Cyclin Dl, we used mice deficient for the Dl cyclin box but sparing exons 4-5. Surprisingly, individual mice have very different phenotypes that we subdivided into four arbitrary groups. Group I mice show the most precocious block in lymphoid lineage differentiation, illustrated by a low cellularity of common lymphoid progenitor cells (CLP). The thymi showed very few CD4*CD8*, double positive (DP) cells, while the CD4 CD8TCR, triple negative (TN) populations were found to be mostly constituted by the early CD44*CD25' (TNI) and few CD44*CD25* (TN2). TNl's early thymocyte progenitors (ETP) were virtually absent. At the B-cell lineage level in the bone marrow (BM) there was a major block in pre-proB differentiation. The number of peripheral T-cells was severely reduced, mainly in LN, since group I T-cells lack CCR7 expression. Group II mice presented moderate thymus atrophy. The block on TN differentiation occurs at a later stage, i.e., in the TN3 to TN4 transition, and the TNI population was characterized by a less severe depletion of the ETP. Group II mice showed a partial pre-proB block and a reduction in pre-B-cells. CLPs were also reduced but to a lesser extent than in group I mice. Group ill and group IV mice appear to have a normal thymocyte population distribution but showed an increase on ETP compartment. Group IV mice displayed thymic hyperplasia while group III mice possessed normal thymus cellularity. B-cell differentiation on both groups appeared to be normal but BM precursors had an increase in both CLP and early haematopoietic progenitor's (LSK) levels as compared with wild type mice. Cyclin Dl involvement in G1 to S transition led us to analyse in vivo division rates. Strikingly, group I mice were virtually devoid of cycling cellsin all lymphoid compartments, explaining why lymphoid lineage cells do not differentiate in these mice. In contrast, in all other groups we observed an increased BrdU incorporation. These contradicting phenotypes correlated with the expression or absence of a truncated Dl protein coded by exons 4-5. The presence of the cyclin Dl truncated mRNA was not found in group I mice but high levels of expression are consistently observed in the remaining groups. In the absence of the Dl truncated protein only trace values of Cyclins D2 and D3 were found, highlighting the role of this protein as a master D cyclin regulator, which further supports the profound aplasia and arrest in lymphoid lineage division on cells that predominantly express Cyclin D2. These results suggest that, while the function of the Dl cyclin box is redundant, the regulatory domain coded by exons 4-5 is fundamental for lymphopoiesis. Full Dl protein was also eliminated by RNA interference both in vitro and in vivo. These experiments reproduced the phenotype of group I mice. We have developed a lentiviral vector with a truncated Dl (exons 4-5) and conditional knockout (KO) mice by floxing exons 4-5 of cyclin Dl. These tools will allow us to show Cyclin Dl Cdk-independent role as a transcription regulator in lymphopoiesis and to attribute this function to exons 4-5. Understanding how exons 4-5 regulate different transcription factors might be a key in...