T lymphocytes play a critical role in adaptive immunity by eliciting and regulating specific immune responses against viral and bacterial pathogens. The development of T cells occurs within the highly specialized thymus and follows a defined set of stage-specific differentiation steps. However, the molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. This was in part due to the inability to obtain substantial numbers of T-lineage cells from hybrid/human fetal thymic organ culture (FTOC) and the inability to recapitulate human T-lymphopoiesis using other systems. To address the molecular and cellular events occurring during early human T-lymphopoiesis, human umbilical cord-blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T-lineage utilizing OP9-DL1 stromal cells. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. In addition, this Thesis demonstrates that in vitro-generated CD34+CD7++ progenitors effectively engrafted the thymus of immunodeficient mice. In addition, two distinct progenitor subsets, CD34+CD45RA+CD7++CD5-CD1a- (proT1) and CD34+CD45RA+CD7++CD5+CD1a- (proT2), were identified with proT2 cells showing a 3-fold enhanced engrafting capacity than the proT1 subset. As proT2 cells exhibit superior engrafting capacity, these cells were tested for their ability to enhance T cell generation following hematopoietic stem cell transplant (HSCT). We observe that when HSCs are coinjected with proT2 cells, a dramatic improvement in HSC-derived T-lymphopoiesis is observed. This Thesis demonstrates that in vitro-derived proT2 cells reorganize the thymus stromal compartment of the host NOD/SCID/γcnull mouse compared to the highly disorganized cortical and medullary compartments in mice not receiving proT cells. This alteration in thymic architecture likely favours the recruitment of BM derived progenitors. Lastly, we address whether functional CD8 T cells can be generated in vitro using hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells and indeed these cells were capable of proliferating, and secreting effector molecules typical of cytotoxic T cells. Taken together, the ability to generate proT cells and mature T cells from Notch-ligand cultures offers a new tool to study human T cell development.

T lymphocytes play a critical role in adaptive immunity by eliciting and regulating specific immune responses against viral and bacterial pathogens. The development of T cells occurs within the highly specialized thymus and follows a defined set of stage-specific differentiation steps. However, the molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. This was in part due to the inability to obtain substantial numbers of T-lineage cells from hybrid/human fetal thymic organ culture (FTOC) and the inability to recapitulate human T-lymphopoiesis using other systems. To address the molecular and cellular events occurring during early human T-lymphopoiesis, human umbilical cord-blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T-lineage utilizing OP9-DL1 stromal cells. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. In addition, this Thesis demonstrates that in vitro-generated CD34+CD7++ progenitors effectively engrafted the thymus of immunodeficient mice. In addition, two distinct progenitor subsets, CD34+CD45RA+CD7++CD5-CD1a- (proT1) and CD34+CD45RA+CD7++CD5+CD1a- (proT2), were identified with proT2 cells showing a 3-fold enhanced engrafting capacity than the proT1 subset. As proT2 cells exhibit superior engrafting capacity, these cells were tested for their ability to enhance T cell generation following hematopoietic stem cell transplant (HSCT). We observe that when HSCs are coinjected with proT2 cells, a dramatic improvement in HSC-derived T-lymphopoiesis is observed. This Thesis demonstrates that in vitro-derived proT2 cells reorganize the thymus stromal compartment of the host NOD/SCID/γcnull mouse compared to the highly disorganized cortical and medullary compartments in mice not receiving proT cells. This alteration in thymic architecture likely favours the recruitment of BM derived progenitors. Lastly, we address whether functional CD8 T cells can be generated in vitro using hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells and indeed these cells were capable of proliferating, and secreting effector molecules typical of cytotoxic T cells. Taken together, the ability to generate proT cells and mature T cells from Notch-ligand cultures offers a new tool to study human T cell development.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22359号 / 医博第4600号 / 新制||医||1042(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 長尾 美紀, 教授 滝田 順子, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Près des deux tiers des patients vivant avec le VIH habitent en Afrique sub-saharienne. Dans ces pays à ressources limitées, la question du niveau optimal de CD4 pour débuter le traitement antirétroviral (ARV) est un enjeu majeur de santé publique. Nous avons étudié la reconstitution immunitaire à long terme sous ARV et son association avec la survie dans quatre projets de prise en charge du VIH soutenus par Médecins Sans Frontières (MSF) en Afrique sub-Saharienne entre 2001 et 2010. Nous avons utilisé des modèles mixtes pour prendre en compte les mesures de CD4 répétées aussi bien dans la modélisation de la réponse immunitaire que dans l'analyse de survie. Dans la première partie modélisant la reconstitution immunitaire, nous avons montré que les femmes ont une meilleure reconstitution immunitaire que les hommes et que la capacité de reconstitution immunitaire est la même quel que soit le taux de CD4 au démarrage des ARV. La deuxième étude a mis en évidence que la survie était améliorée sous ARV chez les patients atteignant un taux de CD4 courant supérieur à 500 cellules/μL par rapport à ceux qui ont un taux de CD4 entre 350 et 499 cellules/μL. Le sexe, l'âge et l'observance ont aussi été identifiés comme prédicteurs indépendants de mortalité après neuf mois sous ARV. En conclusion, nous montrons que les femmes ont une meilleure reconstitution immunitaire et atteignent plus vite un taux de CD4 de 500 cellules/μL qui est associé à une meilleure survie. Ce travail suggère qu'un démarrage plus précoce des ARV, avant que le taux de CD4 ne tombe en dessous de 500 cellules/μl, pourrait améliorer la survie et confirme le besoin d'essais cliniques évaluant les bénéfices individuels d'une telle stratégie / Almost two third of individuals living with HIV are in sub-Saharan Africa. In these resource limited countries, the optimal CD4 cell count level to initiate antiretroviral treatment (ART) is still debated. We studied long term immune reconstitution on ART and its association with survival in four HIV programs supported by Médecins Sans Frontières in sub-Saharan Africa between 2001 and 2010. We used mixed models to account for repeated CD4 measurements in the modelling of the immune reconstitution and the survival analysis. In the first part modelling immune response to ART, we showed that women achieved a better immune reconstitution compared to men and that the immune reconstitution capacity is the same for each level of CD4 at ART initiation. The second study showed that survival is improved for patient with an updated CD4 above 500 cells/μL compared to those with CD4 levels between 350-499 cells/μL. Furthermore, gender, age and adherence are independently associated with mortality. As a conclusion, women have a better immune reconstitution and achieved quicker

Indiana University-Purdue University Indianapolis (IUPUI) / There is an urgent demand for effective medical countermeasures (MCM) in the event of high-dose radiation exposure ranging from nuclear plant disasters to potential nuclear warfare. Victims of lethal-dose radiation exposure face multi-organ injuries including the hematopoietic acute radiation syndrome (H-ARS) and the delayed effects of acute radiation exposure (DEARE) years after irradiation. Defective lymphocyte reconstitution and its subsequent immune insufficiency are some of the most serious consequences of H-ARS and DEARE. In order to investigate potential MCMs to protect or mitigate these radiation injuries, the prolonged tissue-specific immunosuppression at all levels of lymphocyte development in established murine H-ARS and DEARE models was defined, along with unique sex-related and age-related changes present in some tissues but not others. The “double hits” of irradiation and age-related stress on lymphopoiesis led to significant myeloid skew and long-term immune involution. Different kinds and different combinations of hematopoietic growth factors, some in combination with angiotensin converting enzyme inhibitor, were administered to lethally irradiated mice. These radiomitigators were found to significantly increase survival and enhance hematopoiesis in H-ARS, but they did little to alleviate the severity of DEARE including immune insufficiency. 16,16 dimethyl-prostaglandin E2 (dmPGE2), a long-acting formulation of PGE2 with similar biological effects as PGE2, was found to enhance survival and hematopoiesis in lethal-irradiated mice when used as radiomitigator or radioprotectant. The optimum time window for administration of radioprotectant and radiomitigator dmPGE2 was defined, which is -3hr to -15min prior to irradiation and +6hr to +30hr post irradiation. Significant survival efficacy of radioprotectant dmPGE2 was also demonstrated in pediatric and geriatric mice. Using specific PGE2 receptor (EP) agonists, the EP4 receptor was defined as the PGE2 receptor potentially responsible for dmPGE2 radioprotection. Radioprotectant dmPGE2 was also found to prevent radiation-induced thymic involution and to ameliorate the long-term immune suppression in radiation survivors in the DEARE phase via promoting hematopoietic stem cell differentiation towards to the lymphoid lineage. This is the first report of an effective MCM for H-ARS which also targets long-term thymic involution and lymphoid lineage reconstitution.

Allogeneic haematopoietic stem cell transplantation (alloHSCT) is an established therapy for many haematological disorders. Unfortunately, the new donor-derived immune system may damage host cells (graft-versus-host disease (GvHD)), causing significant morbidity and mortality. Since regulatory T cells (Tregs) can modulate immune responses, it was hypothesised that Treg numbers in the haematopoietic stem cell grafts and/or peripheral blood may influence the development of GvHD and other transplant-related complications. In this project, a prospective observational clinical study of putative Tregs in human alloHSCT was performed in Oxford. Flow cytometry and methylation-specific qPCR assays were developed to quantify putative Tregs and lymphocyte populations within the grafts and post-transplant blood samples. Although low CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell numbers were not associated with increased incidence of GvHD, low proportions of CD25(+)FOXP3(+)CD127(-/dim) cells in the graft (as a percentage of total CD4(+) T cells) were independently associated with poor engraftment, increased non-relapse mortality and inferior overall survival. Similarly, falling CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell counts over the first three months post-transplant were associated with higher non-relapse mortality and inferior overall survival. In view of these novel findings, strategies that increase CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T cells in alloHSCT may improve clinical outcomes. One possible route for increasing Tregs is through cellular therapy. This project therefore tested the hypothesis that CD4(+)CD25(+)FOXP3(+) Tregs can be produced in vitro from conventional CD4(+) T cells. In the presence of TGFβ and Azacitidine, FOXP3 was expressed in the majority of activated CD4(+) T cells. These cells also had a demethylated FOXP3 TSDR enhancer which is specific to natural Tregs. However, most of these cells produced pro-inflammatory cytokines, for example, TNFα. Therefore, under these conditions, FOXP3 expression was not sufficient to produce a Treg phenotype. It is proposed that current focus for generating Tregs for human clinical trials should be directed towards improving isolation and expansion of ex vivo isolated Tregs.

O transplante de células-tronco hematopoéticas alogênico (alo-TCTH) é uma opção de tratamento para uma variedade de doenças neoplásicas e não neoplásicas, principalmente de origem hematológica sendo doença do enxerto-contra-hospedeiro (DECH) a sua principal complicação. As células Natural Killer (NK) são os primeiros linfócitos a se recuperarem após o TCTH. Além da capacidade de promover o efeito enxerto-versus-leucemia (EVL), as células NK do doador parecem capazes de promover a pega do enxerto e de prevenir o desenvolvimento da DECH. As células NK compreendem aproximadamente 10% dos linfócitos do sangue periférico e são caracterizadas fenotipicamente pela expressão do antígeno de superfície CD56 (CD, cluster of differentiation) e pela ausência de CD3 (CD56+CD3-). O subtipo de células NK CD56dim (baixa densidade do antígeno) é naturalmente mais citotóxico que o subtipo CD56bright (alta densidade do antígeno) o qual é caracterizado pela capacidade de produção de citocinas. Com base nisso, o objetivo do trabalho é avaliar a presença de células NK nos dias 7, 14, 21 e 28 após o TCTH alogênico e autólogo, caracterizando sua frequência, seu imunofenótipo e a sua capacidade de produzir fatores de crescimento hematopoético e citocinas relacionadas. / Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an option of treatment for a variety of neoplastic and non-neoplastic diseases and graft-versus-host disease (GVHD) is its main complication. Natural Killer cells (NK) are the first lymphocytes to recover after HSCT. In addition to the ability to promote graft versus leukemia effect (GVL), donor NK cells appear to be capable of promoting engraftment and preventing the development of GVHD. NK cells comprise approximately 10% of peripheral blood lymphocytes and are characterized phenotypically by the expression of the CD56 surface antigen and absence of CD3 (CD56 + CD3-). The CD56dim (low density of antigen) NK cell subtype is naturally more cytotoxic than the CD56bright (high density of antigen) subtype which is characterized by the ability to produce cytokines. Based on this, the objective of the study is to evaluate the presence of NK cells on days 7, 14, 21 and 28 after allogeneic and autologous HSCT, characterizing their frequency, their immunophenotype and their capacity to produce hematopoietic growth factors and related cytokines.

O transplante de células-tronco hematopoéticas alogênico (alo-TCTH) é uma opção de tratamento para uma variedade de doenças neoplásicas e não neoplásicas, principalmente de origem hematológica sendo doença do enxerto-contra-hospedeiro (DECH) a sua principal complicação. As células Natural Killer (NK) são os primeiros linfócitos a se recuperarem após o TCTH. Além da capacidade de promover o efeito enxerto-versus-leucemia (EVL), as células NK do doador parecem capazes de promover a pega do enxerto e de prevenir o desenvolvimento da DECH. As células NK compreendem aproximadamente 10% dos linfócitos do sangue periférico e são caracterizadas fenotipicamente pela expressão do antígeno de superfície CD56 (CD, cluster of differentiation) e pela ausência de CD3 (CD56+CD3-). O subtipo de células NK CD56dim (baixa densidade do antígeno) é naturalmente mais citotóxico que o subtipo CD56bright (alta densidade do antígeno) o qual é caracterizado pela capacidade de produção de citocinas. Com base nisso, o objetivo do trabalho é avaliar a presença de células NK nos dias 7, 14, 21 e 28 após o TCTH alogênico e autólogo, caracterizando sua frequência, seu imunofenótipo e a sua capacidade de produzir fatores de crescimento hematopoético e citocinas relacionadas. / Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an option of treatment for a variety of neoplastic and non-neoplastic diseases and graft-versus-host disease (GVHD) is its main complication. Natural Killer cells (NK) are the first lymphocytes to recover after HSCT. In addition to the ability to promote graft versus leukemia effect (GVL), donor NK cells appear to be capable of promoting engraftment and preventing the development of GVHD. NK cells comprise approximately 10% of peripheral blood lymphocytes and are characterized phenotypically by the expression of the CD56 surface antigen and absence of CD3 (CD56 + CD3-). The CD56dim (low density of antigen) NK cell subtype is naturally more cytotoxic than the CD56bright (high density of antigen) subtype which is characterized by the ability to produce cytokines. Based on this, the objective of the study is to evaluate the presence of NK cells on days 7, 14, 21 and 28 after allogeneic and autologous HSCT, characterizing their frequency, their immunophenotype and their capacity to produce hematopoietic growth factors and related cytokines.

O transplante de células-tronco hematopoéticas alogênico (alo-TCTH) é uma opção de tratamento para uma variedade de doenças neoplásicas e não neoplásicas, principalmente de origem hematológica sendo doença do enxerto-contra-hospedeiro (DECH) a sua principal complicação. As células Natural Killer (NK) são os primeiros linfócitos a se recuperarem após o TCTH. Além da capacidade de promover o efeito enxerto-versus-leucemia (EVL), as células NK do doador parecem capazes de promover a pega do enxerto e de prevenir o desenvolvimento da DECH. As células NK compreendem aproximadamente 10% dos linfócitos do sangue periférico e são caracterizadas fenotipicamente pela expressão do antígeno de superfície CD56 (CD, cluster of differentiation) e pela ausência de CD3 (CD56+CD3-). O subtipo de células NK CD56dim (baixa densidade do antígeno) é naturalmente mais citotóxico que o subtipo CD56bright (alta densidade do antígeno) o qual é caracterizado pela capacidade de produção de citocinas. Com base nisso, o objetivo do trabalho é avaliar a presença de células NK nos dias 7, 14, 21 e 28 após o TCTH alogênico e autólogo, caracterizando sua frequência, seu imunofenótipo e a sua capacidade de produzir fatores de crescimento hematopoético e citocinas relacionadas. / Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an option of treatment for a variety of neoplastic and non-neoplastic diseases and graft-versus-host disease (GVHD) is its main complication. Natural Killer cells (NK) are the first lymphocytes to recover after HSCT. In addition to the ability to promote graft versus leukemia effect (GVL), donor NK cells appear to be capable of promoting engraftment and preventing the development of GVHD. NK cells comprise approximately 10% of peripheral blood lymphocytes and are characterized phenotypically by the expression of the CD56 surface antigen and absence of CD3 (CD56 + CD3-). The CD56dim (low density of antigen) NK cell subtype is naturally more cytotoxic than the CD56bright (high density of antigen) subtype which is characterized by the ability to produce cytokines. Based on this, the objective of the study is to evaluate the presence of NK cells on days 7, 14, 21 and 28 after allogeneic and autologous HSCT, characterizing their frequency, their immunophenotype and their capacity to produce hematopoietic growth factors and related cytokines.

La transplantation allogénique de cellules souches hématopoïétiques est une technique très efficace pour traiter différents cancers du sang. Malheureusement la réaction du greffon contre l’hôte (GVHD) demeure la cause principale de morbidité et de mortalité post-greffe. La GVHD entraîne une diminution de la reconstitution immunitaire ce qui accentue considérablement l’immunosuppression associée à ce traitement et de ce fait augmente les risques d’infection et de rechute. Notre laboratoire a démontré précédemment que les niveaux élevés d’IL-7 dans des hôtes lymphopéniques interféraient avec la capacité des cellules dendritiques (DC) à soutenir la prolifération homéostatique (PH) des lymphocytes T CD4+. Puisque les niveaux d’IL-7 sont aussi élevés dans un contexte de GVHD, nous avons émis l’hypothèse que la signalisation de l’IL-7 sur les DC pouvait contribuer à diminuer la reconstitution immunitaire des lymphocytes T CD4+. Pour répondre à cette question, nous avons utilisé le modèle murin de GVHD C57BL/6 (B6) dans B6D2F1. Afin de régénérer une niche hématopoïétique permissive à la PH des lymphocytes T CD4+, nous avons transplanté des souris B6D2F1 avec de la moelle osseuse de souris B6 IL-7Rα-/-. La GVHD a été induite en transférant des lymphocytes T B6 réactifs aux cellules B6D2F1. Dans les souris contrôles, la PH des lymphocytes T CD4+ est maintenue. Par contre, la PH est absente dans les souris en GVHD malgré la présence d’une niche périphérique qui ne répond pas à l’IL-7. L’absence de PH des lymphocytes T CD4+ durant la GVHD est associée à une diminution du nombre de DC. En utilisant un test de cytotoxicité in vivo nous démontrons que les DC B6 générées dans une hôte B6D2F1 sont éliminées par les lymphocytes T B6 alloréactifs. En conclusion, nos résultats démontrent que l’immunosuppression associée à la GVHD est en partie causée par une élimination des DC par les lymphocytes T allogéniques. Nous postulons donc que la perte des DC, et non la signalisation de l’IL-7 sur les DC, est le facteur limitant la PH des lymphocytes T CD4+ durant la GVHD. / Allogeneic hematopoietic stem cell transplantation (SCT) is an effective treatment for numerous types of haematological malignancies. However, graft-versus-host disease (GVHD) remains the major cause of morbidity and mortality following SCT. GVHD is associated with poor immune reconstitution and its adverse effect on T cell regeneration greatly exaggerates the immunodeficiency normally associated with SCT. As a result, patients experiencing GVHD present deficit in T cells that can last for several years. Our laboratory has demonstrated that elevated systemic IL-7 found during lymphopenia can interfere with the capacity of dendritic cells (DC) to support the homeostatic proliferation (HP) of CD4+ T cells. Since IL-7 levels are also elevated during GVHD, we hypothesized that IL-7 signaling in DC could also contribute to diminished T cell reconstitution in this setting. We used the well describe GVHD mouse model C57BL/6 (B6) into B6D2F1 to study the contribution of IL-7 signalling in DC on CD4+ T cell regeneration during GVHD. To regenerate a peripheral niche permissive for CD4+ T cells HP, we transplanted B6D2F1 mice with bone marrow (BM) from B6 IL-7Rα-/- mice. Finally, GVHD was induced with 1x106 of alloreactive B6 T cells. In control mice transplanted with IL-7Rα-/- BM cells, CD4+ T cells HP is efficiently supported. In contrast, CD4+ HP is completely abrogated in GVHD mice despite the presence of a peripheral niche that does not signal IL-7. Loss of CD4+ HP during GVHD was associated with diminished number of DC. Using an in vivo cytotoxic assay, we demonstrated that B6 DC can be eliminated by alloreactive B6 T cells when these cells developed into B6D2F1 hosts. In conclusion, our data demonstrates that the immunosuppression associated with GVHD is in part related to the elimination of donor-derived DC by donor alloreactive T cells. Therefore, we postulate that loss of DC, and not IL-7 signaling in DC, represents the limiting factor that contains CD4+ HP during GVHD.