No description available.

A proposta do nosso estudo foi comparar o número de células apresentadoras de antígenos (CAAs) presentes em biópsias de gengivite (G) e de hiperplasia gengival induzida por ciclosporina (HGIC). Vinte e oito biópsias de pacientes com G e 14 biópsias de HGIC foram analisadas no epitélio de revestimento bucal (ERB), epitélio sulcular (ES) e no tecido conjuntivo (TC). O número de macrófagos (CD68+), de células de Langerhans (CD1a+) e de células dendríticas intersticiais (CDIs) (FXIIIA+) foi investigado por técnica imunoistoquímica. Células CD1a+/mm2 foram significantemente aumentadas na G quando comparada com HGIC, no TC, no ES e no ERB (p<0,05). Em contrapartida, o número de células CD68+ no TC e no ERB, e o número de células FXIIIA+/mm2 no TC foram aumentadas no grupo de HGIC (p<0,05). Ciclosporina A está relacionada com a diminuição de células de Langerhans. Podemos sugerir que este fato aumenta as infecções oportunistas, conseqüentemente, um maior número de macrófagos é necessário para combater os microorganismos. / The aim of this study was to compare the number of antigen-presenting cells (APCs) observed in biopsies of gingivitis (G), and in cyclosporine-induced gingival overgrowth (CIGO). Twenty eight biopsies from patients with G, and 14 with CIGO were analyzed in oral epithelium (OE), sulcular/junctional epithelium (SJE), and connective tissue (CT). The number of macrophages (CD68+), Langerhans’cells (CD1a+), and interstitial dendritic cells (FXIIIA+) was investigated by immunohistochemistry. CD1a+ cells/mm2 were significantly increased in G when compared with CIGO, in CT, in SJE, and in OE (p<0.05). In contrast, the number of CD68+ in CT, and in OE, and FXIIIA+ cells/mm2 in CT were increased in CIGO group (p<0.05). Cyclosporine is related with the diminution of Langerhans’ cells. We can suggest that this fact increase opportunistic infections, consequently, a greater number of macrophages is necessary in order to combat the microorganisms.

A proposta do nosso estudo foi comparar o número de células apresentadoras de antígenos (CAAs) presentes em biópsias de gengivite (G) e de hiperplasia gengival induzida por ciclosporina (HGIC). Vinte e oito biópsias de pacientes com G e 14 biópsias de HGIC foram analisadas no epitélio de revestimento bucal (ERB), epitélio sulcular (ES) e no tecido conjuntivo (TC). O número de macrófagos (CD68+), de células de Langerhans (CD1a+) e de células dendríticas intersticiais (CDIs) (FXIIIA+) foi investigado por técnica imunoistoquímica. Células CD1a+/mm2 foram significantemente aumentadas na G quando comparada com HGIC, no TC, no ES e no ERB (p<0,05). Em contrapartida, o número de células CD68+ no TC e no ERB, e o número de células FXIIIA+/mm2 no TC foram aumentadas no grupo de HGIC (p<0,05). Ciclosporina A está relacionada com a diminuição de células de Langerhans. Podemos sugerir que este fato aumenta as infecções oportunistas, conseqüentemente, um maior número de macrófagos é necessário para combater os microorganismos. / The aim of this study was to compare the number of antigen-presenting cells (APCs) observed in biopsies of gingivitis (G), and in cyclosporine-induced gingival overgrowth (CIGO). Twenty eight biopsies from patients with G, and 14 with CIGO were analyzed in oral epithelium (OE), sulcular/junctional epithelium (SJE), and connective tissue (CT). The number of macrophages (CD68+), Langerhans’cells (CD1a+), and interstitial dendritic cells (FXIIIA+) was investigated by immunohistochemistry. CD1a+ cells/mm2 were significantly increased in G when compared with CIGO, in CT, in SJE, and in OE (p<0.05). In contrast, the number of CD68+ in CT, and in OE, and FXIIIA+ cells/mm2 in CT were increased in CIGO group (p<0.05). Cyclosporine is related with the diminution of Langerhans’ cells. We can suggest that this fact increase opportunistic infections, consequently, a greater number of macrophages is necessary in order to combat the microorganisms.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24515号 / 医博第4957号 / 新制||医||1064(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 森信 暁雄, 教授 竹内 理, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

The eosinophil is a leukocyte whose intracellular mediators are considered to play a central role in the pathogenesis of allergic diseases, including allergic asthma, allergic rhinitis and atopic dermatitis, and which is also involved in immunological responses to parasites. Eosinophil differentiation and maturation from bone marrow progenitors is regulated by interleukin-5 (IL-5), which may be secreted by T helper 2 (Th2) T lymphocytes, and is consistently upregulated in allergic conditions. Eotaxin is a potent chemoattractant for circulating and tissue eosinophils, and the production of this chemokine promotes eosinophil infiltration and accumulation within sites of allergic inflammation.¶ Eosinophils obtained from inflammatory tissues and secretions display an altered phenotype in comparison to peripheral blood eosinophils, with increased surface expression of major histocompatibility complex (MHC) proteins and adhesion molecules (Hansel et al., 1991), and migration across the microvascular endothelium may also increase their capacity to generate an oxidative burst (Walker et al., 1993; Yamamoto et al., 2000). Eosinophils are phagocytic cells, and have been shown to present simple (no requirement for intracellular processing) and complex antigens to MHC-restricted, antigen-specific T lymphocytes (Del Pozo et al., 1992; Weller et al., 1993). Furthermore, eosinophils express the costimulatory molecules required for effective antigen presentation (Tamura et al., 1996), and ligation of costimulatory molecules on the eosinophil cell surface can induce the release of eosinophil derived cytokines (Woerly et al., 1999; Woerly et al., 2002). Therefore the eosinophil may also regulate immune responses.¶ To date, no studies have demonstrated the ability of eosinophils to modulate activated T lymphocyte function via presentation of relevant antigen in the context of MHC class II (MHC-II), concomitant with Th2 cytokine release. In the experiments described in this thesis, murine eosinophils have been observed to rapidly migrate to sites of antigen deposition within the airways mucosa of naïve mice, suggesting a potential role for this granulocyte in the primary response to inhaled antigen. However, human allergic diseases are often diagnosed after the establishment of allergic responses, and symptom development. Therefore, a murine model of allergic airways disease (AAD) was used to investigate the ability for eosinophils to participate as antigen presenting cells (APCs), and thereby modulate activated T lymphocyte function both in vitro and in vivo. Detailed histological analysis of the pulmonary draining lymph nodes following antigen challenge in sensitised mice revealed a rapid infiltration of eosinophils into this tissue, which preceded the accumulation of eosinophils in bronchoalveolar lavage fluid (BALF). This suggested that eosinophils were preferentially translocating to the draining lymph nodes following antigen challenge, and that the subsequent accumulation of these cells in the BALF was a consequence of continued antigen delivery to the lower airways.¶ Eosinophil trafficking to lymphoid tissue via the afferent lymphatics was substantiated using electron microscopy of lymph node sections and the intravenous (i.v.) transfer of fluorescently labeled eosinophils, which did not traffic to lymph nodes via the blood. During the resolution of AAD, eosinophils were noted for their persistence in the pulmonary draining lymph nodes. These observations suggested a continued modulation of T cell function by lymph node dwelling eosinophils during AAD resolution, particularly in light of recent observations for draining lymph node T cell proliferation following instillation of antigen-pulsed eosinophils into the allergic mouse lung (Shi et al., 2000).¶ To further investigate the antigen presenting capacity, eosinophils were obtained from the BALF of mice with AAD, and their surface expression of MHC class II (MHC-II) proteins and costimulatory molecules confirmed using flow cytometric analysis. The ability to acquire and process complex antigen both in vitro and in vivo was also confirmed using naturally quenching fluorescenated ovalbumin (OVA), which is degraded into fluorescent peptides by the action of intracellular proteases. Thus, eosinophil expression of the surface molecules necessary for effective antigen presentation was confirmed, as was their ability to process complex antigen. Further investigations revealed that eosinophils can present complex OVA antigen to CD4+ T lymphocytes obtained from the allergic mouse, and to in vitro derived OVA-specific Th2 cells. In the presence of exogenous antigen, eosinophils co-cultured with T lymphocytes were able to induce Th2 cytokine production, and demonstrated an ability for eosinophils to modulate T lymphocyte function in vitro.¶ The ability for eosinophils to act as antigen presenting cells in vivo was also investigated. Eosinophils obtained from the antigen-saturated lungs of OVA sensitised and challenged mice were transferred to the peritoneal cavities of naïve host mice. When subsequently challenged with aerosolised OVA, eosinophil recipients developed a pulmonary eosinophilia similar to that of OVA sensitised and challenged mice. To validate this finding, the experimental procedure was altered to accommodate the use of non-allergy derived eosinophils, which were pulsed with OVA in vitro, prior to transfer into naïve recipients. When subsequently challenged with aerosolised OVA, eosinophil recipients developed a peripheral blood and pulmonary eosinophilia, and stimulation with OVA induced IL-5 and IL-13 cytokine production from pulmonary draining lymph node cells. Notably, the AAD induced by transfer of antigen pulsed eosinophils did not induce detectable OVA-specific IgG1, which may be attributed to the lack of soluble antigen required for B cell antibody production.¶ During the course of these investigations, an OVA T cell receptor (TCR) transgenic mouse (OT-II) was procured with a view to defining the interaction between eosinophils and activated T lymphocytes (Barnden et al., 1998). Despite having specificity for the OVA323-339 peptide, an immunodominant epitope that skews naïve T cell responses towards Th2 cytokine release (Janssen et al., 2000), T lymphocytes from the OT-II mouse preferentially secreted IFN-γ in response to stimulation with either OVA peptide or OVA. These mice were further characterised in a mouse model of AAD, and found to be refractory to disease induction and progression, which may be attributed to significant IFN-γ secretion by transgenic CD4+ T lymphocytes during antigen sensitisation. Indeed, these cells were noted for their ability to attenuate pulmonary eosinophilia when transferred to OVA sensitised and challenged wild type mice, although serum OVA-specific IgG1, peripheral blood eosinophilia levels and airways response to methacholine challenge remained intact.¶ Knowledge of the biased Th1 phenotype in naïve OT-II provided a unique opportunity to investigate the fate of T lymphocytes bearing high affinity OVA-specific TCRs following neonatal antigen exposure to soluble OVA. In a previous study, subcutaneous (s.c.) administration of soluble OVA to wild type neonatal mice was suspected to have deleted OVA-specific T cells from the T cell repertoire (Hogan et al., 1998a). Using flow cytometry and TCR specific antibody, the delivery of s.c. OVA to OT-II neonates did not alter transgenic T cell populations in adult mice. Instead, it was surprising to find a skewing towards the Th2 phenotype and loss of IFN-γ secretion following OVA sensitisation and challenge in adult mice. A mechanism for this reprogramming of the transgenic T cell from the Th1 to a Th2 phenotype following OT-II neonatal exposure to soluble OVA is proposed, and further experimentation may validate this hypothesis.¶ In conclusion, eosinophils residing in the allergic lung have the capacity to interact with activated T cells, both within this tissue and the draining lymph nodes. Despite their relative inefficiency as antigen presenting cells (Mawhorter et al., 1994), eosinophils may participate en masse in the serial triggering of activated TCRs, and provide appropriate costimulatory signals that modulate T lymphocyte function. Through the elaboration of Th2 cytokines and stimulation of T cell proliferation, antigen presenting eosinophils may transiently prolong or exacerbate the symptoms of allergic diseases. Alternatively, eosinophils presenting relevant antigens may inhibit T cell activity via degranulation, and such activity has recently been observed in a parasite model (Shinkai et al., 2002). Finally, experiments in the OT-II mouse have provided valuable information to suggest that therapies designed to modulate eosinophil numbers in allergic tissues through the secretion of opposing cytokines such as IFN-γ, may be of limited benefit. The results shown here suggest that airways dysfunction remains intact despite significantly reduced pulmonary eosinophilia

L’immunothérapie basée sur le transfert de lymphocytes T (LT) spécifiques de la tumeur est une approche prometteuse contre le cancer. Pour activer et amplifier de tels LT, principale étape limitante de cette approche, des cellules présentatrices d’antigène artificielles (CPAA) ont été développées au laboratoire. Ces CPAA ont été construites à partir de fibroblastes murins NIH/3T3 transduits à l’aide de vecteurs gammarétroviraux afin d’exprimer les principaux éléments nécessaires à l’activation de LT humains. Ces CPAA nous permettent d’obtenir des LT mémoires souches (TSCM : CD95+CD45RA+CD62L+CCR7+), LT très peu différenciés récemment identifiés chez l’homme. Ces TSCM ont été décrits comme étant du plus grand intérêt pour l’immunothérapie en raison de leur capacité d’auto-renouvellement et de leur faculté à se différencier en LT effecteursefficaces. Pour optimiser l’amplification de TSCM spécifiques, nous avons notamment étudié les effets sur les LT de l’expression de différentes molécules de costimulation par nos CPAA (CD80, CD70 et 4-1BBL). Les protéines MART-1 et MELOE-1, surexprimées dans les mélanomes, ont été utilisées comme antigènes modèles pour ces travaux. Les CPAA CD80+CD70+ et CD80+CD70+4-1BBL+ sont les plus prometteuses pour maintenir le phénotype des TSCM. Une étude exhaustive des CPAA CD80+CD70+ a montré que nous pouvions obtenir un plus grand nombre de TSCM fonctionnels spécifiques de MART-1 et de MELOE-1 de manière reproductible avec ces CPAA. Dans une seconde étude, nous avons pu montrer que les CPAA CD80+CD70+4-1BBL+ permettaient d’obtenir le plus grand nombre de LT spécifiques fonctionnels et très peu différenciés après purification et restimulation de LT spécifiques stimulés une première fois par les CPAA CD80+CD70+. Ces travaux devraient nous permettre, après le développement d’un modèle murin, de proposer de nouvelles stratégies d’immunothérapie basées sur l’obtention grâce à nos CPAA optimisées de LT spécifiques anti-tumoraux capables d’assurer une protection à long terme aux patients. / Immunotherapy based on the transfer of tumor-specific T lymphocytes (TLs) is a promising approach against cancer. To activate and amplify such TLs, main limiting step of this approach, artificial antigen presenting cells (AAPCs) have been developed in the laboratory. These AAPCs have been constructed from NIH/3T3 murine fibroblasts transduced with gammaretroviral vectors to express the principal elements required to activate human TLs. With these AAPCs, we can obtain anti-tumor stem cell memory TLs (TSCM: CD95+CD45RA+CD62L+CCR7+), which are very limitedly differentiated TLs recently identified in humans. These TLs have been recently described as cells of great interest for immunotherapy because of their self-renewal capacity and their ability to differentiate into effective effector TLs. To improve the amplification of specific TSCM, we notably studied the effects on TLs of the expression of different co-stimulatory molecules by our AAPCs (CD80, CD70 and 4-1BBL). MART-1 and MELOE-1, proteins that are overexpressed in melanoma, were used as model antigens in this work. CD80+CD70+ and CD80+CD70+4-1BBL+ AAPCs appear to be the most promising ones for maintaining a TSCM phenotype. An exhaustive study of CD80+CD70+ AAPCs showed that we could reproducibly get greater numbers of MART-1- and MELOE-1-specific functional TSCM with these AAPCs. In another study, we have shown that CD80+CD70+4-1BBL+ AAPCs enabled us to get the greatest number of functional and very limitedly differentiated specific TLs after purification and restimulation of specific TLs stimulated first with CD80+CD70+ AAPCs. This work should allow us, after the development of a murine model, to propose new immunotherapy strategies based on the possibility of obtaining with our optimized AAPCs anti-tumor specific TLs capable of ensuring patient long term protection.

CD8+ T cells are critical to the adaptive immune response and are a target for vaccine development. However, the complex dynamics of cell proliferation can vary response success, providing uncertainty when designing vaccines. Computer models can provide clarity by simulating these dynamics, tracking millions of cell-cell interactions, a feat that is impractical experimentally. Our group created the STORE.1 model, a probabilistic simulation of the CD8+ T cell response to vaccination. While able to accurately simulate in vivo mouse T cell clonal expansion, intracellular dynamics are absent. Furthermore, there is no mechanism by which cell division ceases. This work builds upon the STORE.1 model by systematically explaining the division dynamics of CD8+ T cells and providing measures of the extracellular environment. The new STORE.2 model has demonstrated an ability to accurately simulate differences in CD8+ T cell expansion in WT mice and mice lacking type I conventional dendritic cells up to 170 hours after vaccination. It is the first model to simulate individual cell cycle regulator protein counts for millions of cells, and the resulting impact on pH for the extracellular microenvironment. Finally, it provides a partial mechanism behind division cessation, an important element for future models seeking to further simulate the end of the T-cell response. / Thesis / Master of Applied Science (MASc) / T-cells are an important component of the human immune system, but currently, there are no vaccines in clinical use that are designed to target them. This is because there are many different dynamics that underpin how T-cells activate, and to what degree they can replicate into a substantial pool of pathogen-clearing cells. Learning which candidate vaccines can properly elicit a strong T-cell response is time and resource consuming. Mathematical models can therefore speed development of candidate vaccines by virtually testing their T-cell responsiveness. This thesis works to improve on an existing mathematical model by introducing immunological mechanisms that determine how T-cells undergo cell division, change the acidity of their immediate surroundings, and respond to their own growing population. By doing so, this new model can be more representative of the immunological reality and begin to probe new dynamics of the T-cell response.

In zahlreichen Studien konnte an Mausmodellen gezeigt werden, dass dendritische Zellen eine wichtige Rolle im Rahmen der mukosalen Immunabwehr spielen. Eine unkontrollierte Aktivierung immunologischer Effektorzellen durch antigenpräsentierende Zellen ist die Folge, welche die Antigene der luminalen Flora folglich falsch erkennen und damit zu einer Schädigung des Gewebes führen. In der Arbeit wurden humane CD1c+CD11c+CD14-CD19- myeloide dendritische Zellen (mDCs) aus dem peripheren Blut und der intestinalen Mukosa von CED Patienten sowie von gesunden Probanden phänotypisch und funktionell näher charakterisiert. mDCs von Patienten reagieren auf LPS im Gegensatz zu DCs von Gesunden mit der Ausbildung eines aktivierten Phänotyps und der Sekretion pro-inflammatorischer Zytokine. Die Daten lassen vermuten, dass ihre tolerogene Rolle gestört ist und die Zellen so möglicherweise aktiv zum Entzündungsgeschehen durch eine Fehlreaktion auf die kommensale Flora beitragen. Es konnte gezeigt werden, dass zirkulierende mDCs von Erkrankten mehr LPS aufnehmen. Des Weiteren ist die Häufigkeit von mukosalen und aktivierten mDCs bei CED Patienten signifikant erhöht. Die vermehrte Häufigkeit von aktivierten mDCs in der entzündeten Mukosa ist ein Hinweis auf intestinales „homing“, also ein Wiedereinwandern der gereiften Lymphozyten in die Darmwand. Es ist bekannt, dass die Hefe Saccharomyces boulardii (Sb) eine Wirksamkeit bei entzündlichen sowie infektiösen Erkrankungen des Gastrointestinaltraktes hat. Kulturexperimente von mDCs mit Zellkulturüberständen von Sb (SbS) und LPS zeigten eine deutliche Reduzierung in der Expression von CD40 und CD80 sowie des Reifemarkers CD197. SbS reduzierte die Sekretion von TNF- und IL-6. Während es die Sekretion von IL-10 bei gesunden Probanden erhöhte, konnte bei CED Patienten eine leichte Abnahme verzeichnet werden. SbS vermindert die Proliferation von naïven T-Zellen in einer gemischten Lymphozytenreaktion mit gesunden mDCs signifikant. / Various animal studies have provided insights that mucosal dendritic cells play a key role in this process. However, the specific function of certain dendritic cells in IBD is still unknown. Primary CD1c+CD11c+CD14-CD19- myeloid blood (mDCs) and mucosal DCs from IBD patients and healthy controls were compared. More mDCs from IBD patients exhibited an activated phenotype shown by expression of co-stimulatory molecules. mDCs from patients secrete higher levels of pro- and anti-inflammatory cytokines. Circulating mDCs from IBD patients take up more LPS and the frequency of mucosal mDCs and the number of activated, i.e. CD40 and CD80 expressing mucosal mDCs, is significantly greater in CED. The increased frequency of activated mDCs in the inflamed mucosa suggests intestinal homing of mDCs in acute stages of IBD. Further, the data suggests an aberrant LPS response of mDCs in patients suffering from IBD which results in an inflammatory phenotype. The most widely accepted hypothesis for the cause of IBD is a disturbed interaction of the host immune system with commensal microflora and other luminal antigens. The well controlled balance of the intestinal immune system is disturbed and luminal antigens like LPS gain access to the underlying mucosal tissue via the leaky barrier. It was investigated whether the yeast preparation Saccharomyces boulardii (Sb) modulates dendritic cell function which has shown efficacy in inflammatory and infectious disorders of the gastrointestinal tract. Culture experiments of mDCs in the presence of Sb culture supernatant (SbS) significantly reduced the expression of CD40 and CD80 as well as the DC maturation marker CD197 (CCR7) induced by the prototypical microbial antigen LPS. SbS reduced secretion of TNF- and IL-6, while the secretion of anti-inflammatory IL-10 increased. IBD patients showed also a reduction in their secretion level of IL-10. SbS inhibited proliferation of naïve T cells in a mixed lymphocyte reaction with healthy mDCs.

Le respect de l’équilibre entre lymphocytes T effecteurs auto-réactifs et lymphocytes T régulateurs (LTreg) est primordial dans le maintien de la tolérance aux antigènes du soi. Les partenaires cellulaires et les mécanismes moléculaires impliqués dans la rupture de l’équilibre de cette balance ne sont pas ou peu connus dans les maladies auto-immunes. Ainsi, les travaux décrits dans cette thèse portent sur le dérèglement de la balance T effecteurs/ Treg dans deux modèles de maladies auto-immunes chez l’homme: le lupus érythémateux systémique et l’anémie hémolytique auto-immune (AHAI). Nous montrons une augmentation de l’expression de la molécule de costimulation OX40L (CD252, TNFSF4) à la surface des cellules présentatrices d’antigène circulantes et infiltrant les tissus chez les patients lupiques. Cette augmentation est corrélée à l’activité de la maladie chez l’adulte comme chez l’enfant. Elle a pour conséquence l’induction de lymphocytes T effecteurs de type Tfh (T follicular helper) et le blocage des fonctions suppressives des Treg, deux acteurs majeurs dans la physiopathologie du lupus. Dans le second projet, nous montrons une augmentation de la proportion de T8reg circulants chez les patients affectés d’une AHAI à anticorps chauds en phase de rémission. Ces Treg expriment le CD25, le FoxP3 et exercent leur fonction suppressive par un mécanisme faisant intervenir l’IL10. De faibles doses d’IL-2 permettent l’expansion de cette population cellulaire in vitro. Ces résultats apportent de nouvelles connaissances dans la physiopathologie de ces deux maladies et offrent des perspectives thérapeutiques potentielles. / Respect of the balance between autoreactive T cells and regulatory T cells (LTreg) is important to maintain tolerance to self-antigens. Cellular partners and molecular mechanisms involved in the disruption of this balance are not or little known in autoimmune diseases.Thus, the work described in this thesis focuses on the disruption of the T effector/ Treg balance in two models of human autoimmune diseases: systemic lupus erythematosus and autoimmune hemolytic anemia (AIHA). We show an increased expression of the OX40L (CD252, TNFSF4) costimulatory molecule at the surface of both circulating and tissues-infiltrating antigen presenting cells in SLE patients. OX40L expression is correlated with disease activity in adults and in children and results in Tfh (follicular helper T) effector cells induction and Treg suppressive functions inhibition, two key mechanisms in the pathogenesis of lupus. In the second project, we show an increase of the circulating T8reg proportion in patients with a warm AIHA in a non-active state. These Treg express CD25, FoxP3 and exert their suppressive function by a mechanism involving IL-10. Low-dose IL-2 allows the expansion of this cell population in vitro. These results provide new insights into the pathophysiology of these diseases and offer potential therapeutic perspectives.