Moléculas HLA-DR e co-estimulatórias tem papel central na função imune de leucócitos. Diferentes emulsões lipídicas (EL) podem alterar funções imunes de leucócitos. Para avaliar os efeitos de diferentes EL sobre a expressão de moléculas HLA-DR, CD80 e CD86 presentes na superfície de monócitos/macrófagos (MO/M?) e CD28 e CD152 presentes na superfície de linfócitos T auxiliares (L? CD4) humanos, células mononucleares do sangue periférico de voluntários saudáveis (n=10) foram separadas com uso de Ficoll Hypaque (d=1,007) e incubadas por 48 horas (MO/M?) e 72 horas (L?) em meio RPMI 1640 acrescidas ou não (controle negativo) de diferentes EL comerciais ou misturas experimentais na concentração de 1mg/mL. De acordo com o tipo da emulsão lipídica adicionada ao meio de cultura, as células foram divididas em seis grupos experimentais: a) Controle negativo - células mononucleares cultivadas sem o acréscimo de EL b) TCLn-6 - células mononucleares cultivadas com EL a base de óleo de soja rica em ácidos graxos poliinsaturados tipo n-6 (AGPI n-6), c) TCLn-6/TCLn-3 - células mononucleares cultivadas com mistura experimental contendo 80% da EL a base de óleo de soja e 20% de EL a base de óleo de peixe rica em AGPI tipo n-3, d) TCM/TCLn6 - células mononucleares cultivadas com EL composta por 50% óleo de coco, rico em triglicérides de cadeia média e 50% de óleo de soja, e) TCM/TCLn-3 - células mononucleares cultivadas com mistura experimental contendo 80% de EL composta por 50% óleo de coco e 50% de óleo de soja e 20% de EL a base de óleo de peixe, f) SMOF - células mononucleares cultivadas com a nova EL contendo 30% de óleo de soja, 30% de triglicérides de cadeia média, 25% de óleo de oliva e 15% de óleo de peixe. As células mononucleares foram ativadas pelo uso de 10?g/mL de fitohemaglutinina. A expressão das moléculas de superfície foi analisada por citometria de fluxo. A porcentagem de fluorescência, que indica o número de células expressando as moléculas em estudo e a intensidade de fluorescência, que indica de forma indireta o número de moléculas expressas por células, foram medidas. Os resultados obtidos foram submetidos à teste estatístico Friedman e pós-teste Student-Newman-Keuls, adotando-se nível de significância de p<0,05. Devido às diferenças na expressão basal dos doadores, os resultados de intensidade de fluorescência foram transformados em porcentagem relativa ao controle basal (Basal=100). Nos grupos TCLn-6, TCLn-6/TCLn-3, TCM/TCLn-6, TCM/TCLn-3 e SMOF, a intensidade de fluorescência de moléculas HLA-DR expressas na superfície de monócitos/macrófagos diminuiu (medianas = 87,6; 84,0; 81,0; 85,0 e 80,0 respectivamente) em relação ao controle negativo (CN) (mediana=100,0) p=0,01. Todos os grupos tratados com EL aumentaram o número de linfócitos T auxiliares expressando moléculas CD28 (medianas = 90,9; 90,4; 91,5; 92,6 e 90,1 respectivamente) em relação ao CN (mediana=82,8) p=0,001 e também o número de moléculas CD152 expressas por células na superfície de linfócitos T auxiliares (medianas = 120,6; 108,8; 127,7; 114,6 e 121,3 respectivamente) em relação ao CN (mediana=100,0), p=0,03. Não foram encontradas diferenças estatísticas na expressão de moléculas CD80 e CD86 na superfície de monócitos/macrófagos cultivados com diferentes EL. Ainda não foram encontradas diferenças no número de linfócitos T auxiliares expressando CD152. Finalmente a expressão por células de moléculas CD28 na superfície de linfócitos T auxiliares também não mostrou alteração significante com as diferentes emulsões lipídicas. Conclusão: Emulsões lipídicas parenterais in vitro, diminuem a expressão de moléculas HLA-DR na superfície de monócitos/macrófagos e aumentam a expressão de moléculas CD28 e CD152 na superfície de linfócitos T auxiliares humanos. Estas alterações podem ser um dos mecanismos pelos quais as EL modulam funções de células imunes / HLA-DR and co-stimulatory molecules play a central role on leucocytes immune function. Different lipid emulsions (LE) may change leucocytes immune function. It is of interest to study the effect of different LE on HLA-DR and costimulatory molecules expression. To access the effect of LE on the HLA-DR, CD80 and CD86 expression on monocytes/macrophages (MO/MØ) surface and CD28 and CD152 (CD80/CD86 co-stimulatory molecules receptor) expression on human T helper lymphocytes (LØ CD4) surface we obtained mononuclear cells from peripheral blood of healthy volunteers (n=10) by using ficoll hypaque (d=1.077). The cells were cultured for 48 hours (MOMØ) and 72 hours (LØ CD4) and incubated with RPMI 1640 medium without (negative control) or added with 1mg/mL of commercial or experimental mixtures of five LE. Groups: a) NC - negative control without LE, b) LCTn-6 - n-6 polyunsaturated fatty acids (PUFA) rich LE ( soybean oil), c) LCTFO - 80% of LCT and 20% of n-3 PUFA rich LE (FO) (fish oil), d) MCT/LCT - LE containing 50% of medium chain triglycerides and 50% of n-6 PUFA rich LE, e) MCT/LCTFO - 80% of MCT/LCT LE and 20% of FO LE and f) SMOF - a new LE containing 30% of soybean oil, 30% of medium chain triglycerides, 25% of olive oil and 15% of fish oil. Mononuclear cells were activated by using 10?g/mL of phytohemagglutinin. Surface molecules expression was measured by flow cytometry. Percentage and intensity of fluorescence were recorded and the data were submitted to Friedman statistical test and Student-Newman-Keuls post test (p<0,05). Due the differences in basal expression between donors, prior to statistical tests, data from intensity of fluorescence were transformed of percentage relative of basal expression (where basal=100). All LE groups LCT, LCTFO, MCT/LCT, MCT/LCTFO and SMOF decreased HLA-DR intensity of fluorescence on monocytes/macrophages (mean= 87.6, 84.0, 81.0, 85.0, and 80.0 respectively) in relation to negative control (NC) (mean=100.0) cultured without LE (p=0,01). All LE groups increased the percentage of lymphocytes expressing CD28 (means=90.9, 90.4, 91.5, 92.6 and 90.1 respectively) in relation to control (mean=82.8) p=0,001 and CD152 intensity of fluorescence on lymphocytes cultured with all different LE (mean=120,6; 108,8; 127,7; 114,6 and 121,3 respectively) in relation to NC (mean=100,0), p=0,03. No significant differences were found on CD80 and CD86 expression on monocytes/macrophages surface, CD28 intensity of fluorescence and the percentage of lymphocytes expressing CD152 on lymphocytes cultured with the different studied LE. Conclusion: In vitro parenteral LE decreased HLA-DR expression on human monocytes/macrophages surface and increase co-stimulatory molecules receptor expression on human lymphocytes surface. These changes could be one of the mechanisms of LE modulation of immune cells functions

Les molécules du Complexe Majeur d'Histocompatibilité de classe I (CMH I) contrôlent la plasticité synaptique dans le système nerveux central (SNC) et plusieurs travaux expérimentaux suggèrent des interactions antigène-dépendantes entre des neurones infectés par des virus et les lymphocytes T CD8. Cependant, le rôle de la présentation des antigènes par le CMH I des neurones sur la physiopathologie de l'infection par Toxoplasma gondii (T. gondii) n'a pas encore été clarifié. Après la dissémination aigue sous forme de tachyzoites, T. gondii se convertit en bradyzoites, forme persistante dans les neurones du SNC. Chez les individus immunocompétents, la toxoplasmose latente est associée à des variations des fonctions cognitives ainsi qu'à des pathologies neuropsychiatriques. Les sujets dont le système immunitaire est dysfonctionnel peuvent développer une encéphalite létale causée par T. gondii, qui est caractérisée par une réplication du parasite, une infiltration massive et des agrégats leucocytaires et l'activation des cellules gliales. Les lymphocytes T (LT) CD8 et le CMH I sont des facteurs-clés contrôlant la résistance à l'encéphalite. Utiliser les LT CD8 pour éliminer les kystes chez des sujets à risque est une piste thérapeutique intéressante en raison de l'absence d'approches pharmacologiques ciblant les bradyzoites. A ce jour, les mécanismes et la pertinence fonctionnelle de la présentation des antigènes dérivés de T. gondii par les neurones restent à déterminer, ainsi que la contribution des différents stades parasitaires au contrôle de l'infection. L'utilisation de nouveaux parasites exprimant un antigène immunodominant uniquement au stade tachyzoite a permis de montrer que la reconnaissance par les LT CD8 d'antigènes issus des tachyzoites est suffisante pour une protection efficace contre l'encéphalite.[...] / In the Central Nervous System (CNS), Major Histocompatibility Complex class I (MHC I) molecules regulate synaptic plasticity and evidence suggests antigen-specific interactions between virus-infected neurons and CD8 T cells. Yet, little is known about the impact of neuronal MHC I presentation on the pathophysiology of infection by the neurotropic Toxoplasma gondii (T. gondii) parasite. Following acute dissemination as tachyzoites, T. gondii converts into bradyzoites that persist inside cysts within neurons of the CNS. In immunocompetent hosts, latent toxoplasmosis is associated with cognitive changes and neuropsychiatric disorders. Hosts with sub-optimal immune responses may develop a lethal T. gondii Encephalitis (TE), characterized by parasite replication, granuloma-like structures with massive immune cell influx and glial cell activation. CD8 T cells and MHC I are key determinants of TE resistance. Harnessing CD8 T cells in at-risk individuals may turn helpful in the future as we are currently lacking an effective pharmacological approach to eradicate bradyzoites. Yet the mechanisms and functional relevance of neuronal MHC I presentation of T. gondii remain unexplored, as well as which stage of the parasite contributes to efficient control of the infection. Using new T. gondii parasites with restricted expression of the immunodominant antigen at the tachyzoite stage, this work showed that CD8 T cell recognition of tachyzoite antigens at early stages of brain invasion is enough to protect from TE. Interestingly, by comparing situations of toxoplasmosis with varying TE severity and by pioneering antigen presentation assays with T. gondii-infected primary neurons, we revealed that TE susceptibility may be underlied by sub-optimal MHC I presentation of tachyzoites antigens by neurons. At last, we describe a mouse model that allows conditional deletion of a MHC I allele that is essential for TE resistance (H-2Ld). [...]

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

<p>Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator <i>(Aire)</i> gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I).</p><p>Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections.</p><p>These results indicate that Aire has an important function in peripheral tolerance by controlling the phenotype of myeloid-derived APCs and thereby regulating the activation of T and B lymphocytes.</p> / <p>Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator <i>(Aire)</i> gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I).</p><p>Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections.</p><p>These results indicate that Aire has an important function in peripheral tolerance by controlling the phenotype of myeloid-derived APCs and thereby regulating the activation of T and B lymphocytes.</p>

Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These results indicate that Aire has an important function in peripheral tolerance by controlling the phenotype of myeloid-derived APCs and thereby regulating the activation of T and B lymphocytes. / Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These results indicate that Aire has an important function in peripheral tolerance by controlling the phenotype of myeloid-derived APCs and thereby regulating the activation of T and B lymphocytes.

Thesis (Ph.D.)--Mississippi State University. College of Veterinary Medicine. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 92-107).

The overall goal of my thesis research was to understand better the mechanisms that control antigen processing and presentation by class II MHC molecules. Towards this goal I investigated ways in which the physical structure and post-translational modifications of the class II MHC alpha and beta chains and associated molecules might serve to regulate antigen processing and presentation. Specifically, I investigated (1) a hypothesis that Ii might aid binding of foreign antigenic peptides to the class II MHC foreign antigen binding site (desetope), and the application of this hypothesis to the prediction of class II-presented peptides; (2) the proteolytic cleavage of Ii to p25; (3) the proteolytic cleavage of the class II MHC alpha and beta chains, and (4) the phosphorylation of Iiand the alpha and beta chains. In exploring the hypothesis that amphipathic alpha helical peptides digested from foreign antigen, bind to the class II MHC desetope, to be presented to T cell receptors, we found such an extended, amphipathic helix in Ii (Phe146-Val164). A hypothesis was developed that this amphipathic alpha helix of Ii bound to the desetope of class II MHC molecules, and remained there from time of synthesis until catalyzing the charging of the desetope with a foreign peptide. This region of Iicould then be considered to be the prototypic T cell-presented peptide and the "strip-of-helix" algorithm was developed to search the sequences of proteins for similar amphipathic alpha helices. Such peptides might bind to the class II MHC desetope and have a high probability to be presented to the T cell. The strip-of-helix algorithm calculated the mean hydrophobicity (from Kyte-Doolittle values; Kyte and Doolittle, 1982) of sets of amino acids in axial strips down sides of helices for 3 to 6 turns, at positions n, n+4, n+7, n+11, n+14, and n+18. Peptides correlating well with T cell responsiveness had: (1) 12 to 19 amino acids (4-6 turns of an alpha helix), (2) a strip with highly hydrophobic residues, (3) adjacent, moderately hydrophilic strips, and (4) no prolines to break the helix. This algorithm predicted 10 of 12 T cell-presented peptides in 7 well-studied proteins. In a study of the post-translational modifications of Ii, an early proteolytic pathway of the destruction of Ii, resulting in the generation of p25, was described. This 25,000 dalton protein, seen in immunoprecipitates with antibodies to class II MHC molecules or to Ii, was shown to be a C-termina1 fragment of a high mannose form of Ii. The evidence for this conclusion includes the following results. [35S]methionine-1abe1ed Ii and associated molecules were immunoprecipitated, denatured, resolubi1ized and subjected to a second immunoprecipitation with various antibodies. Two antisera to C-termina1 peptides of Ii (183-193 and 192-211), but not an antiserum to an N-termina1 peptide (12-28), immunoprecipitated p25. A monoclonal antibody (mAb) to Ii immunoprecipitated [35S]methionine-1abe1ed p25 but not [35S]cysteine-1abe1ed p25, consistent with the loss of a portion of Ii containing the only cysteine in Ii, Cys28. [35S]methionine pulse-chase labeling demonstrated the maximal appearance of p25 at 20-40 min chase times. p25 molecules were reduced to about 10.5 kD by treatment with endoglycosidases F and H. p25 was, therefore, generated from a high mannose form of Ii in the ER or cis-Golgi. This finding could either implicate that site for class II MHC desetope charging with foreign peptides or reflect a mechanism for degradation of "excess" Ii molecules in the ER. Digestion of class II MHC antigen-Ii complexes with various proteases yielded fragments, migrating at and near p25 in 2-D electrophoretic gels, which were relatively resistant to further digestion. This observation was consistent with the presence of relatively protease-resistant secondary structures (domains) and a relatively protease-sensitive (IgG hinge-like) region in Iinear its insertion into the membrane. In a study of the post-translational modifications of the class II MHC alpha and beta chains, well conserved pairs of basic amino acids in the sequences of these molecules were observed. It was hypothesized these could be sites for proteolytic cleavage, as precedented in other systems (i.e.proinsulin processing). These potential cleavage sites fall in significant locations with respect to the deduced structure of the class II MHC desetope, supporting the hypothesis that these cleavages might either aid or destroy antigen presenting functions. To test this hypothesis we looked for remnant polypeptides of the alpha and beta chains. Polypeptides were observed in gels of immunoprecipitated class II MHC complexes. To identify if such polypeptides were derived from the alpha and beta chains, immunoblotting to electrotransferred polypeptides was attempted, with antisera made to synthesized peptides that mimicked eight regions of the alpha and beta chains. These antisera were produced and characterized by dot blotting, ELISA, western blotting, and immunoprecipitation of native and denatured material. One antiserum, to an alpha chain peptide (77-88), blotted to a polypeptide immunoprecipitated by anti-class II MHC antiserum. This observation supported the hypothesis that the alpha and beta chains undergo proteolytic cleavages, possibly in the control of antigen presentation. It was also demonstrated that Ii and the alpha and beta chains can be phosphorylated under varying culture conditions, but this project was not pursued.

Les nanoparticules (NP) ont été introduites en médecine pour développer des médicaments intelligents ou des agents d'imagerie. En raison de leur petite taille (<200 nm), les NP permettent la mise en place de thérapies ciblées, augmentent la diffusion et l’efficacité des drogues, tout en facilitant les modes d’administration et en diminuant les couts de santé publique. Malgré les promesses que présentent les NP pour la médecine, les risques potentiels pour la santé humaine associés à une exposition aux NP restent mal documentés ; en particulier en ce qui concerne leurs effets sur le système immunitaire. Les cellules présentatrices d’antigène (CPA) (comprenant les macrophages et les cellules dendritiques) sont recrutées au site d’inflammation induite par des pathogènes et constituent une ligne de défense majeure pour notre organisme. Les CPA sont dotées d’une activité de phagocytose et endocytose conduisant à une forte internalisation des NP. Ainsi, ces cellules seront parmi les plus affectées par une exposition aux NP et sont un modèle expérimental pertinent pour l’étude du devenir cellulaire des NP et de leurs effets sur l’hôte.Dans cette étude, nous avons étudié si les fonctions de ces cellules pourraient être modifiées par une exposition aux NP. Comme modèles de NP, nous avons choisi l'or (AuNP) et le gadolinium-polysiloxane (GdSi) utilisés comme agent de contraste ou de théranostique, le poly lactic-co-glycolic acid (PLGA) et une nano émulsion lipidique (LNP) développés comme plateforme de délivrance d’antigènes ou de médicaments. Tout d'abord, en utilisant des microsphères fluorescentes comme sonde, nous avons montré que toutes les NP testées n'altèrent pas la capacité de phagocytose de la lignée cellulaire de macrophages J774. Ensuite, l’activation des cellules a été analysée par cytométrie de flux, basée sur l'expression des marqueurs de surface CD-86 et MHC-II. Nous avons établi que l'exposition aux NP n'active pas les cellules dendritiques dérivées de la moelle osseuse (BMDC). Dans le même sens, aucune de ces NP n’induit par elle-même de sécrétions de cytokines par les BMDC. En outre, l’activation de ces cellules par des activateurs connus, tels que le lipopolysaccharide bactérien (LPS) n'est pas modifiée par les NP. Cependant, l'exposition aux AuNP diminue l'expression des cytokines IL-6, IL-12 et IL-23 par les BMDC activées par LPS. Or, ces cytokines sont impliquées dans la polarisation des lymphocytes T CD4 + vers le phénotype T helper approprié (Th). Nous avons analysé si ces modifications de cytokines pourraient modifier la réponse Th. Dans un modèle in vitro de présentation d'antigène, les BMDC ont été incubés avec un antigène modèle (ovalbumine (OVA)) et co-cultivés avec des cellules T spécifiques de l'OVA. L'exposition aux AuNP a conduit à une augmentation des cytokines spécifiques des lymphocytes T: IL-13 (indiquant un déplacement de la balance Th1 / Th2 classique vers Th2) et IL-17 (permettant de diriger les cellules T vers Th17). L'exposition des BMDC aux autres NP de l'étude ne modifie que très faiblement leurs sécrétions de cytokines inflammatoires, et n'a donc pas d'impact sur le destin des lymphocytes T après la présentation de l'antigène.L’ensemble de ces résultats démontrent que GdSi, PLGA et LNP ne modifient pas la phagocytose, l'activation des DC et la présentation antigénique. Cependant, l'exposition aux AuNP modifie les réponses inflammatoires des DC et le devenir des cellules T vers les phénotypes Th2 et Th17. Ces modifications pourraient entraver la physiologie du système immunitaire et contribuer aux maladies chroniques ou à l'auto-immunité. / Nanoparticles (NP) have been introduced in medicine to develop intelligent drugs or imaging agents. Due to their small size (<200 nm), NPs allow the development of targeted therapies, increase drug diffusion and effectiveness while facilitating modes of administration and decreasing public health costs. Nevertheless, the potential risks for human health associated to NP exposure remain poorly documented; especially about their effects on the immune system. Antigen-presenting cells (APC) (including macrophages and dendritic cells) are recruited at the site of pathogen-induced inflammation and constitute to the maintenance of body integrity, engulfing pathogens and delivering signals to other components of the immune system. Due to their internalization abilities, APC accumulate NP in their cytoplasm. Thus, these cells will be among the most affected by exposure to NP and constitute a relevant experimental model for the study of the cellular fate of NP and their effects on the host.In this study, we investigated whether the functions of these cells could be modified by an exposure to NP. As models of NP, we selected gold (AuNP) and gadolinium-polysiloxane (GdSi) used as contrast agent for therapeutic and diagnostic applications, and poly lactic-co-glycolic acid (PLGA) and lipid nano emulsion (LNP) developed as a platform for the delivery of antigens or drugs.First, using fluorescent microspheres as probe, we have shown that all of the tested NP did not alter the phagocytosis capacity of the J774 macrophage cell line. Then, cell activation was analyzed by flow cytometry, based on the expression of the surface markers CD-86 and MHC-II. We have established that NP exposure did not activate bone marrow derived dendritic cells (BMDC). In this way, none of these NP induced cytokine secretions by the BMDC. Furthermore, activation of these cells by known activators, such as bacterial lipopolysaccharide (LPS) was not modified by NP.However, in this case, the cytokine response was altered by AuNP exposure, showing reduced inflammatory cytokine production such as IL-6, IL-12 and IL-23. Interestingly, these cytokines are involved in the polarization of CD4 + T lymphocytes to the appropriate T helper phenotype (Th). In a model of antigen presentation in vitro, this cytokine profile resulted into an altered development of specific immune responses. AuNP exposure increased T cell specific cytokines: IL-13 and IL-4 (indicating a shift of classical Th1/Th2 balance towards Th2) and IL-17 (standing for an alteration of T-cell fate towards Th17). The exposure of BMDC to the other NP of the study only very slightly altered their inflammatory cytokine secretions and therefore did not affect the fate of T lymphocytes after antigen presentation.All together, these results demonstrate that GdSi, PLGA and LNP do not modify phagocytosis, DC activation and antigen presentation. However, exposure to AuNP alters the DC induced inflammatory responses and polarizes the T cell fate towards Th2 and Th17 phenotypes. These changes could impair the physiology of the immune system and contribute to chronic diseases or autoimmunity.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapeutic option for many malignant diseases. However, the efficacy of allo-HSCT is limited by the occurrence of destructive graft-versus-host disease (GVHD). Since allogeneic T cells are the driving force in the development of GVHD, their activation, proliferation, and differentiation are key factors to understanding GVHD pathogenesis. On the other hand, antigen-presenting cells (APCs) are essential for allogeneic T-cell priming and the development of GVHD. The T-box transcription factor T-bet is a master regulator for IFN-γ production and Th1 differentiation. T-bet also regulates the functions of APCs including dendritic cells (DCs) and B cells. Therefore, we investigated the role of T-bet in T cell responses, as well as on APC functions, in acute GVHD (aGVHD) using murine models of allogenic bone marrow transplantation (allo-BMT). In Chapter 2, we evaluated the roles of T-bet and IFN-γ in T-cell responses. T-bet-/- T cells induced significantly less GVHD compared with either wild-type (WT) or IFN-γ-/- counterparts in CD4-driven major histocompatibility complex (MHC)- or minor histocompatibility antigen (miHA)-mismatched models. We defined several T-bet-dependent but IFN-γ-independent molecules that may account for this distinct outcome. Further study indicates that T-bet also controls the optimal activity of Th17 cells to induce GVHD. Moreover, the compromised graft-versus-leukemia (GVL) effect of T-bet-/- T cells could be essentially reversed by IL-17 neutralization. Thus, targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD in the clinic. In Chapter 3, we evaluated the role of T-bet on APCs and found that T-bet-/- recipients developed much milder GVHD than their WT counterparts in MHC-mismatched or CD4-depedent miHA-mismatched models. As the functional readout of APCs, allogeneic donor T cells, particular CD4 subpopulation, significantly reduced IFN-γ production, proliferation and migration, and caused less damage in liver and gut in T-bet-/- recipients. We further observed that T-bet on recipient hematopoietic APCs, particular DCs, was primarily responsible for donor T-cell response and pathogenicity in GVHD. In fact, Trail/DR5 interaction served as a major signaling pathway responsible for donor T-cell apoptosis and impaired GVHD development in T-bet-/- recipients. Furthermore, T-bet expression on the host is largely dispensable for the GVL effect. Taken together, we propose that T-bet is a potential therapeutic target for the control of GVHD through regulating T cells as well as APCs. We believe further exploration and understanding of the immunobiology of T-bet in controlling the activities of T cells and APCs in GVHD will expand therapeutic options for the continuing success of allo-HSCT.