O nervo vago exerce um papel importante na regulação da homeostase, e seus ramos eferentes emitem sinais capazes de atenuar a resposta inflamatória através da via do reflexo inflamatório. Nossa hipótese foi de que esta via pode também alterar a função das células dendríticas (DCs), uma vez que estas células possuem receptores colinérgicos e podem ser reguladas pelas citocinas inflamatórias. Sendo assim, DCs esplênicas e DCs geradas a partir de células precursoras da medula óssea de animais vagotomizados unilateralmente foram analisadas quanto aos marcadores fenotípicos CD11c e MHC-II e as moléculas co-estimuladoras CD80 e CD86. As DCs derivadas da medula óssea ainda foram avaliadas quanto a função fagocítica, a apresentação de antígenos, bem como, a produção de citocinas do cocultivo destas com linfócitos de camundongos OT-II. Um ensaio de reação de hiperensibilidade tardia (DTH) para se avaliar a resposta imune celular também foi realizado, mas não mostrou alterações significantes. A análise dos marcadores fenotípicos revelou um aumento da expressão de CD80 em DCs esplênicas de animais vagotomizados unilateralmente, mas não houveram alterações nas DCs derivadas da medula óssea. A avaliação da capacidade fagocítica revelou uma diminuição significante em DCs derivadas da medula óssea de animais vagotomizados unilateralmente enquanto no ensaio de proliferação de linfócitos OTII não houve efeito significante. Entre as citocinas avaliadas, a IL-6 e IFN-ϒ se apresentaram aumentadas nos animais vagotomizados unilateralmente. Portanto, o presente trabalho foi capaz de evidenciar que as DCs também estão sujeitas à modulação do sistema nervoso parassimpático, e que, uma vez moduladas, estas células são capazes de exercer influências sobre outras células do sistema imunológico. / The vagus nerve plays an important role in the regulation of homeostasis and its efferent branches emit signals capable of attenuating the inflammatory response via the inflammatory reflex. Our hypothesis was that this pathway may also alter the function of dendritic cells (DCs), since these cells have cholinergic receptors and can be regulated by the inflammatory cytokines. Thus, splenic DCs and DCs generated from bone marrow precursor cells unilaterally vagotomized animals were analyzed for phenotypic markers CD11c and MHC-II and co-stimulatory molecules CD80 and CD86. The bone marrow-derived DCs were also evaluated for phagocytic function, antigen presentation, as well as the production of cytokines such co-culture with lymphocytes from mice OT-II. A delayed type hypersensitivity test (DTH) twas also performed to evaluate the cellular immune response was also performed, but showed no significant changes. The analysis of phenotypic markers showed increased CD80 expression in splenic DCs from unilaterally vagotomized animals but there were no changes in bone marrow-derived DCs. The evaluation of phagocytic ability showed a significant decrease in DCs from unilaterally vagotomized animals while in the OT-II lymphocyte proliferation assay there was no significant effect. Among the cytokines evaluated, IL-6 and IFN-ϒ were increased in unilaterally vagotomized animals. Therefore, this study was able to demonstrate that DCs are also subject to modulation of the parasympathetic nervous system, which, once modulated, these cells are able to exert influence on other cells.

Os efeitos do estresse, que são fatores constantes e componentes importantes na vida de um indivíduo, provocam alterações sobre o sistema imunológico. Nossa hipótese foi de que o estresse agudo de contenção aplicado em três sessões em dias alternados pode alterar a expressão de marcadores de membrana e a função de células dendríticas (DCs) de camundongos Balb/c. Investigar os efeitos do estresse sobre as DC nos pareceu relevante uma vez que estas são importantes elementos de ligação entre as respostas imune inata e adaptativa, ou seja, são células apresentadoras de antígenos altamente especializadas com uma capacidade única para ativar linfócitos T. Inicialmente, foram realizados experimentos comportamentais e dosagens de hormônios relacionados à resposta ao estresse, a fim de caracterizar e validar o modelo de estresse agudo por contenção utilizado. Em seguida, DCs esplênicas e DCs geradas a partir de células precursoras da medula óssea foram analisadas quanto a expressão de marcadores fenotípicos CD11c, MHC-II, CD80, CD86, CD40 e CCR-7. O modelo de estresse agudo de contenção mostrou-se capaz de produzir alterações no comportamento e de ativar o eixo HPA e o SNS. A análise dos marcadores fenotípicos revelou um aumento da expressão de CD40 em DCs esplênicas de animais estressados, porém, não houve alterações nas DCs derivadas da medula óssea. O ensaio de proliferação demonstrou uma alteração na função de DCs na proporção de 1:1 no co-cultivo com esplenócitos. O presente trabalho, portanto, demonstrou que o estresse agudo de contenção, realizado em três sessões em dias alternados, é capaz de alterar alguns parâmetros relacionados ao fenótipo e à função de DCs esplênicas. / The effects of stress which are constant factors and important components in an individual\'s life cause changes on the immune system. Our hypothesis was acute restraint stress in three sessions on alternate days can change the expression of membrane markers and the function of dendritic cells (DCs) in BALB/c mice. Evaluate the effects of stress on the DC seemed to be relevant since these are important elements linking innate and adaptive immune responses, i,e., cells are highly specialized antigen-presenting with a unique capacity to activate T cells. First of all, behavioral assessment and dosages of hormones related to stress response were conducted to characterize and to validate the model of acute restraint stress. Then, splenic DCs and DCs generated from bone marrow were analyzed for expression of phenotypic markers CD11c, MHC-II, CD80, CD86, CD40 and CCR-7. The model of acute restraint stress changed the behavior and activate the HPA axis and the SNS. The analysis of phenotypic markers revealed an increased expression of CD40 on splenic DCs from stressed animals, however, there were no changes in BM-dDCs. The proliferation assay showed a change in the function of DCs in the coculture with splenocytes in a ratio of 1:1. Therefore, the present study indicated that acute restraint stress, conducted in three sessions on alternate days, is able to change some parameters related to the phenotype and function of splenic DCs.

Despite the actions of central tolerance during thymic selection, it is clear that the peripheral T cell repertoire contains significant numbers of self-reactive T cells. The immune system needs to curtail the risk of autoimmune disease by controlling the activity of these self-reactive T cells. Various mechanisms are in place to achieve this control (peripheral tolerance). Activation of CD4+ T cells requires two signals; engagement of the T cell receptor (TCR) with an appropriate peptide:MHC complex (signal 1), and the aggregate effect of multiple signals generated following ligation of costimulatory and coinhibitory molecules (signal 2). Both signals are required for the generation of a productive T cell response and both are provided by the professional antigen presenting cell, the dendritic cell (DC). T cells are fully activated upon receiving both signal 1 and 2, but are rendered tolerant when they receive only signal 1. This can be exploited therapeutically through the administration of peptides to induce tolerance in peptidereactive T cells. Administration of peptide with an adjuvant provides both signal 1 and 2, and leads to a sustained T cell response against the administered peptide (immunity). However, if the same peptide is administered in soluble form, only signal 1 is provided, leading to the establishment of T cell tolerance. The studies in this thesis explore the role of both signal 1 and signal 2 in peptide-induced T cell tolerance. Previous data from our laboratory have highlighted PD-1 and RANKL as costimulatory molecules which could play a role in peptide-induced T cell tolerance. Here we show that PD-1, an important coinhibitory molecule, plays a vital role in restraining peripheral T cell expansion under conditions leading to T cell immunity. However, in contrast to data from other studies, we demonstrate that PD-1 plays no role in the induction, establishment or maintenance of peptide-induced T cell tolerance. We show that the costimulatory receptor ligand pair RANK:RANKL plays a role in the balance between T cell tolerance and immunity; as administration of anti-RANKL was seen to potentiate both tolerance and immunity. We also explored the effect of altering the affinity of a peptide for MHC on the induction of peptide tolerance. We demonstrate that use of a peptide with a high-affinity for MHC induces tolerance via a novel, non-deletional mechanism of peptide-tolerance induction. Importantly, we show that the high-affinity peptide can form peptide- MHC complexes which persist in a biologically relevant form for fourteen days following peptide administration. We suggest that this leads to chronic stimulation of peptide-reactive T cells which promotes acquisition of a novel tolerant phenotype. Collectively the work described in this thesis demonstrates the important roles both signal 1 and 2 play in therapeutic-tolerance induction and how the qualitative and quantitative alteration of these signals can alter T cell fate and/or responsiveness.

Identificamos em 12 pacientes deficientes de CD40L uma incidência de infecções fúngicas de 83%, incluindo os primeiros casos de paracoccidioidomicose (PCM) e aspergilose. As células dendríticas imaturas (DCs) dos pacientes apresentaram expressão reduzida das moléculas HLA-DR, CD80 e CD86 e as DCs maduras (mDCs) pulsadas com os fungos P. brasiliensis ou C albicans apresentaram baixa expressão das moléculas HLA-DR e CD80. As mDCs dos pacientes secretaram baixa concentração de IL-12 e alta de IL-10. Nas co-culturas das mDCs com os linfócitos autólogos, observamos que a produção de IFN-<font face=\"Symbol\">g foi reduzida e a produção de IL-4 e IL-5 foi aumentada. Após tratamento com CD40L solúvel os defeitos observados foram revertidos. Os neutrófilos dos pacientes falharam na atividade fungicida em resposta ao P. brasiliensis, reduzida produção de H2O2 e sinalização via TLR1/TLR2 e TLR2/TLR6 foram observadas. Falhas na resposta de DCs e neutrófilos sugerem que esses são mecanismos imunopatológicos subjacentes à suscetibilidade às infecções fúngicas em pacientes deficientes de CD40L. / We identified 12 CD40L-deficient patients with an incidence of fungal infections of 83%, including the first case of paracoccidioidomycosis and aspergillosis. The immature DCs (iDCs) from the patients present reduced protein expression of HLA-DR, CD80 and CD86 and mature DCs (mDCs) pulsed with P. brasiliensis (mDCs) presented low expression of HLA-DR and CD80 molecules The mDCs from the patients secreted low levels of IL-12 and high of IL-10. In co-cultures of mDCs and autologous T lymphocytes we found low production of IFN-<font face=\"Symbol\">g and high production of IL-4 and IL-5. After treatment with soluble CD40L, the defects were reversed. The neutrophils from these patients showed impaired fungicidal activity in response P. brasiliensis and low production of H2O2. Additionally defective TLR1/TLR2 and TLR2/TLR6 signaling were observed. Failure in DCs and neutrophils responses are imunophatological mechanisms underlying the suscetibility to fungal infections in CD40L-deficient patients.

A análise da expressão da enzima indoleamina 2,3-dioxigenase (IDO), envolvida na regulação da resposta imune, em vacinas de células dendríticas (DCs) como imunoterapia para tratamento de indivíduos HIV+, pode fornecer informações sobre o perfil dessas células e auxiliar o aperfeiçoamento das técnicas atualmente utilizadas. A avaliação da expressão de IDO, por citometria de fluxo, foi realizada em monócitos e DCs de indivíduos sadios e HIV+. A expressão do RNAm de IDO foi analisada por PCR e a capacidade das DCs em estimular linfoproliferação e apresentar antígenos de HIV a linfócitos autólogos foi avaliada por ensaio de cocultivo. DCs ativadas de indivíduos HIV+ demonstraram expressão mais elevada de IDO tanto em relação às DCs imaturas quanto em relação às DCs dos indivíduos sadios. DCs foram capazes de induzir resposta proliferativa e polifuncional de linfócitos autólogos. Nossos resultados sugerem uma expressão diferencial de IDO entre indivíduos sadios e HIV+, indicando um importante papel da enzima no controle da resposta imune e na patogênese da AIDS. / The evaluation of indoleamine 2,3-dioxygenase (IDO) levels, a regulatory enzyme, in the context of DCs vaccines as a therapeutic alternative for the HIV+ patients, can bring information about DCs profiles that can improve current techniques of vaccine production. IDO expression was evaluated by flow cytometry in monocytes and DCs from healthy subjects and HIV+ patients. Expression of IDO mRNA was performed by real-time PCR and the ability in stimulate lymphoproliferation and presenting HIV antigens to autologous lymphocytes was evaluated in coculture assays. Comparison between immature and activated DCs showed an increased IDO expression in activated DCs in patients group. DCs derived from HIV+ patients showed an increased IDO expression when compared to healthy donors. DCs were able to induce lymphofoproliferation and polyfunctional response in autologous lymphocytes. Our results suggest a differential expression of IDO between health subjects and HIV+ patients, indicating an important role of IDO in the control of the immune response and in the HIV pathogenesis.

Dendritic cells (DCs) are the most potent professional antigen presenting cells (pAPCs) of the immune system and play a fundamental role in coordinating innate and adaptive immune responses. Through the expression of a wide array of pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), DCs recognize a variety of microbial pathogens and infectious stimuli. Stimulation of DCs through TLR ligation results in a rapid series of activation-associated events, termed "maturation," which include the upregulation of surface co-stimulatory molecule expression, inflammatory cytokine secretion, and stimulation of naïve T cells via antigen presentation by MHC molecules. Activation of DCs through TLRs is coupled with an increased metabolic demand fulfilled by a rapid change in DC glucose metabolism and characterized by increased aerobic glycolysis rates. TLR-driven glycolytic reprogramming plays an essential role in generating building blocks required for high level protein synthesis associated with maturation. Although glucose imported from extracellular environments has been broadly considered as the major driver of glycolytic metabolism in immune cells, the contributions of intracellular glucose stores to these processes are not well-defined. The role of intracellular stores of glucose, in the form of glycogen, is widely appreciated in non-immune systems. However, very little is known about the implication of glycogen metabolism in DC immune responses. This work unveils the role and potential regulatory mechanisms of glycogen metabolism in support of DC effector function. The first part of this work primarily focuses on our characterization of the role of glycogen metabolism in early DC activation responses; while in the last chapter, we describe a potential regulatory mechanism of DC glycogen metabolism by activation-associated nitric oxide (NO) production. In this work, we tested the overarching hypothesis that DC-intrinsic glycogen metabolism supports the early glycolytic reprogramming required for effector responses and that nitric oxide can regulate this metabolism. We demonstrate that DCs possess the enzymes required for glycogen metabolic machinery and that glycogen metabolism supports DC immune effector response, particularly during early activation and in nutrient-limited environments. More importantly, we uncover a very intriguing metabolic phenomenon, in which DCs engage in the differential metabolic pathways driven by carbons derived distinctively from glycogen and free glucose. Our studies present the fundamental role and regulatory mechanisms of DC-intrinsic glycogen metabolism and underline the differential utilization of glycogen and glucose metabolism to support their effector responses. Overall, this work adds to a growing field of immuno-metabolism an improved understanding of an intricate layer of metabolic mechanisms that immune cells undertake in response to immune stimuli.

We have recently demonstrated in a model of influenza A virus (IAV) infection that the absence of specific pulmonary DC subsets, including plasmacytoid DC (pDC) and CD8a+ DC, from the lungs leads to a significant decrease in the number of virus-specific CD8 T cells. Reconstitution of the lungs with physiologic numbers of pDC or CD8a+ DC is able to restore the pulmonary IAV-specific CD8 T cell response to near normal levels via a mechanism that is dependent upon direct DC:T cell interactions, DC-expressed MHC I and the presence of viral antigen. Interestingly, however, this rescue is DC subset specific, as reconstitution with purified alveolar and airway DC or alveolar macrophages was unable to rescue the virus-specific CD8 T cell response. Following IAV infection there is an abundance of IAV antigen and MHC I expressing cells present in the lungs, including infected epithelial cells. Given this fact and the inability of all DC subsets to rescue the virus-specific CD8 T cell response, it suggested that there were additional, undefined requirements for pDC- and CD8a+ DC-mediated rescue of the T cell response in the lungs. Further, although it was known that the reduction in virus-specific CD8 T cells in the lungs was a result of increased T cell apoptosis, it remained unclear what pathways of apoptosis were contributing to the increased cell death, and what mechanism pulmonary DC subsets were utilizing to rescue this defect. Here, we demonstrate that in the absence of lung-resident DC subsets, virus-specific CD8 T cells undergo significantly increased levels of apoptosis via both extrinsic activation induced cell death and intrinsic activated cell-autonomous death pathways. Reconstitution of aDC depleted lungs with pulmonary pDC and CD8a+ DC promotes increased T cell expression of the pro-survival molecule Bcl-2 and hence, increased T cell survival and accumulation in the lungs. Our studies herein demonstrate that pulmonary DC subsets utilize a variety of mechanisms to promote the rescue of virus-specific CD8 T cells in the lungs. Blockade of the costimulatory molecules CD70, and in some cases, 4-1BBL and OX40L, ablates the pulmonary DC mediated rescue of CD8 T cell numbers in the lungs, suggesting that late costimulation is one essential mechanism that pulmonary DC use to regulate CD8 T cell immunity following IAV infection. Further, we demonstrate that the absence of DC following IAV infection results in significantly reduced levels of IL-15 in the lungs and that pulmonary DC-mediated rescue of virus-specific CD8 T cell responses in the lungs requires the trans-presentation of IL-15 via DC-expressed IL-15Ra. In addition to the role of pulmonary DC mediated costimulation and IL-15 trans-presentation, we further demonstrate a previously unrecognized role for viral antigen in regulating the accumulation of both pulmonary DC and virus-specific CD8 T cells in the lungs, suggesting that viral load can dictate the nature of the inflammatory environment in the lungs and thus, regulate the character of the ensuing IAV-specific immune response. Collectively, the results detailed here demonstrate a previously unrecognized role for pulmonary DC in regulating primary IAV-specific CD8 T cell immunity, and hence, promoting enhanced viral clearance and recovery from disease.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.