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.

A paracoccidioidomicose (PCM) é um a micose profunda de natureza granulomatosa, causada pelo fungo Paracoccidioides brasiliensis (P. brasiliensis) e que compromete preferencialmente o tecido pulmonar. O P. brasiliensis sintetiza várias substâncias, dentre estas, destaca-se a glicoproteína de 43 kDa (gp43), a qual é considerada o principal componente antigênico do fungo. Dada a importância da gp43, anticorpos monoclonais (Mabs) contra esta glicoproteína foram obtidos e posteriormente caracterizados. Baseado na hipótese da rede idiotípica, Jerne (1974) propôs que cada anticorpo, quando produzido em resposta a um antígeno, induz a formação de outros anticorpos dirigidos contra sua região variável, na qual é única. Anticorpos anti-idiotípicos, que reconhecem o paratopo, contendo a imagem interna do antígeno, são denominados de Ab2-β e seu potencial terapêutico tem sido explorado em diferentes sistemas. Apesar da molécula de anticorpo ser complexa estruturalmente, sabemos que a região Fab, é a responsável pelo mimetismo do antígeno. Então, nosso grupo de pesquisa construiu uma nova molécula de anticorpo à partir do Mab anti-idiotípico Ab2-β, que mimetiza o antígeno gp43 de P. brasiliensis, denominada de fragmento variável de cadeia única (scFv). A transfecção dessa molécula em células dendríticas (DCs) mostrou ser promissora na PCM experimental, visto que estes transfectomas foram eficientes em apresentar a proteína scFv às células dos linfonodos, induzindo linfoproliferação, além de diminuírem a carga fúngica pulmonar. Visto que a molécula de scFv que possui a região de reconhecimento e ativação de linfócitos T mostrou eficiência no modelo de terapia na PCM experimental, o principal objetivo deste trabalho foi analisar os mecanismos pelos quais os transfectomas de DCs ativam a resposta imune em camundongos infectados com o fungo, para que possamos entender melhor a capacidade destas células em modular a resposta imune na PCM experimental. Dessa maneira, avaliamos a capacidade das DCs trasfectadas com pMAC/PS-scFv em migrar para os linfonodos regionais e induzirem uma resposta protetora com produção de IgG, bem como, analisamos o fenótipo destas células e produção de citocinas. Nosso modelo de terapia com pMAC/PS-scFv mostrou-se eficiente, uma vez que observamos diminuição de células T regulatórias nos linfonodos regionais, bem como aumento da produção de citocinas como IFN-γ e IL-12. Ainda, observamos aumento do isotipo IgG2b, sugerindo a modulação de resposta imune para o tipo Th1, importante na proteção da PCM. / The paracoccidioidomycosis (PCM) is the nature of granulomatous mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis) and preferentially compromises lung tissue. The P. brasiliensis synthesizes various substances, among these, there is a 43 kDa glycoprotein (gp43), which is considered a major antigenic component of the fungus. Given the importance of gp43 monoclonal antibodies (Mabs) against this glycoprotein were obtained and further characterized. Based on the idiotypic network hypothesis, Jerne (1974) proposed that each antibody, when produced in response to an antigen, induces the several of other antibodies production against its variable region, which is unique. Anti-idiotypic antibodies that recognize the paratope containing the internal image of the antigen are called Ab2 β and their therapeutic potential has been explored in different systems. Although the antibody molecule is structurally complex, we know that the Fab region, is responsible for antigen mimicking. Therefore, our research group has engineered a new molecule from the antibody Mab-anti-idiotypic Ab2 β that mimics the antigen gp43 of P. brasiliensis, known as single chain variable fragment (scFv). Transfection of this molecule on dendritic cells (DCs) has shown promise in experimental PCM, since these transfection were effective scFv protein present in the cells of the lymph nodes, inducing lymphoproliferation, in addition to reducing fungal burden in the lung. Since the scFv molecule that has the region recognition and activation of T lymphocytes showed efficiency in therapy model in experimental PCM, the main objective of this study was to analyze the mechanisms by which transfected DCs activate the immune response in infected mice with the fungus, so that we can better understand the ability of these cells to modulate the immune response in experimental PCM. Thus, the capacity of transfected DCs with pMAC / PS-scFv to migrate to regional lymph nodes and induce a protective response with IgG production, as well as analyze the phenotype of these cells and cytokine production. Our therapy model with pMAC / PS-scFv was efficient, since we observed decrease of regulatory T cells in regional lymph nodes, as well as increased production of cytokines such as IFN-γ and IL-12. Furthermore, we observed increased IgG2b isotype, suggesting modulation of the immune response to a Th1 response, the protect model of experimental PCM.

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.

A isquemia e reperfusão (IR) é a principal causa de insuficiência renal aguda. Evidências mostram a participação de linfócitos T e células dendríticas (DC) na lesão por IR. Entretanto, os mecanismos envolvidos não estão claros. A enzima heme oxigenase (HO)-1 está relacionada à diminuição de respostas inflamatórias. Neste trabalho, investigamos a participação de linfócitos T CD4+ e DC na proteção da lesão por IR induzida pela HO-1. Injetamos em camundongos um indutor de HO-1 (Hemin), e realizamos a IR. Avaliamos a lesão pela uréia e creatinina no soro, a expressão de citocinas por RT-PCR, nível de proteínas por bioplex e perfil celular por FACS. Observamos que a HO-1 protegeu da lesão por IR. A diminuição de INFg com a HO-1 sugeriu uma menor ativação de linfócitos T. No entanto, a transferência de células CD4+ tratadas com Hemin não apresentou diferença. Vimos que a HO-1 alterou o fenótipo das DC após IR e suprimiu a produção de TNFa pelas mesmas. Concluímos que a proteção pela HO-1 é capaz de modular a resposta inflamatória de DC, diminuindo o TNFa. / The ischemia and reperfusion (IR) is the main acute kidney injury. Evidences have shown the role of CD4+ T cells and dendritic cells (DC) in the IR injury. However, the mechanisms involved in the participation of these cells are not clear. The heme oxygenase (HO)-1 enzyme is associated to decrease in inflammatory responses. In this work, we investigated the role of CD4+ T cells and DC in renal injury protection induced by HO-1. We injected in mice a HO-1 inducer (Hemin), and we did the IR. Renal injury was evaluated by serum levels of urea and creatinine, cytokines expression by RT-PCR, proteins level by bioplex and cell profile by FACS. We observed that HO-1 lead to IR injury protection. The IFNg decrease with HO-1 suggested less T cells activation. However, transfer of hemin treated CD4+ cells did not differ from the other group. We observed that HO-1 altered DC phenotype after IR and suppressed TNFa production by these cells. We concluded that the protection by HO-1is able to modulate DC inflammatory response, diminishing TNFa.

Dendritic cells are a key population in the immune system. There are two main subsets: conventional DC (sometimes called classical DC, cDCs), which survey tissues for pathogens and activate naive T cells, and plasmacytoid dendritic cells (pDCs), which produce high amounts of IFNα in response to viral products. Both subsets begin development in the bone marrow from common dendritic progenitor (CDP), and driven by Flt3 signaling induced by the growth factor Flt3L. The CDP gives rise to a cDC committed preDC which exits the bone marrow and seeds peripheral organs to give rise to cDCs, while pDCs finish development in the bone marrow and migrate into the periphery as mature cells. pDC development is directed by E2-2, a member of the E protein family of basic helix-loop-helix transcription factors that are obligate dimers and bind an E-box sequence. E proteins are antagonized by Id proteins, and Id family member Id2 is required for cDC development. The apparently cell intrinsic choice of pDC vs. cDC fate is determined by the net activity of E proteins. Loss of E2-2 affects only pDC development, but its expression is not as specific, as E2-2 is also expressed in cDCs, macrophages and B cells. E2-2 expression in cDCs was particularly puzzling, considering the dependence of cDCs on Id2, the E2-2 antagonist. We strived to address the discrepancy between the very specific activity of E2-2 in pDC development and its broader expression. This work shows that the E2-2 locus encodes two independently regulated isoforms. E2-2Long, the more active isoform is expressed only in pDCs, and E2-2Short, the less transcriptionally active isoform, is expressed more broadly. Moreover, E2-2Long is required for optimal pDC development. Homozygous loss of just E2-2Long leads to lower pDC frequency in the spleen and phenotypically affected pDCs in the periphery. Although E2-2 is essential for pDC development, no signal has yet been identified that induces E2-2 expression to influence pDC over cDC fate. Given the essential nature of E2-2 in pDC development, we aimed to understand better how E2-2 is regulated during pDC fate specification. We examined an E2-2Long reporter and found that E2-2Long expression precedes pDC commitment in early progenitors. E2-2 is only upregulated in committed pDCs, and actively shut down in cDCs through the expression of Id2. Analysis of E2-2 in an in vitro time controlled model of dendritic cell development showed that all dendritic cells (both cDCs and pDC) go through an E2-2 expressing stage only to resolve into E2-2- cDCs and E2-2+ pDCs. Early E2-2 expression and E2-2 upregulation upon pDC commitment hinted at the presence of an E2-2 controlled cis-regulatory module. ChIP-Seq data showed only one peak of E2-2 binding located 150 kb downstream of the TCF4 gene. Heterozygous deletion of this region in the in vitro DC development model led to impaired pDC development and a fail to undergo the E2-2+ stage described above. The regulatory element is essential for E2-2 upregulation during DC development. This work describes two new mechanisms of E protein regulation during dendritic cell development: cell specific differential isoform usage and a distal cis regulatory element responsible for enforcing and upregulating E2-2 expression. These new mechanisms can lead to better understanding of how this family of broadly expressed and pleiotropic transcription factors is regulated, with implications in overall development, not only dendritic cell fate decision.

The origin of human dendritic cells (DCs) has long been debated. DCs are a subset of innate immune cells that are essential for initiating adaptive immune responses. Determining their ontogeny is critical for vaccine development and for unveiling the molecular mechanism of DC insufficiency, which underlies many primary immune deficiency disorders and leukemia. Like all blood cells, human DCs develop from hematopoietic stem cells through a sequence of increasingly restricted progenitors. Initially it was assumed that DCs should derive exclusively from myeloid progenitors. However, during the past few decades, a number of myeloid and lymphoid progenitors have been described and shown to have DC potential, instigating the debate of the myeloid vs. lymphoid origin of DCs. Hindering the resolution of this debate, human DC-restricted progenitors had not been identified. Further, the potential of known progenitors could not be interrogated due to the lack of a culture system that supports simultaneous differentiation of all human DCs subsets, in conjunction with other myeloid and lymphoid cells. In this work, we establish a culture system that supports the development of the three major subsets of DCs (plasmacytoid DCs or pDCs, and the two classical DCs, cDCs) as well as granulocytes, monocytes and lymphocytes. This system combines mitomycin C-treated murine stromal cell lines, MS5 and OP9, together with human recombinant cytokines, FLT3L, SCF and GM-CSF, and supports the differentiation of progenitor cells at a population and single cell level. Using this culture in combination with a phenotypic characterization of CD34+ progenitor cells, we identify four consecutive DC progenitors with increasing degree of commitment to DCs and describe their anatomical location of development. We show that DCs develop from a granulocyte-monocyte-DC progenitor (GMDP), which produces granulocytes and a monocyte-DC progenitor (MDP), which then generates monocytes and a common DC progenitor (CDP), which produces pDCs and a cDC precursor (pre-cDC), which finally produces cDCs only. Lastly, we establish a staining panel that allows the phenotypic identification and separation of newly found DC progenitors as well as all previously described myeloid and lymphoid progenitors. We investigate their inter-developmental relationship and DC potential at the single cell level. We show that each progenitor population with homogeneous phenotype is heterogeneous in developmental potential and prove that cell surface marker expression cannot be directly equated to a specific developmental potential. In order to resolve the unreliability of phenotype to draw developmental pathways, we propose to use the quantitative clonal output in order to delineate the DC developmental pathway. In summary, our studies provide a new tool to determine DC potential in vitro, identify new stages of DC development, and propose a new method for tracing the developmental pathway for DC lineage. This will generate a new model of dendritic cell hematopoiesis that can explain and reconcile the conflicts of data on DC origin and development.

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.