archives@tulane.edu / Effective adaptive immune responses depend on the presentation to CD4+ T cells antigen peptides bound to major histocompatibility complex class II proteins. The structure of an antigen strongly influences its processing within the endolysosome and potentially controls the identity and abundance of peptides that are presented to T cells. The dissertation presented here sought to expand our understanding of how antigen structure and stability influence adaptive immune responses for two model antigens. Pseudomonas exotoxin A domain III (PE-III) functions as an ADP-ribosyltransferase with significant cellular toxicity and has been incorporated into a recombinant immunotoxin for the treatment of cancer. The bacterial component of the PE-III immunotoxin is highly immunogenic and generates neutralizing antibodies that render subsequent treatments ineffective. A group of six single-amino-acid substitutions in PE-III that were predicted to disrupt CD4+ T-cell epitopes have been shown to reduce antibody responses in mice. Here we demonstrate that only one of the substitutions, R494A, exhibits reduced folding stability and proteolytic resistance through the removal of a hydrogen bond. This destabilization significantly reduces its antibody immunogenicity while generating CD4+ T-cell epitopes that are indistinguishable from those of wildtype PE-III. PE-III specific B cells isolated from R494A-immunized animals contained fewer somatic mutations, which are associated with affinity maturation, and exhibited a weaker germinal-center gene signature, compared to B cells from wildtype-immunized animals. Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possess a protease-sensitive reactive center loop that lies adjacent to the OT-II epitope. We took advantage of the previously described single-substitution-variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins to study how changes in loop size and protein stability influences CD4+ T-cell priming in vivo. We observed that OVA R339T loop-insertion increases overall stability and protease resistance and significantly shortens the reactive center loop. This results in reduced CD4+ T-cell priming of the OT-II epitope in SJL mice. These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools. / 1 / Daniel Moss

Sphingolipid metabolites play a role in the initiation and perpetuation of inflammatory responses. Since intestinal inflammation is a driving force in the development of colon cancer, in the present study, we investigated the suppression of dextran sodium sulfate (DSS)-induced colitis by dietary sphingomyelin in mice that lack functional peroxisome proliferator-activated receptor γ (PPAR-γ) in intestinal epithelial and immune cells. Dietary spingomyelin decreased colonic inflammation in mice of both genotypes but more efficiently in mice expressing PPAR-γ. Using a real-time polymerase chain reaction array, we detected an up-regulation in genes involved in Th1 (interferon γ) and Th17 (interleukin [IL]-17 and IL-23) responses despite the reduced inflammation. However, the genes involved in Th2 (IL-4, IL-13 and IL-13ra2) and Treg (IL-10rb) anti-inflammatory responses were up-regulated in a PPAR-γ-dependent manner. In order to direct mechanistic studies of how PPAR-γ expression is involved in SM-induced suppression of DSS colitis, we investigated the effect of dietary SM in DSS-treated mice that lack PPAR-γ in the CD4+ T-cells. While the pathogenesis of colitis was independent of PPAR-γ expression in CD4+ T-cells, dietary SM decreased disease activity and colonic inflammation in mice of both genotypes but more efficiently in mice expressing PPAR-γ, indicating both PPAR-γ dependent and independent signaling pathways. In conclusion, in contrast to endogenous sphingolipid metabolites, dietary SM modulated both pro- and anti-inflammatory responses at the early stages of the disease in a partially PPAR-γ dependent manner resulting in a suppression of inflammation that may be critical for the suppression of inflammation-driven colon cancer. / Master of Science

CD4+ T cells are a group of lymphocytes that play critical roles in the immune system. By releasing cytokines, CD4+ T cells regulate other immune cells for maximizing the efficiency of the system. Naive CD4+ T cells are activated and become mature upon engagement with antigens, and the mature CD4+ T cells have several subsets, which play diverse regulatory functions. For the past two decades, our understanding of CD4+ T cells has been advanced through the studies on the differentiation process and the lineage specification of various subsets of these cells. Although in most experimental studies of CD4+ T cells, researchers focused on how transcription factors and signaling molecules influence the differentiation of a particular subset of these cells, many evidence have shown that the differentiation of CD4+ T cells can be heterogeneous in terms of the phenotypes of the cells involved. This dissertation describes a framework that uses mathematical models of the dynamics of the signaling pathways to explain heterogeneous differentiation. We show that the mutual inhibitions among the master regulators govern the formation of multi-stability behavior, which in turn gives rise to heterogeneous differentiation. The framework can be applied to systems with two or more master regulators, and models based on the framework can make specific predictions about heterogeneous differentiations. In addition, this dissertation describes an experimental study on CD4+ T cell differentiation. Being part of the adaptive immune system, the differentiation of CD4+ T cells was previously known to be induced by the signals from the innate immune cells. However, the expression of Toll-like receptor in CD4+ T cells suggests that microbial products can also influence the differentiation directly. Using an in vitro cell differentiation approach, we show that the differentiation and proliferation of CD4+ T cells can be influenced by lipopolysaccharide under the condition that would favor the differentiation of induced regulatory T cells. These theoretical and experimental studies give novel insights on how CD4+ T cells differentiate in response to pathogenic challenges, and help to gain deeper understanding of regulatory mechanisms of the complex immune system. / Ph. D.

CD4+ T cells mediate and orchestrate a tremendous panoply of lymphoid cell subsets in the human immune system. CD4+ T cells are able to differentiate into either effector pro-inflammatory or regulatory anti-inflammatory subsets depending on the cytokine milieu in their environment. This complex process is mediated through a variety of cytokines and soluble factors. Yet, the mechanisms of action underlying the process of differentiation and plasticity of this interesting immune subset are incompletely understood. To gain a better understanding of the CD4+ T cell differentiation and function, here we present an array of different strategies to model and validate CD4+ T cell differentiation and heterogeneity. The approaches presented here vary from ordinary-differential equation-based to agent-based simulations, from data-driven to theory-based approaches, and from intracellular mathematical to tissue-level or cellular modeling. The knowledge generated throughout this dissertation exemplifies how a combination of computational modeling with experimental immunology can efficiently advance the scene on CD4+ T cell differentiation. In this thesis I present i) an overview on CD4+ T cell differentiation and an introduction to which computational strategies have been adopted in the field to tackle with this problem, ii) ODE-based modeling and predictions on Th17 plasticity modulated by PPARγ, iii) ODE- and ABM-based cellular level modeling of immune responses towards Helicobacter pylori and the role of CD4+ T cell subsets on it, iv) Intracellular strategies to validate a potential therapeutic target within a CD4+ T cell to treat H. pylori infection, and finally v) data-driven strategies to model Th17 differentiation based on sequencing or microarray data to generate novel predictions on specific components. I present both mathematical and computational work as well as experimental work, in vitro and in vivo with animal models, to demonstrate how computational immunology and immunoinformatics can help, not only in understanding this complex process, but also in the development of immune therapeutics for infectious, allergic and immune-mediated diseases. / Ph. D.

CD4+-T-Helferzellen sind in großer Zahl in der entzündeten Synovialmembran bei rheumatoider Arthritis (RA) sowie in Arthritismodellen vorhanden und spielen mit großer Wahrscheinlichkeit eine bedeutende Rolle in der Pathogenese von Arthritiden. Bei der präventiven Behandlung mit drei verschiedenen Anti-CD4 monoklonalen Antikörpern (mAk) im Modell der Adjuvansarthritis der Ratte (AA) wurden abhängig von dem jeweils eingesetzten mAk unterschiedliche klinische Verbesserungen beobachtet. Im Mittelpunkt der Untersuchungen stand deshalb die Suche nach Parallelen zwischen der unterschiedlichen klinischen Effizienz der Anti-CD4 mAk W3/25, OX35 (klinisch effizient) und RIB5/2 (klinisch ineffizient) bei der präventiven Therapie der AA und ihren in vitro Effekten auf TZell-Funktionen als Erklärung für die unterschiedlichen Therapieeffekte. Keine klaren Parallelen zur differentiellen klinischen Effizienz ergaben sich bei den folgenden Untersuchungen: 1.) Bestimmung der Affinitäten der mAk zum CD4-Molekül. 2.) Inhibition der Proliferation in der primären gemischten Lymphozytenkultur (1° MLC) mit CD4+-T-Zellen und CD8+-T-Zellen durch die drei mAk 3.) Beeinflussung der Produktion der Zytokine IL-2, IFNg, IL-10 und IL-4 in verschiedenen experimentellen Ansätzen (sekundäre MLC nach Anwesenheit der mAk in der 1° MLC, Kreuzvernetzung des CD4-Moleküls mittels der mAk nach bzw. vor einer Stimulation von CD4+-T-Zellen über den TZR). 4.) Einfluss der drei Anti-CD4 mAk auf die TZR-vermittelte Apoptose. 5.) Mobilisierung von intrazellulärem Kalzium durch CD4-Kreuzvernetzung mittels der mAk. 6.) Aktivität der Tyrosinkinasen p56lck und p59fyn nach CD4-Kreuzvernetzung mittels der mAk. 7) Phosphorylierung des Shc-Adaptermoleküls durch CD4-Kreuzvernetzung mittels der drei mAk. 8.) Effekte der drei mAk auf die Aktivität der Transkriptionsfaktoren NF-AT und AP-1. Dagegen ergaben sich bei den Untersuchungen zur Produktion von TNFa und zur Aktivität des Transkriptionsfaktors NF-kB eindeutige Parallelen zur differentiellen klinischen Effizienz: 1.) Die Kreuzvernetzung des CD4-Moleküls mittels des mAk RIB5/2 nach bzw. vor einer Stimulation von CD4+-T-Zellen über den TZR induzierte eine signifikant höhere Sekretion von TNFa als mit den mAk W3/25 und OX35. 2.) Die Kreuzvernetzung des CD4-Moleküls mittels des mAk RIB5/2 vor einer Stimulation von CD4+-T-Zellen über den TZR führte zu einer signifikant stärkeren Erhöhung der Aktivität von NF-kB als mit den mAk W3/25 und OX35. Beide differentiellen Effekte könnten daher die Erklärung für die unterschiedliche klinische Effizienz der drei Anti-CD4 mAk darstellen.

L’autophagie est un mécanisme de l’immunité innée contre le VIH-1 déclenché très rapidement dans les cellules T CD4 par les protéines d’enveloppe du virus (Env). Dans les cellules T CD4 appelées « bystander », c’est-à-dire où l’infection est abortive, l’autophagie entraine l’apoptose. Dans les cellules T CD4 infectées de façon productive, l’autophagie est inhibée, empêchant ainsi à la fois la dégradation du virus et/ou des protéines virales et l’apoptose médiée par Env. Le but de cette étude de doctorat est de comprendre comment l’autophagie est bloquée dans les cellules T CD4 infectées par le VIH-1. Durant la première année de ma thèse, j’ai contribué au travail montrant que Vif nouvellement synthétisé bloque l’autophagie dans les lymphocytes T CD4 infectés. Les données ont été publiées en Janvier 2015 dans AIDS sous le titre «HIV-1 viral infectivity factor interacts with microtubule-associated protein light chain 3 and inhibits autophagy». Ensuite, j’ai montré que Vpr contribue aussi à l’inhibition de l’autophagie. En effet, Vpr ectopique diminue grandement le nombre des autophagosomes présents dans les cellules T CD4 quand l’autophagie est induite par un inhibiteur de mTOR. De même, Vpr incorporé dans les virions diminue le nombre des autophagosomes présents dans les cellules T CD4 très rapidement après leur infection productive (4h et 8h). Dans le but de définir le mécanisme par lequel la protéine Vpr entraine ce blocage, j’ai fait des expériences de GST pull-down et j’ai identifié que Vpr interagit avec BNIP3, une protéine pro-autophagique. De plus, le niveau d’expression de BNIP3 est augmenté dans les cellules T CD4 après le contact avec Env, suggérant que BNIP3 pourrait être impliqué dans l’induction de l’autophagie médiée par l’Env. Vpr co-localise avec BNIP3 et Vpr incorporé dans les virions induit une diminution drastique du niveau d’expression de BNIP3 après 8 heures d’infection. En conclusion, j’ai montré qu’au moins deux protéines du VIH-1 sont séquentiellement impliquées dans l’inhibition de l’autophagie dans les cellules T CD4 infectées par le VIH-1, Vpr qui contrôle l’autophagie durant la phase précoce de l’infection, et Vif néo-synthétisé qui inhibe l’autophagie après la transcription du génome viral. La compréhension complète des mécanismes par lesquels le VIH-1 inhibe l’autophagie devrait à terme permettre l’élaboration de nouvelles stratégies thérapeutiques pour lutter contre ce virus. / Autophagy is a potent anti-HIV-1 mechanism. It is triggered in CD4 T cells by the viral envelope (Env) upon HIV-1 entry. In bystander CD4 T cells, autophagy leads to apoptosis. In productively infected CD4 T cells, autophagy is inhibited, preventing thus HIV-1 virophagy and Env-mediated apoptosis. The aim of my PhD study was to understand how autophagy is blocked in the HIV-1-infected CD4 T cells. During the first year of my thesis, I contributed to the work demonstrating that Vif neosynthesized blocks autophagy in the infected CD4 T lymphocytes. The data were published in January 2015 in AIDS under the title “HIV-1 viral infectivity factor interacts with microtubule-associated protein light chain 3 and inhibits autophagy”. Then, I demonstrated that Vpr contributes also to the inhibition of autophagy. Indeed, both ectopic expression of Vpr and Vpr incorporated into the virions decrease the number of autophagosomes in CD4 T cells when autophagy is induced by an inhibitor of mTOR and Env, respectively. To define the mechanism by which HIV-1 Vpr inhibits autophagy, I performed GST pull-down experiments and identified that Vpr interacts with BNIP3, a pro-autophagic protein. Importantly, BNIP3 expression level is increased in CD4 T cells upon Env contact, suggesting that BNIP3 could be responsible for the Env-mediated induction of autophagy. Furthermore, Vpr co-localizes with BNIP3 and viral incorporated Vpr decreases BNIP3 levels after 8 hours of infection. In conclusion, I demonstrated that at least two HIV-1 proteins are sequentially involved in the inhibition of autophagy in HIV-1-infected CD4 T cells, Vpr that controls autophagy during the early phase of infection, and then neo-synthesized Vif that inhibits autophagy after HIV-1 genome transcription. The complete understanding of the mechanisms by which HIV-1 inhibits autophagy should lead to the rising of new molecular strategies to fight against this virus.

CD4 T cells differentiate into multiple effector subsets that mediate pathogen clearance. ThCTL are anti-viral effectors with MHC-II restricted cytotoxicity. The factors regulating ThCTL generation are unclear, in part due to a lack of a signature marker. I show here that in mice, NKG2C/E identifies ThCTL that develop in the lung during influenza A virus (IAV) infection. ThCTL phenotype indicates they are highly activated effectors with high levels of binding to P-selectin, T-bet, IFNγ production, and degranulation. ThCTL express increased levels of granzymes and perforin and lower levels of genes associated with memory and recirculation compared to non-ThCTL lung effectors. ThCTL are also restricted to the site of infection, the lung in IAV and systemically in LCMV. ThCTL require Blimp-1 for their differentiation, suggesting a unique effector CD4 population. As ThCTL are highly activated, they also require antigen signaling post priming during IAV infection. Late antigen was necessary and sufficient for the differentiation of ThCTL. In the context of late antigen encounter, ThCTL surprisingly do not require CD80 and CD86 costimulation for their differentiation. Additionally ThCTL do not require late IL-2 for their differentiation and instead require late IL-15 signals for their efficient generation. Thus these data suggest ThCTL are marked by the expression of NKG2C/E and represent a unique CD4 effector population specialized for cytotoxicity.

<p>Understanding the mechanisms that control T cell function and differentiation is crucial to develop new strategies to modulate immune function and prevent autoimmune and inflammatory disease. The balance between effector (Teff; Th1, Th2 and Th17) and regulatory (Treg) T cells is critical to provide an appropriate, but not excessive, immune response and therapies to induce Treg or inhibit Teff are likely promising treatment strategies. It has recently become clear that T cell metabolism is important in both T cell activation and differentiation. T cells undergo a metabolic reprogramming upon activation and not all differentiated T cell subsets utilize the same metabolic fuels or programs.</p><p>These metabolic differences are not trivial, as T cell metabolism is tightly</p><p>regulated and dysregulation can lead to cell death or reduced immunity. An</p><p>understanding of the metabolic differences between Teff and Treg may lead to a new direction for treating inflammatory diseases by modulating the Teff:Treg balance through metabolic inhibition. Previous studies have shown that Teff express higher levels of the glucose transporter Glut1 than Treg, however the role of Glut1, and importantly, the cell-intrinsic role of glucose metabolism in T cell differentiation and inflammation was not previously examined. The work presented here examines the role of Glut1 in T cell differentiation. We show that effector CD4 T cells were dependent on Glut1 for proliferation and function both in vitro and in vivo. In contrast, Treg were Glut1-independent and capable of suppressing colitis in the absence of Glut1 expression.</p><p>Additionally, previous studies have shown broad metabolic differences between Teff and Treg, however the specific metabolic profiles of Teff and Treg are poorly understood. Here, Teff and Treg metabolism is examined to test if dependence on distinct metabolic pathways will allow selective targeting of different T cell populations. We show that pyruvate dehydrogenase kinase 1 (PDHK1) is differentially expressed in the T cell subsets and inhibition of PDHK1 selectively suppresses Th17 and promotes Treg differentiation and function. Because Teff and Treg have distinct metabolic profiles, we hypothesized that the Treg-­specific transcription factor FoxP3 may drive the Treg oxidative metabolic program. We therefore examined the role of FoxP3 in T cell metabolism and determined that FoxP3 promotes glucose and lipid oxidation and suppresses glycolytic metabolism. Importantly, we show that promoting glycolysis with transgenic expression of Glut1 inhibits Treg suppressive capacity. Together, these data suggest that FoxP3 drives an oxidative metabolic program that is critical to Treg function. Overall, this work examines the metabolic phenotypes and regulation of Teff and Treg and potential metabolic targets that could be used to treat autoimmune and inflammatory disease.</p> / Dissertation

A lectina ArtinM, extraída de sementes de Artocarpus heterophyllus e caracterizada como um homotetrâmero constituído de subunidades de 16 kDa, tem alta afinidade de ligação a manotriose Man? 1-3 [Man? 1-6] Man, que constitui o core de N-glicanas. ArtinM é dotada de interessantes propriedades biológicas: (1) ativa neutrófilos a partir do reconhecimento de N-glicanas dos receptores CXCR2 e TLR2; (2) induz a desgranulação de mastócitos por interagir com N-glicanas de Fc?R ou com N-glicanas de IgE ligadas a Fc?R; (3) estimula a produção de IL-12, por reconhecer N-glicanas contidas no ectodomínio de TLR2 da superfície de células apresentadoras de antígeno (APCs); (4) exerce atividade imunomoduladora, que direciona o padrão de resposta para o perfil Th1; (5) confere resistência a infecções por patógenos intracelulares, como Paracoccidioides brasiliensis, Leishmania amazonensis e Leishmania major, Neospora caninum e Candida albicans Células T CD4+ participam de funções essenciais do sistema imune; durante o estabelecimento de uma resposta imune, podem ser desenvolvidas subpopulações de células T CD4+ adequadas para gerar respostas eficientes de combate a patógenos, manutenção da tolerância e regulação da imunidade. A ativação das células T CD4+ depende de um primeiro sinal, desencadeado pelo complexo TCR/CD3, e de um segundo sinal, oriundo de moléculas coestimulatórias como CD28. A ativação e expansão de células T CD4+ são limitadas pela ação de moléculas inibitórias, principalmente por CTLA-4. Lectinas podem ativar as células T, sendo a fitohemaglutinina (PHA) e a Concanavalin A (ConA) os exemplos mais conhecidos. Além disso, está bem caracterizado que o alvo de reconhecimento de ConA localiza-se no complexo TCR/CD3. No presente estudo buscou-se caracterizar os efeitos da lectina ArtinM sobre células T CD4+ murinas e investigar os possíveis mecanismos responsáveis pelos efeitos exercidos. Foram avaliados, inicialmente, os efeitos diretos de ArtinM sobre as células T CD4+, no que se refere à produção de citocinas, expressão de moléculas coestimulatórias e inibitórias e indução de diferenciação celular. Passou-se então à identificação de possíveis receptores de superfície reconhecidos por ArtinM e responsáveis pelo desencadeamento da ativação celular. Finalmente, buscou-se apontar moléculas sinalizadoras envolvidas nos efeitos diretos de ArtinM. A primeira evidência da interação direta de ArtinM com células T CD4+ foi proporcionada por aglutinação celular. Uma curva dose-resposta revelou que 5µg/ml foi a melhor concentração para adquirir significativa produção de citocinas Th1 (IL-2 e IFN-?) e Th17 (IL-6 e IL-17A) pelas células T CD4+. O estímulo com a concentração ótima de ArtinM mostrou que após 12 horas de incubação houve um significativo aumento nos níveis de IL-2, IFN-?, IL-6 e IL-17A no sobrenadante celular; persistindo no curso de 48 horas de observação. A secreção concomitante de IFN-? e IL-17A motivou a avaliação, por citometria de fluxo, da ocorrência de dupla marcação intracelular dessas citocinas. O estímulo, por 24 horas, com ArtinM, levou a importante aumento da frequência de células duplo-positivas para IFN-? e IL-17. Uma vez comprovado pelo padrão de citocinas secretadas que ArtinM promove a ativação das células T CD4+, investigou-se a expressão das moléculas CD25 e CTLA-4. ArtinM aumentou a expressão de ambas as moléculas, de maneira dose-dependente. Curiosamente, a detecção tanto de CD28, como de CTLA-4, foi precoce e persistente, diferindo do padrão temporal de expressão proporcionado por outros ativadores de células T CD4+. Com vistas a determinar o mecanismo através do qual ArtinM atua nas células T CD4+, alvos potenciais de reconhecimento foram ensaiados: CD3?, CD3??, CD28, CD45 e CD4. Esses receptores foram selecionados com base em predição de potenciais sítios Nglicosilados. Dessa forma, anticorpos específicos para essas moléculas foram utilizados para analisar a sua capacidade de inibir a atividade de ArtinM de induzir as células T CD4+ a produzir citocinas, como IL-2, IFN-?, IL-6 e IL-17A. Apenas o anticorpo anti-CD3?? foi capaz de impedir a secreção das citocinas induzidas por ArtinM. Além disso, esse anticorpo inibiu a marcação de células T CD4+ por ArtinM biotinilada. Esses dados indicam que ArtinM exerce sua atividade sobre células T CD4+ através do reconhecimento de glicanas na cadeia ? do receptor CD3, não excluindo-se, entretanto, a ocorrência da interação de ArtinM com outras glicoproteínas na superfície de linfócitos T CD4+. Também foi verificado que ArtinM possui alta especificidade por glicanas na superfície dessas células, pois foram necessárias elevadas concentrações de manotriose para inibir em 50% a ligação de ArtinM à superfície das células T CD4+. Através do uso de inibidores específicos para moléculas sinalizadoras, constatou-se que PI3K, PTK, p42/44MAPK, p38MAPK, JNK e PKC estão implicadas na sinalização para a produção das citocinas de perfis Th1 e Th17, induzida por ArtinM. Esse conjunto de resultados indica que ArtinM é um potente e rápido ativador de células T CD4+. A ativação celular induzida por ArtinM está relacionada com a ligação à cadeia ? do receptor CD3 e se associa à alta expressão de moléculas coestimuladoras e inibitórias. Ademais, demonstrou-se que ArtinM promove a diferenciação das células T CD4+ naive em células Th1 e Th17, utilizando moléculas sinalizadoras que são conhecidas como críticas para a indução de citocinas que caracterizam essas subpopulações celulares. / The lectin ArtinM, extracted from seeds of Artocarpus heterophyllus and characterized as a homotetramer consisted of 16 kDa subunits, has high binding affinity to the manotriose Man? 1-3 [Man? 1-6] Man, which is the core of N-glycans. ArtinM is endowed with interesting biological properties: (1) it activates neutrophils through the recognition of Nglycans attached to CXCR2 and TLR2 receptors; (2) induces degranulation of mast cells by interacting with N-glycans of Fc?R or to N-glycans of IgE bound to Fc?R; (3) stimulates the production of IL-12 through the recognition of N-glycans of the TLR2 ectodomain, expressed on the surface of antigen presenting cells (APCs); (4) exerts immunomodulatory activity, which accounts for Th1 immunity (5) confers resistance to intracellular pathogens, such as P. brasiliensis, Leishmania amazonensis and Leishmania major, Neospora caninum e Candida albicans. CD4+ T cells participate in essential functions of the immune system. During the development of an immune response, CD4+ T cells are activated and give origin to subpopulations of cells that are suitable for establishing effective responses to combat pathogens, for tolerance maintenance, and for adequate immuneregulation. The activation of CD4+ T cells depends on a first signal, triggered by the TCR/CD3 complex, and a second signal, provided by costimulatory molecules. The activation and expansion of CD4+ T cells is limited by the action of inhibitory molecules. Lectins may activate T cells, and Phytohemagglutinin (PHA) and Concanavalin A (ConA) are the best know examples. Furthermore, it is well characterized that the target for ConA recognition is localized in the TCR/CD3 complex. The present study was delineated to characterize the effects of the lectin ArtinM on murine CD4+ T cells and to investigate the possible mechanisms accounting for the observed effects. It was investigated the ArtinM direct effects on CD4+ T cells, concerning its ability to induce the production of cytokines, the expression of costimulatory and inhibitory molecules and cell differentiation. In addition, the possible surface receptors recognized by ArtinM and responsible for triggering cell activation were also assessed. Finally, signaling molecules involved in the direct effects of ArtinM were approached. The first evidence of direct interaction of ArtinM with CD4+ T cells was provided by cell agglutination. A dose-response curve has revealed that 5µg/ml was the best ArtinM concentration to achieve significant production of Th1 (IL-2 and IFN-?) and Th17 (IL-6 and IL-17A) cytokines by TCD4+ cells. Stimulus with the optimum ArtinM concentration has showed that after 12 hours incubation there was a significant augmentation of IL-2, IFN-?, IL- 6 and IL-17A levels in the cell supernatant; which has persisted in the course of 48 hours observation. The concomitant secretion of IFN-? and IL-17A led us to evaluate, by flow cytometry, the intracellular expression of these cytokines. After 24 hours stimulation with ArtinM, there was a significant increase in the frequency of cells IFN-?+IL-17+. Once the cytokines detection indicated that CD4+ T cells have been activated by ArtinM, the expression of CD25 and CTLA-4 molecules was assessed. ArtinM increased the expression of both molecules, in a dose-dependent manner. Interestingly, both cell surface molecules, CD25 and CTLA-4, were early and persistently detected a temporal pattern that is distinct from the provided by other inducers of CD4+ T cell activation. In order to determine the mechanism by which ArtinM acts on CD4+ T cells, potential targets of recognition were assessed: CD3??, CD3?, CD28, CD45 and CD4. These receptors were selected on the basis of prediction of N-glycosylation sites. Specific antibodies for these molecules were assayed regarding their ability to inhibit the ArtinM of inducing TCD4+ cells to produce cytokines, such as IL-2, IFN-?, IL-6 and IL-17A. Only anti-CD3 antibody was able to prevent the cytokines secretion induced by ArtinM. In addition, anti-CD3 antibody has inhibited the T CD4+ cell labeling by biotynil-ArtinM. These data indicate that ArtinM exerts its biological activity on T CD4+ cells through recognition of CD3 receptor ? chain glycans, without excluding the occurrence of ArtinM interactions with other glycoproteins on the surface of T CD4+ lymphocytes. The interaction of ArtinM with glycans at the surface of these cells was found to occur with great specificity, since high concentrations of the manotriose - Man? 1-3 [Man? 1-6] Man - were required to inhibit the binding. By using specific inhibitors of signaling molecules, we have found that PI3K, PTK and p42/44MAPK are relevant cytokine production profiles of Th1 and Th17 cells after stimulation with ArtinM. All toghether, these results indicate that ArtinM is a potent and rapid activator of CD4+ T cells. The activation induced by ArtinM is triggered by its binding to the CD3 receptor ? chain, which induces high expression of costimulator and inhibitory molecules. Moreover, it was demonstrated that ArtinM promotes the differentiation of naive CD4+ T cells into Th1 and Th17 cells by committing signaling molecules that are known as critical for the induction of cytokines that characterize these subpopulations of cells.

A lectina ArtinM, extraída de sementes de Artocarpus heterophyllus e caracterizada como um homotetrâmero constituído de subunidades de 16 kDa, tem alta afinidade de ligação a manotriose Man? 1-3 [Man? 1-6] Man, que constitui o core de N-glicanas. ArtinM é dotada de interessantes propriedades biológicas: (1) ativa neutrófilos a partir do reconhecimento de N-glicanas dos receptores CXCR2 e TLR2; (2) induz a desgranulação de mastócitos por interagir com N-glicanas de Fc?R ou com N-glicanas de IgE ligadas a Fc?R; (3) estimula a produção de IL-12, por reconhecer N-glicanas contidas no ectodomínio de TLR2 da superfície de células apresentadoras de antígeno (APCs); (4) exerce atividade imunomoduladora, que direciona o padrão de resposta para o perfil Th1; (5) confere resistência a infecções por patógenos intracelulares, como Paracoccidioides brasiliensis, Leishmania amazonensis e Leishmania major, Neospora caninum e Candida albicans Células T CD4+ participam de funções essenciais do sistema imune; durante o estabelecimento de uma resposta imune, podem ser desenvolvidas subpopulações de células T CD4+ adequadas para gerar respostas eficientes de combate a patógenos, manutenção da tolerância e regulação da imunidade. A ativação das células T CD4+ depende de um primeiro sinal, desencadeado pelo complexo TCR/CD3, e de um segundo sinal, oriundo de moléculas coestimulatórias como CD28. A ativação e expansão de células T CD4+ são limitadas pela ação de moléculas inibitórias, principalmente por CTLA-4. Lectinas podem ativar as células T, sendo a fitohemaglutinina (PHA) e a Concanavalin A (ConA) os exemplos mais conhecidos. Além disso, está bem caracterizado que o alvo de reconhecimento de ConA localiza-se no complexo TCR/CD3. No presente estudo buscou-se caracterizar os efeitos da lectina ArtinM sobre células T CD4+ murinas e investigar os possíveis mecanismos responsáveis pelos efeitos exercidos. Foram avaliados, inicialmente, os efeitos diretos de ArtinM sobre as células T CD4+, no que se refere à produção de citocinas, expressão de moléculas coestimulatórias e inibitórias e indução de diferenciação celular. Passou-se então à identificação de possíveis receptores de superfície reconhecidos por ArtinM e responsáveis pelo desencadeamento da ativação celular. Finalmente, buscou-se apontar moléculas sinalizadoras envolvidas nos efeitos diretos de ArtinM. A primeira evidência da interação direta de ArtinM com células T CD4+ foi proporcionada por aglutinação celular. Uma curva dose-resposta revelou que 5µg/ml foi a melhor concentração para adquirir significativa produção de citocinas Th1 (IL-2 e IFN-?) e Th17 (IL-6 e IL-17A) pelas células T CD4+. O estímulo com a concentração ótima de ArtinM mostrou que após 12 horas de incubação houve um significativo aumento nos níveis de IL-2, IFN-?, IL-6 e IL-17A no sobrenadante celular; persistindo no curso de 48 horas de observação. A secreção concomitante de IFN-? e IL-17A motivou a avaliação, por citometria de fluxo, da ocorrência de dupla marcação intracelular dessas citocinas. O estímulo, por 24 horas, com ArtinM, levou a importante aumento da frequência de células duplo-positivas para IFN-? e IL-17. Uma vez comprovado pelo padrão de citocinas secretadas que ArtinM promove a ativação das células T CD4+, investigou-se a expressão das moléculas CD25 e CTLA-4. ArtinM aumentou a expressão de ambas as moléculas, de maneira dose-dependente. Curiosamente, a detecção tanto de CD28, como de CTLA-4, foi precoce e persistente, diferindo do padrão temporal de expressão proporcionado por outros ativadores de células T CD4+. Com vistas a determinar o mecanismo através do qual ArtinM atua nas células T CD4+, alvos potenciais de reconhecimento foram ensaiados: CD3?, CD3??, CD28, CD45 e CD4. Esses receptores foram selecionados com base em predição de potenciais sítios Nglicosilados. Dessa forma, anticorpos específicos para essas moléculas foram utilizados para analisar a sua capacidade de inibir a atividade de ArtinM de induzir as células T CD4+ a produzir citocinas, como IL-2, IFN-?, IL-6 e IL-17A. Apenas o anticorpo anti-CD3?? foi capaz de impedir a secreção das citocinas induzidas por ArtinM. Além disso, esse anticorpo inibiu a marcação de células T CD4+ por ArtinM biotinilada. Esses dados indicam que ArtinM exerce sua atividade sobre células T CD4+ através do reconhecimento de glicanas na cadeia ? do receptor CD3, não excluindo-se, entretanto, a ocorrência da interação de ArtinM com outras glicoproteínas na superfície de linfócitos T CD4+. Também foi verificado que ArtinM possui alta especificidade por glicanas na superfície dessas células, pois foram necessárias elevadas concentrações de manotriose para inibir em 50% a ligação de ArtinM à superfície das células T CD4+. Através do uso de inibidores específicos para moléculas sinalizadoras, constatou-se que PI3K, PTK, p42/44MAPK, p38MAPK, JNK e PKC estão implicadas na sinalização para a produção das citocinas de perfis Th1 e Th17, induzida por ArtinM. Esse conjunto de resultados indica que ArtinM é um potente e rápido ativador de células T CD4+. A ativação celular induzida por ArtinM está relacionada com a ligação à cadeia ? do receptor CD3 e se associa à alta expressão de moléculas coestimuladoras e inibitórias. Ademais, demonstrou-se que ArtinM promove a diferenciação das células T CD4+ naive em células Th1 e Th17, utilizando moléculas sinalizadoras que são conhecidas como críticas para a indução de citocinas que caracterizam essas subpopulações celulares. / The lectin ArtinM, extracted from seeds of Artocarpus heterophyllus and characterized as a homotetramer consisted of 16 kDa subunits, has high binding affinity to the manotriose Man? 1-3 [Man? 1-6] Man, which is the core of N-glycans. ArtinM is endowed with interesting biological properties: (1) it activates neutrophils through the recognition of Nglycans attached to CXCR2 and TLR2 receptors; (2) induces degranulation of mast cells by interacting with N-glycans of Fc?R or to N-glycans of IgE bound to Fc?R; (3) stimulates the production of IL-12 through the recognition of N-glycans of the TLR2 ectodomain, expressed on the surface of antigen presenting cells (APCs); (4) exerts immunomodulatory activity, which accounts for Th1 immunity (5) confers resistance to intracellular pathogens, such as P. brasiliensis, Leishmania amazonensis and Leishmania major, Neospora caninum e Candida albicans. CD4+ T cells participate in essential functions of the immune system. During the development of an immune response, CD4+ T cells are activated and give origin to subpopulations of cells that are suitable for establishing effective responses to combat pathogens, for tolerance maintenance, and for adequate immuneregulation. The activation of CD4+ T cells depends on a first signal, triggered by the TCR/CD3 complex, and a second signal, provided by costimulatory molecules. The activation and expansion of CD4+ T cells is limited by the action of inhibitory molecules. Lectins may activate T cells, and Phytohemagglutinin (PHA) and Concanavalin A (ConA) are the best know examples. Furthermore, it is well characterized that the target for ConA recognition is localized in the TCR/CD3 complex. The present study was delineated to characterize the effects of the lectin ArtinM on murine CD4+ T cells and to investigate the possible mechanisms accounting for the observed effects. It was investigated the ArtinM direct effects on CD4+ T cells, concerning its ability to induce the production of cytokines, the expression of costimulatory and inhibitory molecules and cell differentiation. In addition, the possible surface receptors recognized by ArtinM and responsible for triggering cell activation were also assessed. Finally, signaling molecules involved in the direct effects of ArtinM were approached. The first evidence of direct interaction of ArtinM with CD4+ T cells was provided by cell agglutination. A dose-response curve has revealed that 5µg/ml was the best ArtinM concentration to achieve significant production of Th1 (IL-2 and IFN-?) and Th17 (IL-6 and IL-17A) cytokines by TCD4+ cells. Stimulus with the optimum ArtinM concentration has showed that after 12 hours incubation there was a significant augmentation of IL-2, IFN-?, IL- 6 and IL-17A levels in the cell supernatant; which has persisted in the course of 48 hours observation. The concomitant secretion of IFN-? and IL-17A led us to evaluate, by flow cytometry, the intracellular expression of these cytokines. After 24 hours stimulation with ArtinM, there was a significant increase in the frequency of cells IFN-?+IL-17+. Once the cytokines detection indicated that CD4+ T cells have been activated by ArtinM, the expression of CD25 and CTLA-4 molecules was assessed. ArtinM increased the expression of both molecules, in a dose-dependent manner. Interestingly, both cell surface molecules, CD25 and CTLA-4, were early and persistently detected a temporal pattern that is distinct from the provided by other inducers of CD4+ T cell activation. In order to determine the mechanism by which ArtinM acts on CD4+ T cells, potential targets of recognition were assessed: CD3??, CD3?, CD28, CD45 and CD4. These receptors were selected on the basis of prediction of N-glycosylation sites. Specific antibodies for these molecules were assayed regarding their ability to inhibit the ArtinM of inducing TCD4+ cells to produce cytokines, such as IL-2, IFN-?, IL-6 and IL-17A. Only anti-CD3 antibody was able to prevent the cytokines secretion induced by ArtinM. In addition, anti-CD3 antibody has inhibited the T CD4+ cell labeling by biotynil-ArtinM. These data indicate that ArtinM exerts its biological activity on T CD4+ cells through recognition of CD3 receptor ? chain glycans, without excluding the occurrence of ArtinM interactions with other glycoproteins on the surface of T CD4+ lymphocytes. The interaction of ArtinM with glycans at the surface of these cells was found to occur with great specificity, since high concentrations of the manotriose - Man? 1-3 [Man? 1-6] Man - were required to inhibit the binding. By using specific inhibitors of signaling molecules, we have found that PI3K, PTK and p42/44MAPK are relevant cytokine production profiles of Th1 and Th17 cells after stimulation with ArtinM. All toghether, these results indicate that ArtinM is a potent and rapid activator of CD4+ T cells. The activation induced by ArtinM is triggered by its binding to the CD3 receptor ? chain, which induces high expression of costimulator and inhibitory molecules. Moreover, it was demonstrated that ArtinM promotes the differentiation of naive CD4+ T cells into Th1 and Th17 cells by committing signaling molecules that are known as critical for the induction of cytokines that characterize these subpopulations of cells.