A uveíte autoimune é uma doença inflamatória crônica, caracterizada pela resposta imune a antígenos oculares. É mediada por linfócitos T CD4+ com perfil TH1, e responsável por uma parcela significativa de casos de deficiências visuais e cegueira. Embora efetivos, os tratamentos disponíveis estão associados a efeitos adversos importantes. Logo, a busca de novos alvos terapêuticos mais específicos tem sido o objetivo principal no campo da imunoterapia. Nesse trabalho foi avaliada a eficiência do antagonista seletivo de CD28, mPEG PV1-Fab´(PV1), no tratamento da uveíte autoimune experimental (EAU). Camundongos tratados com PV1 exibiram menores graus de doença quando comparados a controles não tratados. Tal achado foi acompanhado de uma diminuição da ativação de linfócitos T, tanto nos olhos quanto nos órgãos linfoides periféricos desses animais. Mais ainda, o tratamento com PV1 levou a uma diminuição da população de linfócitos T reguladores e de células do tipo TH1. Portanto, concluiu-se que PV1 é eficaz no tratamento da EAU por agir em linfócitos T efetores. / Autoimmune uveitis is a T-cell mediated disease that targets mainly the posterior eye pole. Similar to human uveitis, experimental autoimmune uveitis (EAU) is mostly dependent on T cells with a TH1 phenotype. Although many treatment strategies are available, most of them focus on general immunossuppression, resulting in undesirable side effects. Thus, the development of more specific therapies is the major aim in the field of immunotherapy. Here we evaluated the efficacy of mPEG PV-1-Fab´ (PV1), a specific CD28 antagonist, in the treatment of EAU. Our results indicate that PV1 blocks T cell activation by decreasing expression of different costimulatory molecules. Furthermore, PV1 treatment led to a decrease of Treg cell population in peripheral lymphoid organs. Also, IFN-g production by CD4+ cells and TH1 lymphocytes population were decreased. Altogether, our results raise this CD28 blockade strategy as a potential tool for the treatment of autoimmune disorders in the eye, and indicate that mPEG PV1-Fab acts mainly on IFN-g production and TH1 polarization.

Les anticorps monoclonaux (AcM) ont été utilisés pour traiter des cancers dès le début des années 1980, en particulier lors du travail pionnier de l’équipe de Ronald Levy dans le traitement des lymphomes. Ces traitements ont pendant longtemps été considérés comme une sérothérapie passive à effet immédiat et à court terme. Cependant, au cours de ces dernières années, le concept d’un effet « vaccinal » des anticorps à usage thérapeutique en oncologie a peu à peu vu le jour du fait de réponses cliniques à long terme observées chez certains patients et de différentes études précliniques. En 2010, notre équipe a démontré que des souris immunocompétentes injectées avec les cellules tumorales EL4-huCD20 et traitées avec un AcM anti-huCD20 générait une réponse immunitaire anti-tumorale à long-terme par le biais de mécanismes dépendants de la région constante de l’anticorps et de lymphocytes T CD4+. Mon travail de thèse a donc porté sur l’analyse des mécanismes cellulaires et moléculaires par lesquels le traitement par un AcM anti-CD20 génère une immunité cellulaire adaptative anti-tumorale. J’ai ainsi pu montrer que le traitement des souris avec l’AcM anti-CD20 conduit à une expansion de lymphocytes Th1 producteurs d’IFN-γ, à l’apparition de lymphocytes T CD4+ effecteurs mémoires spécifiques des cellules tumorales CD20+, et au blocage de l’expansion de lymphocytes Tregs induite par les cellules tumorales. Le rôle central dans la protection anti-tumorale et la genèse d’une réponse adaptative anti-tumorale joué par l’axe IL-12/IFN-γ et leurs principales sources cellulaires, cellules dendritiques (DCs) et cellules NK, a été démontré par des expériences de neutralisation de ces cytokines, qui provoque une importante diminution du nombre de Th1 spléniques, de déplétion des cellules NK, ainsi que par des analyses phénotypiques qui ont permis d’identifier des DCs activées par le traitement - comme le montre l’expression accrue des molécules de classe II du CMH et de co-stimulation CD80 et CD86 - comme une importante source cellulaire de l’IL-12. Enfin, nous avons pu montrer qu’un variant de l’IL-2, liant préférentiellement le récepteur de l’IL-2By et faiblement le récepteur de l’IL-2aBy exprimé majoritairement par les Tregs, permettait l’obtention d’une protection anti-tumorale accrue d’animaux porteurs de tumeurs et traités par l’AcM anti-CD20. En conclusion, nous avons démontré qu’un contexte immunitaire pro-tumoral façonné par la présence d’une tumeur en développement peut être inversé par le traitement par un anticorps anti-tumoral, aboutissant à un contexte anti-tumoral. Qu’une telle réponse immunitaire adaptative cellulaire puisse être observée chez des patients atteints de lymphomes, traités par un anticorps anti-CD20, reste encore à être déterminé. / Monoclonal antibodies have been used to treat cancers since the early 1980s, in particular with the pioneer work of Ronald Levy for the treatment of lymphomas. Those treatments have been considered for a long time as a passive serotherapy with immediate and short term actions. Yet, recently, the idea of a vaccine effect of therapeutic antibodies in oncology have appeared, after preclinical studies and clinic observations suggesting a long term immune response in patients. In 2010, our team demonstrated that immunocompetent mice injected with EL4-huCD20 tumor cells and treated with anti-huCD20 monoclonal antibody generated a long term anti-tumor immune response linked with mechanisms dependent on constant part of antibodies and CD4+ T cells. My PhD work was based on the analysis of cellular and molecular mechanisms by which the treatment by an anti-CD20 mAb generates a cellar adaptive anti-tumor immunity. I could show that the treatment of mice with anti-CD20 antibody lead to the expansion of Th1 lymphocytes IFN-γ producers, to the apparition of effector memory CD4+ T cells specific for CD20 antigen, and to the blockade of the expansion of Treg cells induced by tumor cells. The key role of an adaptive anti-tumor immune response played by IL-12/IFN- γ and their main cellular sources, dendritic cells and NK cells, in the anti-tumor protection and genesis, has been demonstrated by experiments of cytokine neutralization, provoking an important decrease of splenic Th1 number, by NK depletion and by phenotypic analysis that allowed the identification of DCs activated by the treatment – as it is shown by the increased expression of MHC-II and CD80 and CD86 costimulation molecules, - as an important cellular source of IL-12. Finally, we could show that a variant of IL-2, binding preferentially IL-2By with a lower affinity for the IL-2aBy receptor mainly expressed by Tregs, could induce an increased anti-tumor protection of tumor-bearing animals treated with anti-CD20 mAb. In conclusion, we have demonstrated that a pro-tumor immune contexture affected by a growing tumor can be modified by an anti-tumor antibody leading to an anti-tumor contexture. That such cellular adaptive immune response could be observed in lymphoma patients treated with anti-CD20 still need to be determined.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 5, 2010). Vita. Thesis advisor: Habib Zaghouani. Includes bibliographical references.

Submitted by FERNANDO CHRISTIANO GABRIEL MORELLI null (fcgmorelli@yahoo.com.br) on 2016-02-12T17:49:35Z No. of bitstreams: 1 Fernando - Tese versão final Repositório UNESP.pdf: 1900802 bytes, checksum: a8c9044a1f5a46e203d3432398fc0a07 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-02-15T11:32:07Z (GMT) No. of bitstreams: 1 morelli_fcg_dr_araca.pdf: 1900802 bytes, checksum: a8c9044a1f5a46e203d3432398fc0a07 (MD5) / Made available in DSpace on 2016-02-15T11:32:07Z (GMT). No. of bitstreams: 1 morelli_fcg_dr_araca.pdf: 1900802 bytes, checksum: a8c9044a1f5a46e203d3432398fc0a07 (MD5) Previous issue date: 2016-02-01 / Erosões e úlceras são achados comuns no abomaso e causam preocupação econômica nos mais variados sistemas de produção de gado. Muitos fatores podem predispor ao aparecimento de úlceras e acúmulo de gases no abomaso, incluindo alimentos grosseiros, estresse ambiental, deficiências de vitaminas e minerais e infecções bacterianas. Essas úlceras podem ser subclínicas, sendo descobertas nas necropsias ou após o abate do animal, ou levarem à redução da motilidade do órgão, prejudicando o fluxo do seu conteúdo e causando transtornos digestivos graves e até ao aparecimento de síndromes semelhantes à indigestão vagal. Existem informações a respeito da resposta do sistema imune na maior parte das mucosas do trato gastrintestinal de não-ruminantes e ruminantes, porém são raras a respeito do abomaso. Os objetivos desse estudo foram detectar os níveis de citocinas (IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-2) no conteúdo abomasal em bovinos de corte, determinar o perfil Th1 ou Th2 dessas citocinas em animais com úlceras de grau 1 e 2 na região cárdica abomasal e comparar esses valores com os níveis de citocinas de animais sem úlceras (controle), em amostras colhidas em abatedouro, para auxiliar na compreensão da fisiopatologia do processo inflamatório local. A avaliação macroscópica e a classificação das úlceras foi realizada por meio de exames visual e histológico em amostras de tecidos da parede da região cárdica abomasal ulcerada. Os níveis de citocinas produzidas do líquido abomasal dos animais com ou sem úlceras foram avaliados por citometria de fluxo (método Cytometric Bead Array). As citocinas citadas foram detectadas no líquido do abomaso dos bovinos. Não houve diferença na liberação das citocinas entre os grupos com úlceras e o grupo sem úlcera, indicando um equilíbrio entre perfis Th1 e Th2 da resposta inflamatória. / Erosions and ulcers are common findings in the abomasum and cause economic concern in several livestock production systems. Many factors may predispose to ulcers and bloat in the abomasum, including roughage, environmental stress, deficiencies of vitamins and minerals and bacterial infections. These ulcers may be subclinical and are found during necropsy or after slaughter, or lead to reduction of abomasal motility, hindering the flow of your content and causing serious digestive disorders and even the appearance of syndromes similar to vagal indigestion. There are some studies evaluating the immune system response in most of the mucous membranes of the gastrointestinal tract of non-ruminants and ruminants, but rarely related to the abomasum. The aims of this study were to investigate the levels of cytokines (IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-2) in the abomasal fluid of beef cattle, to determine the Th1 or Th2 profile of these cytokines in animals with types 1 or 2 ulcers located in the abomasal cardic region and to compare these levels with those of animals without ulcers (controls), in samples collected in an abbatoir, to help to the understand the pathophysiology of the local inflammatory process. Ulcers from the abomasal cardic region were macroscopicaly evaluated, then classified by histology. Cytokine levels in the abomasal fluid from animals with or without ulcers were evaluated by flow cytometry (Cytometric Bead Array). Cytokines were detected in the abomasum fluid of cattle. There was no difference in the release of cytokines between groups, indicating a balance between Th1 and Th2 profiles of the inflammatory response.

Orientador: Juliana Regina Peiró / Banca: Lina Maria Wehrle Gomide / Banca:Fernanda Bovino / Banca: José Paes de Oliveira Filho / Banca:Glenda Nicioli da Silva / Resumo: Erosões e úlceras são achados comuns no abomaso e causam preocupação econômica nos mais variados sistemas de produção de gado. Muitos fatores podem predispor ao aparecimento de úlceras e acúmulo de gases no abomaso, incluindo alimentos grosseiros, estresse ambiental, deficiências de vitaminas e minerais e infecções bacterianas. Essas úlceras podem ser subclínicas, sendo descobertas nas necropsias ou após o abate do animal, ou levarem à redução da motilidade do órgão, prejudicando o fluxo do seu conteúdo e causando transtornos digestivos graves e até ao aparecimento de síndromes semelhantes à indigestão vagal. Existem informações a respeito da resposta do sistema imune na maior parte das mucosas do trato gastrintestinal de não-ruminantes e ruminantes, porém são raras a respeito do abomaso. Os objetivos desse estudo foram detectar os níveis de citocinas (IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-2) no conteúdo abomasal em bovinos de corte, determinar o perfil Th1 ou Th2 dessas citocinas em animais com úlceras de grau 1 e 2 na região cárdica abomasal e comparar esses valores com os níveis de citocinas de animais sem úlceras (controle), em amostras colhidas em abatedouro, para auxiliar na compreensão da fisiopatologia do processo inflamatório local. A avaliação macroscópica e a classificação das úlceras foi realizada por meio de exames visual e histológico em amostras de tecidos da parede da região cárdica abomasal ulcerada. Os níveis de citocinas produzidas do líquido abomasal... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Erosions and ulcers are common findings in the abomasum and cause economic concern in several livestock production systems. Many factors may predispose to ulcers and bloat in the abomasum, including roughage, environmental stress, deficiencies of vitamins and minerals and bacterial infections. These ulcers may be subclinical and are found during necropsy or after slaughter, or lead to reduction of abomasal motility, hindering the flow of your content and causing serious digestive disorders and even the appearance of syndromes similar to vagal indigestion. There are some studies evaluating the immune system response in most of the mucous membranes of the gastrointestinal tract of non-ruminants and ruminants, but rarely related to the abomasum. The aims of this study were to investigate the levels of cytokines (IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-2) in the abomasal fluid of beef cattle, to determine the Th1 or Th2 profile of these cytokines in animals with types 1 or 2 ulcers located in the abomasal cardic region and to compare these levels with those of animals without ulcers (controls), in samples collected in an abbatoir, to help to the understand the pathophysiology of the local inflammatory process. Ulcers from the abomasal cardic region were macroscopicaly evaluated, then classified by histology. Cytokine levels in the abomasal fluid from animals with or without ulcers were evaluated by flow cytometry (Cytometric Bead Array). Cytokines were detected in the abomasu... (Complete abstract click electronic access below) / Doutor

A uveíte autoimune é uma doença inflamatória crônica, caracterizada pela resposta imune a antígenos oculares. É mediada por linfócitos T CD4+ com perfil TH1, e responsável por uma parcela significativa de casos de deficiências visuais e cegueira. Embora efetivos, os tratamentos disponíveis estão associados a efeitos adversos importantes. Logo, a busca de novos alvos terapêuticos mais específicos tem sido o objetivo principal no campo da imunoterapia. Nesse trabalho foi avaliada a eficiência do antagonista seletivo de CD28, mPEG PV1-Fab´(PV1), no tratamento da uveíte autoimune experimental (EAU). Camundongos tratados com PV1 exibiram menores graus de doença quando comparados a controles não tratados. Tal achado foi acompanhado de uma diminuição da ativação de linfócitos T, tanto nos olhos quanto nos órgãos linfoides periféricos desses animais. Mais ainda, o tratamento com PV1 levou a uma diminuição da população de linfócitos T reguladores e de células do tipo TH1. Portanto, concluiu-se que PV1 é eficaz no tratamento da EAU por agir em linfócitos T efetores. / Autoimmune uveitis is a T-cell mediated disease that targets mainly the posterior eye pole. Similar to human uveitis, experimental autoimmune uveitis (EAU) is mostly dependent on T cells with a TH1 phenotype. Although many treatment strategies are available, most of them focus on general immunossuppression, resulting in undesirable side effects. Thus, the development of more specific therapies is the major aim in the field of immunotherapy. Here we evaluated the efficacy of mPEG PV-1-Fab´ (PV1), a specific CD28 antagonist, in the treatment of EAU. Our results indicate that PV1 blocks T cell activation by decreasing expression of different costimulatory molecules. Furthermore, PV1 treatment led to a decrease of Treg cell population in peripheral lymphoid organs. Also, IFN-g production by CD4+ cells and TH1 lymphocytes population were decreased. Altogether, our results raise this CD28 blockade strategy as a potential tool for the treatment of autoimmune disorders in the eye, and indicate that mPEG PV1-Fab acts mainly on IFN-g production and TH1 polarization.

Les lymphocytes T CD4 naïfs sont des cellules plastiques, capables de moduler finement leur programmation selon les signaux environnementaux qu'ils intègrent. Ils adaptent ainsi leur phénotype et leur fonction au type de danger Lors d'une infection par un agent pathogène intracellulaire par exemple, ils acquièrent un phénotype Th1 sous l'influence de médiateurs solubles tels que l'IL-12 et l' IFN-γ. Ces signaux mobilisent un set restreint de facteurs de transcription, coordonné par Tbet, qui programment la cellule afin qu'elle induise l'élimination du danger par des mécanismes impliquant une production massive d'IFN-γ. En réponse à des allergènes ou à des parasites extracellulaires, les lymphocytes T peuvent aussi acquérir un phénotype Th2, caractérisé par l'expression du facteur de transcription Gata-3 et par la production d'IL-4, d'IL-5 et d'IL-13. Afin de garantir la stabilité des lignages, ces processus de différenciation peuvent s'accompagner d'une perte de potentialité. Contrairement aux cellules T naïves, les cellules Th1 sont par exemple incapables d'allumer le programme d'expression génique Th2 en présence d'IL-4, et les lymphocytes Th2 verrouillent le programme Th1. Si nous savons aujourd'hui que l'acquisition des fonctions effectrices, comme l'équilibre entre détermination cellulaire et plasticité, sont régulés par des mécanismes épigénétiques, la plupart des acteurs moléculaires qui contrôlent la programmation des lymphocytes T au niveau de la chromatine reste encore à identifier. Durant ma thèse, j'ai étudié le rôle de la lysine méthyltransférase SETDB1, qui catalyse la di- ou tri-méthylation de la lysine 9 de l'histone 3 (H3K9me3), dans la différenciation des lymphocytes T CD4. Il avait déjà été proposé qu'H3K9me3 ait un impact sur la programmation de ces cellules en réponse aux signaux de l'environnement, mais personne n'avait encore étudié le rôle de SETDB1 dans ces processus lorsque j'ai commencé ma thèse. A l'aide d'une lignée murine déficiente pour SETDB1 spécifiquement dans les lymphocytes T, nous avons montré in vitro et in vivo que la balance Th1/Th2 est fortement augmentée en l'absence de l'enzyme, et que cette dérégulation résulte d'une perte de répression du réseau génique Th1. [...] / Upon activation, naïve CD4 T cells differentiate into distinct helper or regulatory T cell subsets depending on environmental signals received. This process relies on complex and lineage-specific gene expression programs whose dynamics and stability are regulated at the level of the chromatin. The epigenetic pathways involved, however, remain largely unknown. Here, we report that the histone methyltransferase SETDB1 critically controls the Th1 gene expression program. SETDB1-deficient naïve CD4 T cells show exacerbated Th1 priming, and when exposed to a Th1-instructive signal, SETDB1-deficient Th2 cells cross lineage boundaries and transdifferentiate into Th1 cells. Surprisingly, SETDB1 does not appear to control Th1 gene promoter activity. Instead, it deposits the repressive H3K9me3 mark at a restricted and cell-type specific set of endogenous retroviruses (ERVs) strongly associated with genes involved in immune processes. Refined bioinformatic analyses indicated that these retrotransposons either flank and repress Th1 gene cis-regulatory elements or behave themselves as Th1 gene enhancers. In conclusion, H3K9me3 deposition by SETDB1 ensures T cell lineage integrity by repressing a repertoire of ERVs that have been exapted into cis-regulatory modules to shape and control the Th1 gene network.

Indiana University-Purdue University Indianapolis (IUPUI) / Immune response enables the body to resist foreign invasions. The Inflammatory response is an important aspect in the immune response which is articulated by elements such as cytokines, APC, T-cell and B-cell, effector cell or natural killer. Of these elements, T-cells especially T-helper cells; a sub class of T-cells plays a pivotal role in stimulating the immune response by participating in various biological reactions such as, the transcription regulatory network. Transcriptional regulatory mechanisms are mediated by a set of transcription factors (TFs), that bind to a specific region (motifs or transcription factor binding sites, TFBS), on the target gene(s) controlling the expression of genes that are involved in T-helper cell mediated immune response. Eukaryotic regulatory motifs, referred to as cis regulatory modules (CRMs) or cistrome, co-occur with the regulated gene’s transcription start site (TSS) thus, providing all the essential components for building the transcriptional regulatory networks that depends on the relevant TF-TFBS interactions. Here, we study IL-12 stimulated transcriptional regulators in STAT4 mediated T helper 1 (Th1) cell development by focusing on the identification of TFBS and CRMs using a set of Stat4 ChIP-on-chip target genes. A region containing 2000 bases of Mus musculus sequences with the Stat4 binding site, derived from the ChIP-on-chip data, has been characterized for enrichment of other motifs and, thus CRMs. Our experiments identify some potential motifs, (such as NF-κB and PPARγ/RXR) being enriched in the Stat4 binding sequences compared to neighboring background sequences. Furthermore, these predicted CRMs were observed to be associated with biologically relevant target genes in the ChIP-on-chip data set by meaningful gene ontology annotations. These analyses will enable us to comprehend the complicated transcription regulatory network and at the same time categorically analyze the IL-12 stimulated Stat4 mediated Th1 cell differentiation.

L’injection de cellules immunologiquement compétentes à un hôte histo-incompatible amène une réaction qui peut se traduire par la maladie du greffon-contre-l’hôte (GVHD). La GVHD demeure une barrière importante à une utilisation plus répandue de la greffe allogénique de cellules hématopoïétiques (AHCT), pourtant un traitement efficace pour traiter de nombreuses maladies. Une meilleure compréhension des mécanismes qui sous-tendent cette pathologie pourrait en faciliter le traitement et la prévention. L’Interféron-gamma (IFN-γ) et le Transforming Growth Factor-béta (TGF-β) sont deux cytokines maîtresses de l’immunité impliquées dans la fonction et l’homéostasie des cellules greffées. Nous démontrons chez la souris que l’IFN-γ limite la reconstitution lympho-hématopoïétique de façon dose-dépendante en mobilisant des mécanismes d’apoptose et en inhibant la prolifération cellulaire. Le TGF-β est quant à lui généralement connu comme un immunosuppresseur qui contrôle l’immunité en utilisant plusieurs voies de signalisation. Le rôle relatif de ces voies en AHCT est inconnu. Nous avons étudié une de ces voies en greffant des cellules provenant de donneurs déficients pour le gène SMAD3 (SMAD3-KO), un médiateur central de la voie canonique du TGF-β, à des souris histo-incompatibles. Bien que l’absence de SMAD3 ne cause aucune maladie chez nos souris donneuses, l’injection de cellules SMAD3-KO amène une GVHD du colon sévère chez le receveur. Cette atteinte est caractérisée par une différenciation Th1 et une infiltration massive de granulocytes témoignant d’un rôle central de SMAD3 dans la physiologie des lymphocytes T CD4 et des cellules myéloïdes. Nous avons focalisé ensuite nos efforts sur le rôle de SMAD3 chez les lymphocytes T CD4 en sachant que SMAD3 était actif chez les lymphocytes T CD4 tolérants. Nous avons découvert que SMAD3 était rapidement inactivé après une activation des cellules T, suggérant que l’inactivation de SMAD3 était fonctionnellement importante pour briser l’état de tolérance. Des études de micro-puces d’ADNc nous ont montré que SMAD3 contrôlait en effet l’expression de nombreux transcrits de gènes connus comme étant reliés à la tolérance et/ou à des processus biologiques dont les rôles dans le maintien de la tolérance sont plausibles. / The injection of immuno-competent cells into a histo-incompatible host can result in the development of Graft-versus-Host disease (GVHD). GVHD is the most significant barrier to a more widespread use of allogeneic hematopoietic cell transplantation (AHCT), a potent treatment for several diseases. A better understanding of the pathophysiological underpinnings of GVHD would facilitate the design of rational approaches to treat and prevent this complication of AHCT. Gamma-interferon (IFN-γ) and Transforming Growth Factor-beta (TGF-β) are master cytokines of immunity and have a role in the function and homeostasis of transplanted cells. Using a murine model, we show that IFN-γ curtails lympho-hamatopoitic reconstitution in a dose-dependent fashion by increasing apoptosis and by limiting donor cell proliferation. TGF-β is an immunosuppressive cytokine that controls immune cells through multiple signaling pathways. The relative contribution of these pathways in AHCT is unknown. We specifically studied the role of one of these pathways by transplanting SMAD3 deficient cells (SMAD3-KO) in histo-incompatible hosts. SMAD3 is a key mediator of the so-called canonical TGF-β signaling pathway. Although SMAD3-KO donor mice are healthy, the injection of SMAD3-KO cells leads to severe GVHD in the hosts, characterized by intestinal involvement associated with Th1 skewing and massive granulocyte infiltration. These findings hint at a crucial role for SMAD3 in CD4 T-cell and myeloid cell biology. We then focalized on the role of SMAD3 in CD4 T cells knowing that SMAD3 is active in tolerant, resting CD4 T cells. We found that SMAD3 was rapidly inactivated upon T cell activation, suggesting that SMAD3 inactivation was functionally important to break the state of tolerance. Our cDNA microarray experiments show that indeed, SMAD3 regulates the transcript levels of multiple genes known to be involved in T cell tolerance and in biological processes plausibly related to immune tolerance.

No presente trabalho investigamos a relevância dos mediadores lipídicos (Leucotrienos) gerados pela enzima 5-Lipoxigenase (5-LO) na susceptibilidade ou resistência de camundongos ao Melanoma experimental com células tumorais B16-F0, utilizando como modelo camundongos produtores de leucotrienos (129_WT) e camundongos geneticamente deficientes \"knockout\" de 5-LO (129_5-LO KO). Primeiramente, verificamos que leucócitos peritoneais provenientes de animais WT implantados com melanoma B16-F0, apresentam aumento da expressão do gene para 5-LO (Alox5). Nossos resultados mostram que animais 5-LO KO, deficientes de 5-LO são mais eficientes no controle da progressão do tumor e apresentam significativo aumento na sobrevivência, quando comparados a animais WT, produtores de 5-LO. A nossa análise do perfil imunológico em células esplênicas indicam que a maior eficiência dos camundongos 5-LO KO no controle do crescimento de células tumorais B16-F0 estariam associados à presença numérica aumentada de neutrófilos (Gr-1+), células apresentadoras de antígeno (I-Ab+) majoritariamente CD19+CD80+ e esplenócitos capacitados para produção de altos níveis de citocinas pró-inflamatórias/efetoras como a IL-6, TNF?, IFN-? e baixos níveis de citocinas regulatórias como IL-10, 15 dias pós-implantação do tumor; a rápida geração da resposta imune polarizada para produção elevada de citocinas Th1 (IFN-?), mas não, citocinas Th2 (IL-10) e presença de maiores números de linfócitos T CD4+ e CD8+ efetoras, expressando o fenótipo CD44high ou CD44highCD62Llow. Ainda, verificamos que a deficiência genética da 5-LO ou a inibição da 5-LO pelo MK886 em células LAK, aumenta significativamente sua atividade citotóxica em células do melanoma B16-F0. Nossos resultados em conjunto, indicam que leucotrienos gerados pela enzima 5-LO, modulam negativamente a geração de resposta imune protetora em camundongos para o Melanoma B16-F0. / In the present work we examine the contribution of 5-lipoxigenase-derived lipid mediators during experimental melanoma (B16-F0) in 5-LO gene knockout (KO) mice and wild-type (WT) mice. The 5-LO KO mice presented delayed tumor growth, lesser tumor volume and delayed mortality. The greater resistance of 5-LO KO mice correlated with the following: High splenic Gr-1+ leukocytes counts, High and dominant presence of splenic IAb+CD19+CD80+ antigen-presenting cells counts and capacity of spleen cell to produce high levels of IL-6, TNF-?, IFN-? and lower levels of IL-10 early after tumor cells implantation; rapid T-cell polarization to secret high quantities of Th1 type cytokine IFN-? and low quantities of Th2 type cytokine IL-10; rapid generation and greater numbers of CD4+ and CD8+ activated T cells expressing CD45RB or CD44 markers; and also CD4+ and CD8+ CD44high or CD44highCD62Llow effector T cells. Herein, IL-2 induced splenic LAK cells from 5-LO KO mice, compared with splenic LAK cells from WT mice, were more efficient at killing B16-F0 melanoma cells. The increased B16-F0 melanoma cells killing activity were also found by treatment of splenic LAK cells from WT mice with a 5-LO activity inhibitor, MK886. Our findings suggest that 5-LO deficiency altered antigen-presenting cells profile, IFN-? and IL-10 production during skin cancer disease favoring the generation of protective immune responses and also provide evidence that 5-LO-derived LTs negatively affect the host survival during experimental B16-F0 melanoma.