P>Dendritic cells (DCs) play an important role in the clearance of apoptotic cells. The removal of apoptotic cells leads to peripheral tolerance, although their role is still not clear. We show that the uptake of apoptotic thymocytes by DCs converts these cells into tolerogenic DCs resistant to maturation by lipopolysaccharide, modulating the production of interleukin-12 and up-regulating the expression of transforming growth factor-beta(1) latency associated peptide. We also observed that DCs pulsed with apoptotic cells in the allogeneic context were more efficient in the expansion of regulatory T cells (Tregs), and that this expansion requires contact between DCs and the T cell. The Tregs sorted from in vitro culture suppressed the proliferation of splenocytes in vitro in a specific and non-specific manner. In the in vivo model, the transfer of CD4+ CD25- cells to Nude mice induced autoimmunity, with cell infiltrate found in the stomach, colon, liver and kidneys. The co-transfer of CD4+ CD25- and CD4+ CD25+ prevented the presence of cell infiltrates in several organs and increased the total cell count in lymph nodes. Our data indicate that apoptotic cells have an important role in peripheral tolerance via induction of tolerogenic DCs and CD4+ CD25+ Foxp3+ cells that present regulatory functions.; National Council for Scientific and Technologic Development (CNPq); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Carrapatos são artrópodes hematófagos de distribuição cosmopolita que têm grande importância médica e veterinária devido ao efeito deletério direto causado por se fixarem e sugarem seus hospedeiros, como também por serem importantes vetores de doenças para o homem e para os animais domésticos. Sabendo que carrapatos permanecem fixos em seus hospedeiros por longos períodos de tempo sem serem rejeitados, é possível inferir que esses ácaros possuam um arsenal de mecanismos que atuem no controle da resposta imune do hospedeiro. De fato, diversos trabalhos têm demonstrado que carrapatos são capazes de modular a resposta imune de seus hospedeiros através de componentes presentes na saliva, que são inoculados durante o repasto sangüíneo. Assim, este trabalho procurou investigar se carrapatos exercem a modulação da resposta imune do hospedeiro através do recrutamento de células T regulatórias CD4+CD25+ (Tregs), com a intenção de conter uma resposta inflamatória / imune prejudicial à sua alimentação. Para isso, células isoladas de amostras de pele e linfonodos de camundongos BALB/c infestados com carrapatos Rhipicephalus sanguineus foram analisadas quanto à expressão das moléculas de superfície CD4...

Dentre as infecções bacterianas, a tuberculose é responsável pelo maior número de casos no mundo. É uma doença freqüentemente fatal quando associada com cepas resistentes e extremamente resistentes às drogas, ao abandono de tratamento e imunossupressão. Dependendo da natureza e da magnitude da resposta do hospedeiro, uma inflamação excessiva, comumente não protetora nos indivíduos susceptíveis, acompanha a progressão da infecção. Nesse sentido, é de grande interesse identificar mecanismos que não somente caracterizem a evolução da infecção como também aqueles que participam no controle do dano tecidual. Neste estudo, nós usamos linhagens de animais com susceptibilidade distinta à infecção por M. tuberculosis com o objetivo de avaliar se a freqüência e a atividade das células T reguladoras são influenciadas por características genéticas do hospedeiro e se essas diferenças poderiam representar um fator de susceptibilidade durante a tuberculose experimental. Nossos resultados mostram que tanto a freqüência como a atividade supressora das células T reguladoras de animais BALB/c estava aumentada, inibindo a produção de IFN-g e IL-2 por células efetoras CD4+CD25-. Essa atividade supressora não parece ser dependente de IL-10 ou TGF-b. Do contrário...

Paracoccidioides brasiliensis é um fungo dimórfico patogênico agente etiológico da paracoccidioidomicose (PCM), uma micose endêmica no Brasil. A busca por alternativas para reduzir o tempo de tratamento da PCM levou ao desenvolvimento de uma vacina de DNA contendo a sequência do peptídeo P10 de P. brasiliensis. Neste trabalho, avaliamos a geração de células T de memória e células T reguladoras em camundongos imunizados com esta vacina de DNA antes e após o desafio com o fungo, através da análise de seus esplenócitos e linfócitos pulmonares por citometria de fluxo. Os resultados mostram um aumento no percentual de células T reguladoras e de memória no baço e pulmões dos animais imunizados antes e depois de 30, 60 e 120 dias do desafio em comparação com os grupos controle e não imunizado. Outro experimento revelou que o modelo experimental da PCM in vivo é capaz de induzir a expressão de RORγt. Este estudo mostra que nossa vacina de DNA contra a PCM gera células com fenótipo de reguladoras e de memória, caracterizando seu potencial para o tratamento desta micose.; Paracoccidioides brasiliensis is a dimorphic fungal pathogen that is the etiological agent of paracoccidioidomycosis (PCM), a mycosis endemic in Brazil. The search for new alternatives to reduce the duration of PCM treatment led to the development of a DNA vaccine encoding the peptide P10 of P. brasiliensis. Presently...

Imunização terapêutica utilizando células dendríticas derivadas de monócitos (MoDCs) pulsadas com antígenos de HIV constitui um meio promissor de potencializar a resposta imune específica anti-HIV em pacientes infectados. Neste contexto, é importante ressaltar que células dendríticas além de estimular a resposta imune específica, podem ser capazes de promover a tolerância periférica em linfócitos T CD4+ e T CD8+ ao induzir deleção, anergia ou através da expansão de células T reguladoras (T regs). Experimentos in vitro foram conduzidos para avaliar a capacidade de MoDCs pulsadas com HIV autólogo inativado em induzir apoptose celular, respostas celulares específicas e a geração de T regs. Os pacientes avaliados neste estudo foram indivíduos infectados pelo HIV, sem uso de tratamento antirretroviral (n = 14) com número de células T CD4+ acima de 350 células/L. MoDCs foram geradas a partir de células mononucleares de sangue periférico e em seguida foram pulsadas com vírus autólogo inativado por Aldrithiol-2, tratadas com estímulo para maturação e então cultivadas com linfócitos autólogos. A apoptose de linfócitos T e MoDCs e a frequência de células efetoras e reguladoras foram avaliadas por citometria de fluxo. Os resultados obtidos mostraram que não houve diferença nos níveis de apoptose de células T CD4+...

A cromoblastomicose é uma infecção fúngica crônica que acomete pele e tecido subcutâneo. As lesões podem ser classificadas em tumoral, verrucosa, cicatricial e do tipo placa. A resposta imune é principalmente celular e a forma grave da doença correlaciona-se com citocinas de perfil Th2. Nós exploramos populações celulares do tipo T reguladoras e Th17. Foram utilizadas vinte e três biópsias da forma verrucosa obtidas de pacientes com diagnóstico clínico e histopatológico de cromoblastomicose, sem tratamento. Foi realizado o método de imunohistoquímica para detectar Foxp3, CD25, TGF-beta, IL-6, IL-17 e IL-23. A IL-17 predominou sobre os outros marcadores, embora haja número regular de Foxp3. TGF-beta, IL-6 e IL-23 raramente foram visualizados. A constituição de uma resposta imune local com alta expressão de IL-17 e baixa expressão de outras citocinas pode ser, ao menos em parte, uma tentativa de ajudar o sistema imunológico contra infecções fúngicas. Células Foxp3 poderiam ser capazes de interferir na resposta imune eficiente contra fungos, mas também beneficiar o hospedeiro, através da capacidade de reduzir os danos do tecido que seguem uma resposta imune local. Esses elementos celulares podem contribuir para a cronicidade que caracteriza esta doença; Chromoblastomycosis is a chronic fungal infection that affects skin and subcutaneous tissue. Lesions can be classified in tumorous...

T-cell immunoglobulin mucin 3 (TIM3) is an inhibitory molecule that has emerged as a key regulator of dysfunctional or exhausted CD8+ T cells arising in chronic diseases such as cancer. In addition to exhausted CD8+ T cells, highly suppressive regulatory T cells (Tregs) represent a significant barrier against the induction of antitumor immunity. We have found that the majority of intratumoral FOXP3+ Tregs express TIM3. TIM3+ Tregs co-express PD-1, are highly suppressive and comprise a specialized subset of tissue Tregs that are rarely observed in the peripheral tissues or blood of tumor-bearing mice. The co-blockade of the TIM3 and PD-1 signaling pathways in vivo results in the downregulation of molecules associated with TIM3+ Treg suppressor functions. This suggests that the potent clinical efficacy of co-blocking TIM3 and PD-1 signal transduction cascades likely stems from the reversal of T-cell exhaustion combined with the inhibition of regulatory T-cell function in tumor tissues. Interestingly, we find that TIM3+ Tregs accumulate in the tumor tissue prior to the appearance of exhausted CD8+ T cells, and that the depletion of Tregs at this stage interferes with the development of the exhausted phenotype by CD8+ T cells. Collectively...

The immune system requires a network of regulatory mechanisms that enable the host to maintain immune regulation, homeostasis and tolerance. A functionally committed CD4+CD25+FOXP3+ T cells subset (Treg cells) have a key role in determining the outcomes of protective immunity to a spectrum of foreign antigens while maintaining tolerance to self-antigens and suppressing excessive inflammation that can cause pathology. The transcription factor forkhead P3 (FOXP3) is highly expressed in Treg cells and it is critical for their suppressive function. The importance of FOXP3 is demonstrated in humans with a severe autoimmunity disease called immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) caused by mutations in FOXP3 gene. Therefore, there is an increasing interest in manipulating FOXP3 function and/or using CD4+CD25+FOXP3+ Treg cells as cell therapy to modify immune responses in cancer, autoimmunity and transplantation. The transcription factor FOXP3 has been shown to regulate negatively some genes such as Il2 and positively others, such as Cd25 and Ctla4. To better understand the function of FOXP3 as transcriptional activator, the regulation of CD25, the IL-2Rα chain, by FOXP3 was investigated (Part I). Analyzing a regulatory region of Cd25 promoter...

BACKGROUND: Adaptation of the maternal immune response to accommodate the semi-allogeneic fetus is necessary for pregnancy success, and disturbances in maternal tolerance are implicated in infertility and reproductive pathologies. T regulatory (Treg) cells are a recently discovered subset of T-lymphocytes with potent suppressive activity and pivotal roles in curtailing destructive immune responses and preventing autoimmune disease. METHODS: A systematic review was undertaken of the published literature on Treg cells in the ovary, testes, uterus and gestational tissues in pregnancy, and their link with infertility, miscarriage and pathologies of pregnancy. An overview of current knowledge on the generation, activation and modes of action of Treg cells in controlling immune responses is provided, and strategies for manipulating regulatory T-cells for potential applications in reproductive medicine are discussed. RESULTS: Studies in mouse models show that Treg cells are essential for maternal tolerance of the conceptus, and that expansion of the Treg cell pool through antigen-specific and antigen non-specific pathways allows their suppressive actions to be exerted in the critical peri-implantation phase of pregnancy. In women, Treg cells accumulate in the decidua and are elevated in maternal blood from early in the first trimester. Inadequate numbers of Treg cells or their functional deficiency are linked with infertility...

Dendritic cells (DC) and regulatory T cells (T(regs) ) are vital to the development of transplant tolerance. Curcumin is a novel biological agent extracted from Curcuma longa (turmeric), with anti-inflammatory and anti-oxidant activity mediated via nuclear factor (NF)-κB inhibition. We investigated the immunomodulatory effects of curcumin on human monocyte-derived and murine DC. Human monocyte-derived DC (hu-Mo-DC) were generated in the presence (CurcDC) or absence (matDC) of 25 µM curcumin, and matured using lipopolysaccharide (1 µg/ml). DC phenotype and allostimulatory capacity was assessed. CD11c(+) DC were isolated from C57BL/6 mice, pretreated with curcumin and injected into BALB/c mice, followed by evaluation of in vivo T cell populations and alloproliferative response. Curcumin induced DC differentiation towards maturation-arrest. CurcDC demonstrated minimal CD83 expression (<2%), down-regulation of CD80 and CD86 (50% and 30%, respectively) and reduction (10%) in both major histocompatibility complex (MHC) class II and CD40 expression compared to matDC. CurcDC also displayed decreased RelB and interleukin (IL)-12 mRNA and protein expression. Functionally, CurcDC allostimulatory capacity was decreased by up to 60% (P < 0·001) and intracellular interferon (IFN-γ) expression in the responding T cell population were reduced by 50% (P < 0·05). T cell hyporesponsiveness was due to generation of CD4(+) CD25(hi) CD127(lo) forkhead box P3 (FoxP3)(+) T(regs) that exerted suppressive functions on naïve syngeneic T cells...

For successful pregnancy, the maternal immune system must tolerate the presence of a fetus that expresses alloantigens. The appropriate and timely acquisition of this state of tolerance is critical and emerging evidence suggests that it needs to be present from the time the embryo implants into the uterus. Recently it has been demonstrated that a subpopulation of lymphocytes termed CD4⁺CD25⁺ regulatory T cells (Treg cells) are required for immune tolerance of the fetus during pregnancy. Despite their importance the factors that control regulatory T cells during pregnancy, and in particular in the peri-implantation period, are poorly understood. Using mouse models we have assessed the role of the ejaculate and its components (sperm and seminal plasma) in coordinating Treg cells in the period prior to embryo implantation. We have also used mice with a null mutation in the interleukin 10 (IL-10) gene to assess the role of this cytokine in coordination of Treg cell populations in later pregnancy. Experiments in the peri-implantation period just prior to implantation (day 3.5 postcoitum) showed that there was a significant increase (approximately 2-fold; p<0.05) in the total number of (CD4⁺Foxp3⁺) Treg cells in the iliac lymph nodes (LNs) that drain the uterus...

The autoimmune regulator (Aire) is involved in the prevention of autoimmunity by promoting thymic expression of tissue restricted antigens which leads to elimination of self-reactive T cells. We found that Aire knockout (KO) mice as well as mouse strains that are susceptible to experimental autoimmune myasthenia gravis (EAMG) have lower thymic expression of acetylcholine receptor (AChR- the main autoantigen in MG), compared to wild type (WT) mice and EAMG-resistant mouse strains, respectively. We demonstrated that Aire KO mice have a significant and reproducible lower frequency of CD4+Foxp3+ cells and a higher expression of Th17 markers in their thymus, compared to wild type (WT) mice. These findings led us to expect that Aire KO mice would display increased susceptibility to EAMG. Surprisingly, when EAMG was induced in young (2 month-old) mice, EAMG was milder in Aire KO than in WT mice for several weeks until the age of about 5 months. However, when EAMG was induced in relatively aged (6 month-old) mice, Aire KO mice presented higher disease severity than WT controls. This age-related change in susceptibility to EAMG correlated with an elevated proportion of Treg cells in the spleens of young but not old KO, compared to WT mice, suggesting a role for peripheral Treg cells in the course of disease. Our observations point to a possible link between Aire and Treg cells and suggest an involvement for both in the pathogenesis of myasthenia.; Revital Aricha...

The mechanism behind the apparent lack of effective antiviral immune responses in chronic hepatitis C virus (HCV) patients is poorly understood. It remains unclear if natural regulatory T cells (Treg) contribute to the induction and maintenance of HCV persistence. We herein report for the first time that CD25highIFN-italic gamma-FOXP3high Tregs can be rapidly induced by culturing peripheral blood mononuclear cells (PBMCs) of HCV-positive patients with HCV protein-derived peptides. The HCV-specific Tregs, generally CD4+CD45RO+, did not proliferate in response to HCV peptide and failed to produce interferon (IFN)-italic gamma, in distinct contrast to antiviral effector cells. Stimulation of healthy donor PBMCs with HCV peptides did not result in CD25 and FOXP3 upregulation above non-antigen background. To further investigate the antigen specificity of these potentially disease-associated natural Tregs, CD25+ cells were isolated from PBMCs, labeled with carboxyfluorescein diacetate succinimidylester and added back to CD25-depleted PBMCs, and the co-cultures were then stimulated with individual peptides derived from the HCV core protein. We found that the actual peptide that can stimulate Treg varied between patients, but within any given subject only a small number of the peptides were able to stimulate Treg...

Regulatory T cells are a purported lineage of CD4⁺ cells that inhibit the proliferation and effector functions of other T cells to prevent the development of autoimmune disease. However, little is known about how they arise, their lifespan and their patterns of recirculation. Furthermore, the mechanisms through which they inhibit other T cells remain unclear. In order to address these issues, we investigated the relationship between regulatory T cells and recent thymic emigrants (RTE) which are newly formed T cells released into the periphery from the thymus. The CD25⁺ Foxp3⁺ regulatory T cell subset was found to be closely associated with RTE, and generated the CD25⁻ Foxp3⁺ T regulatory T cell subset by unidirectional differentiation. This process was exploited to mature flow sorted CD4⁺ CD25bright[bright in superscript]Foxp3⁺ T cells into CD25⁻ Foxp3⁺ T cells and determine that they retain their functional suppressive activity. The phenotype and physiology of the CD25⁺ Foxp3⁺ and CD25⁻ Foxp3⁺ regulatory T cell subsets were characterised and compared to conventional T cell subsets, revealing the differential expression of numerous key molecules. The high expression of CD62L and LFA-1 by CD25⁺ Foxp3⁺ regulatory T cells was consistent with both their relative enrichment within secondary lymphoid tissues and their sessile nature. The profile of adhesion molecules on the surface of CD25⁻ Foxp3⁺ cells suggested they may tend to localise to sites of inflammation other than the lamina propria...

Purpose of review: Regulatory T cells exert a dominant effect in controlling autoimmunity and maintaining peripheral tolerance. Regulatory T cells are also involved in preventing allograft rejection and graft versus host disease. Cellular therapy with expanded regulatory T cells represents a promising approach to control T-cell mediated pathology. In this review we will summarize the efforts to design new methods for expanding regulatory T cells and exploit their regulatory function as cellular therapy for the treatment of graft versus host disease after hematopoietic stem cell transplantation. Recent findings: Among CD4+ T cells, the best described are the naturally occurring CD4+CD25+ regulatory T cells and type 1 regulatory T cells. Recent progress has been made in the characterization of both subsets in terms of isolation and induction, respectively. However, a clear definition of their mechanisms of action has still to be achieved. Summary: Better understanding of the mechanisms of suppression mediated by regulatory T cells might enable their use to modulate specific immune responses. Moreover, the recent development of methods allowing the ex-vivo expansion of regulatory T cells, to provide sufficient number of cells for in-vivo infusion...

Die Experimentelle Autoimmune Enzephalomyelitis (EAE) ist das Tiermodell der Multiplen Sklerose. Die EAE verläuft schubförmig und ist durch eine Entzündung gegen Myelinscheiden und Axone des Zentralen Nervensystem (ZNS) gekennzeichnet. Diese Entzündung verursacht schwere, aber reversible Lähmungen aller Extremitäten. Die wesentliche pathogene Zellpopulation in der EAE scheinen Myelin-spezifische CD4+-T Zellen mit einer Th1-Polarisierung zu sein. Die EAE kann in geeigneten Mausstämmen durch Immunisierung der Tiere mit Myelin-Komponenten im Freundschen Adjuvans ausgelöst werden. In der vorliegenden Arbeit wurden SJL-Mäuse gewählt, die mit einem Epitop (PLP139-151) des Proteolipidproteins immunisiert wurden. In dieser Arbeit wurden nun Mechanismen antigenspezifischer Immuntherapien untersucht. Hierfür wurden die Mäuse mit rekombinanten Invarianten Ketten, deren CLIP-Fragment durch das Epitop PLP139-151 ersetzt wurde, behandelt. Wie vor kurzem in unserer Arbeitsgruppe gezeigt wurde, kann mit diesen rekombinanten Invarianten Ketten (Ii-PLP) die EAE vollständig unterdrückt werden, wobei die zellulären Mechanismen dieser Immuntherapie bisher nicht untersucht wurden. Mit Hilfe der neu etablierten MHC Klasse II Tetramere konnten in der vorliegenden Arbeit autoreaktive CD4+-T Zellen in antigenspezifischer Weise untersucht werden. In der vorliegenden Arbeit wurde auf diese Weise gezeigt...

Inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and coeliac disease are debilitating gastrointestinal diseases that seriously affect the quality of life of those affected. Under normal circumstances, the intestinal immune system is maintained in a state of controlled inflammation, whereby balance exists between protective immunity, mediated by effector cells, and tolerance mediated by cells with regulatory function. However, an aberrant immune response is believed to contribute to the intestinal inflammation present in individuals afflicted by these diseases. This thesis investigated the involvement of CD4⁺ CD25[superscript]high Foxp3⁺ Regulatory T cells (Treg) and Th17 Effector cells in both inflammatory bowel disease (IBD) and coeliac disease. The reciprocal relationship between Treg and Th17 cells under certain cytokine conditions, has prompted the exploration of these two cell types in IBD and coeliac disease. Previous studies have examined these factors individually in a range of diseases, however, to our knowledge the study of both Treg and Th17 in IBD and coeliac disease subjects represents a novel area of research. Crohn’s disease (CD), ulcerative colitis (UC) and coeliac disease subjects were recruited through the Department of Gastroenterology and Hepatology at The Queen Elizabeth Hospital (QEH) in Adelaide...

The development of an effective vaccine against HIV has proved to be difficult. Many factors including natural regulatory T cells (Treg cells) can dampen the CD8 T-cell immunogenicity. In this study, we aimed to understand how Treg cells control CD8+ T-cell immune responses during DNA prime-boost immunization. Animals were immunized with plasmid HIV IIIB gp120 DNA following elimination of Treg cells by administration of anti-CD25 neutralizing antibody. Results demonstrated that the pool size of CD4+ T cells producing both IL-2 and/or IFN-γ (CD4+/IL-2+/IFN-γ+) was increased solely during the priming phase. An increment of tetramer binding and intracellular cytokine IFN-γ expression, however, were elevated in both primary and secondary stages in CD8+ T cells. The speed of antigen clearance was also investigated by using DNA luciferase. Surprisingly, DNA luciferase expression was declined to basal level over the ensuing observation period when Treg cells were depleted. Importantly, we found for the first time that DNA expression pattern in Treg-depleted animals was similar to that of the regular memory phase. Moreover, in mice that were exposed to antigen over 5 days prior to Treg cell depletion, CD8+ T-cell memory response was not affected. Thus...

This is thefinal version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S0896841114001462; Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcR? binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained...