Orientadores: Wirla Maria da Silva Cunha Tamashiro. Patrícia Ucelli Simioni / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:08:57Z (GMT). No. of bitstreams: 1 Thome_Rodolfo_M.pdf: 2705250 bytes, checksum: 05d8c21f84c00f9da679ff148b082292 (MD5) Previous issue date: 2011 / Resumo: O camundongo BALB/c, linhagem geneticamente resistente à artrite induzida por colágeno (CIA), pode desenvolver um quadro similar ao de camundongos susceptíveis quando uma proteína não relacionada ao próprio, como a ovalbumina (OVA), é associada a colágeno tipo II (CII). Utilizando esse modelo, avaliamos se a tolerância oral a OVA poderia interferir nas respostas imunes contra CII, bem como o efeito da transferência adotiva de células dendríticas (DCs) tolerogênicas para camundongos artríticos. Para avaliação dos efeitos da tolerância oral sobre o desenvolvimento de artrite em BALB/c, os camundongos foram alimentados com OVA misturada à água de beber na concentração de 4mg/mL, por sete dias consecutivos, antes ou depois do desafio com CII+OVA (100?g/mL de cada antígeno). Para avaliar a participação de células dendríticas (DCs) tolerogênicas na modulação da artrite em BALB/c, células CD11c+ foram isoladas de baços de animais tolerantes à OVA e transferidas adotivamente para camundongos naïve, que foram subsequentemente imunizados com CII+OVA (100?g de cada antígeno). Para acompanhamento da evolução dos quadros de artrite, foram avaliados: o edema de patas, tomando-se regularmente as medidas de espessura de patas; realizadas análises histológicas dos tecidos articulares de joelhos e; conduzidas avaliações ex-vivo dos níveis séricos de anticorpos anti-CII e de respostas proliferativas e produção de citocinas de linfócitos T esplênicos. O tratamento com OVA antes da indução de CIA preveniu o desenvolvimento da artrite em todos os parâmetros analisados, enquanto que o tratamento com OVA após o estabelecimento da doença reduziu significativamente a inflamação e a produção de anticorpos anti-CII. Observamos ainda que a transferência de DCs tolerogênicas preveniu o aparecimento dos sinais clínicos da doença e o aumento dos níveis de anticorpos específicos no soro e reduziu significativamente a proliferação de linfócitos T CII-específicos. Enquanto a frequência de células CD4+CD25+Foxp3+ foi maior nas culturas de células de animais recipientes de DCs tolerogênicas, houve redução significativa na frequência de células produtoras de IFN? e IL-17. Os níveis de TGF-?, IL-4 e IL-10 foram significativamente mais elevados nas culturas de células esplênicas de animais recipientes de DCs tolerogênicas, enquanto que os de IFN-?, IL-6 e TNF-? foram mais reduzidos. Tomados em conjunto, nossos resultados indicam que a tolerância oral a um antígeno não relacionado ao próprio modifica o curso da artrite experimental em resposta ao colágeno, e que células dendríticas com perfil tolerogênico estão envolvidas nos fenômenos observados / Abstract: BALB/c mice, genetically resistant to collagen-induced arthritis (CIA), can develop a inflammatory condition resembling what is observed in susceptible strains when a non-related protein, such as ovalbumin (OVA), is associated with type II collagen (CII). Using this model, we evaluated whether oral tolerance to OVA could interfere in the immune response against CII, as well as the effect of adoptive transfer of tolerogenic dendritic cells (DCs) to arthritic mice. In order to evaluate the effect of oral tolerance over arthritis development in BALB/c mice, animals were fed with OVA in the drinking water at a 4mg/mL concentration, for seven consecutive days, before or after challenge with CII+OVA (100?g of each antigen). In order to evaluate the participation of tolerogenic DCs in the modulation of arthritis, splenic CD11c+ cells were isolated from OVA tolerant mice and adoptively transferred to naïve mice, which were subsequently immunized with CII+OVA. In order to monitor the evolution of the severity of arthritis, we evaluated paw edema, taking paw thickness regularly measured; performed histological analyses of articular knee tissues and, conducted ex-vivo evaluation of serum specific antibody levels and proliferation and cytokine secretion of splenic T lymphocytes. The treatment with OVA before CIA induction prevented the development of arthritis in all analyzed parameters, while the treatment after disease onset significantly reduced inflammation and CII-specific antibody production. We also observed that tolerogenic DC transfer prevented the appearance of clinical signs of arthritis, the increase of serum specific antibody levels and significantly reduced CII-specific T lymphocytes proliferation. While the frequency of CD4+CD25+Foxp3+ cells were higher in cell culture from tolerogenic DC recipient mice, frequency of IFN?- and IL-17- producing cells were significantly reduced. We observed that levels of TGF-?, IL-4 and IL-10 were significantly higher in cultures of splenic cells from mice recipient of tolerogenic DC, while levels of IFN-?, IL-6 and TNF-? were reduced. Taken together, our results indicate that oral tolerance to a non-related antigen modifies the course of experimental arthritis in response to collagen, and that dendritic cells with a tolerogenic profile are involved in the observed phenomena / Mestrado / Mestre em Genética e Biologia Molecular

Myeloid‐derived suppressor cells (MDSC s) and microRNA s (miRNA s) contribute to attenuating immune responses during chronic viral infection; however, the precise mechanisms underlying their suppressive activities remain incompletely understood. We have recently shown marked expansion of MDSC s that promote regulatory T (Treg) cell development in patients with chronic hepatitis C virus (HCV ) infection. Here we further investigated whether the HCV ‐induced expansion of MDSC s and Treg cells is regulated by an miRNA ‐mediated mechanism. The RNA array analysis revealed that six miRNA s were up‐regulated and six miRNA s were down‐regulated significantly in myeloid cells during HCV infection. Real‐time RT ‐PCR confirmed the down‐regulation of miR‐124 in MDSC s from HCV patients. Bioinformatic analysis suggested that miR‐124 may be involved in the regulation of signal transducer and activator of transcription 3 (STAT ‐3), which was overexpressed in MDSC s from HCV patients. Notably, silencing of STAT ‐3 significantly increased the miR‐124 expression, whereas reconstituting miR‐124 decreased the levels of STAT ‐3, as well as interleukin‐10 and transforming growth factor‐β , which were overexpressed in MDCS s, and reduced the frequencies of Foxp3+ Treg cells that were developed during chronic HCV infection. These results suggest that reciprocal regulation of miR‐124 and STAT ‐3 in MDSC s promotes Treg cell development, thus uncovering a novel mechanism for the expansion of MDSC and Treg cells during HCV infection.

A key step in the development of an adaptive immune response is the activation of naive T cells by dendritic cells (DCs). DCs sample antigens in the periphery and migrate to the lymphoid organs were they provide different signals to T cells: they present antigenic peptides in the context of MHC molecules, express costimulatory or coinhibitory ligands and produce cytokines that influence T cell fate. The integration of these signals will either induce tolerance or lead to the activation and expansion of effector T cells which will mediate the immune response.<p>The costimulatory CD70/CD27 pathway plays important roles in the development of pro-inflammatory Th1 and CTL responses. CD70 expression on DCs has also been described as a molecular switch from tolerance to immunity. Accordingly, its activity is tightly regulated in vivo. The aim of this work was to investigate the mechanisms controlling the expression of CD70 on dendritic cells and CD27 on T lymphocytes.<p>First, we described a cell-extrinsic mechanism of inhibition exerted on DCs by regulatory T cells (Tregs). Indeed, Tregs controlled Th1 priming in vivo and in vitro by downregulating CD70 on DCs. This control involved a transfer of the CD27 receptor to DCs, possibly via the production of CD27-bearing microvesicles by T cells at the immunological synapse. Acquisition of CD27 by DCs induced the internalization of both CD27 and CD70 and probably their lysosomal degradation. As a consequence, DCs were impaired in their ability to efficiently prime Th1 cells. Second, we analyzed CD70 and CD27 expression in the periphery and provided evidence for a cell-intrinsic control of CD27 expression by ectodomain shedding in the gastrointestinal tract.<p>While they efficiently clear infections, inflammatory responses can also be deleterious to the organism. By restraining CD70 expression on DCs, Tregs would promote tolerance and limit inflammation. Interestingly, tolerance is particularly important in the intestines, which are in constant contact with dietary antigens and the commensal microbiota. Accordingly, we propose that a second layer of control of CD27-driven costimulation takes place in the gut :by shedding CD27, T cells would be desensitized for any potential CD70-dependent costimulation.<p>To further investigate the physiological significance of the mechanisms described above, the immune response will be monitored in animals specifically lacking CD27 expression in the Treg population or expressing a nonsheddable CD27 receptor.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21451号 / 医博第4418号 / 新制||医||1032(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 椛島 健治, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Objective: Regulatory T cells (Tregs) contribute to HIV-1 disease progression by impairing antiviral immunity; however, the precise mechanisms responsible for the development of Tregs in the setting of HIV-1 infection are incompletely understood. Design: In this study, we provide evidence that HIV-induced expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) promote the differentiation of Foxp3+ Tregs. Methods: We measured MDSC induction and cytokine expression by flow cytometry and analyzed their functions by coculturing experiments. Results: We observed a dramatic increase in M-MDSC frequencies in the peripheral blood of HIV-1 seropositive (HIV-1+) individuals, even in those on antiretroviral therapy with undetectable viremia, when compared with healthy participants. We also observed increases in M-MDSCs after incubating healthy peripheral mononuclear cells (PBMCs) with HIV-1 proteins (gp120 or Tat) or Toll-like receptor 4 ligand lipopolysaccharides in vitro, an effect that could be abrogated in the presence of the phosphorylated signal transducer and activator of transcription 3 inhibitor, STA-21. Functional analyses indicated that M-MDSCs from HIV-1+ individuals express higher levels of IL-10, tumor growth factor-β, IL-4 receptor α, p47phex, programmed death-ligand 1, and phosphorylated signal transducer and activator of transcription 3 – all of which are known mediators of myelopoiesis and immunosuppression. Importantly, incubation of healthy CD4+ T cells with MDSCs derived from HIV-1+ individuals significantly increased differentiation of Foxp3+ Tregs. In addition, depletion of MDSCs from PBMCs of HIV-1+ individuals led to a significant reduction of Foxp3+ Tregs and increase of IFNγ production by CD4+ T effector cells. Conclusions: These results suggest that HIV-induced MDSCs promote Treg cell development and inhibit T cell function – a hallmark of many chronic infectious diseases.

Background: Patients treated with an inhibitor of the mechanistic target of rapamycin (mTORI) in a calcineurin inhibitor (CNI)-free immunosuppressive regimen after heart transplantation (HTx) show a higher risk for transplant rejection. We developed an immunological monitoring tool that may improve the identification of mTORI-treated patients at risk for rejection. Methods: Circulating dendritic cells (DCs) and regulatory T cells (Tregs) were analysed in 19 mTORI- and 20 CNI-treated HTx patients by flow cytometry. Principal component and cluster analysis were used to identify patients at risk for transplant rejection. Results: The percentages of total Tregs (p = 0.02) and CD39+ Tregs (p = 0.05) were higher in mTORI-treated patients than in CNI-treated patients. The principal component analysis revealed that BDCA1+, BDCA2+ and BDCA4+ DCs as well as total Tregs could distinguish between non-rejecting and rejecting mTORI-treated patients. Most mTORI-treated rejectors showed higher levels of BDCA2+ and BDCA4+ plasmacytoid DCs and lower levels of BDCA1+ myeloid DCs and Tregs than mTORI non-rejectors. Conclusion: An mTORI-based immunosuppressive regimen induced a sufficient, tolerance-promoting reaction in Tregs, but an insufficient, adverse effect in DCs. On the basis of patient-specific immunological profiles, we established a flow cytometry-based monitoring tool that may be helpful in identifying patients at risk for rejection.

Background: Extracorporeal photopheresis (ECP) induces immunological changes that lead to a reduced risk of transplant rejection. The aim of the present study was to determine optimum conditions for ECP treatment by analyzing a variety of toleranceinducing immune cells to optimize the treatment. Methods: Ten ECP treatments were applied to each of 17 heart-transplant patients from month 3 to month 9 post-HTx. Blood samples were taken at baseline, three times during treatment, and four months after the last ECP treatment. The abundance of subsets of tolerance-inducing regulatory T cells (Tregs) and dendritic cells (DCs) in the samples was determined by flow cytometry. A multivariate statistical model describing the immunological status of rejection-free heart transplanted patients was used to visualize the patient-specific immunological improvement induced by ECP. Results: All BDCA+ DC subsets (BDCA1+ DCs: p < 0.01, BDCA2+ DCs: p < 0.01, BDCA3+ DCs: p < 0.01, BDCA4+ DCs: p < 0.01) as well as total Tregs (p < 0.01) and CD39+ Tregs (p < 0.01) increased during ECP treatment, while CD62L+ Tregs decreased (p < 0.01). The cell surface expression level of BDCA1 (p < 0.01) and BDCA4 (p < 0.01) on DCs as well as of CD120b (p < 0.01) on Tregs increased during the study period, while CD62L expression on Tregs decreased significantly (p = 0.04). The cell surface expression level of BDCA2 (p = 0.47) and BDCA3 (p = 0.22) on DCs as well as of CD39 (p = 0.14) and CD147 (p = 0.08) on Tregs remained constant during the study period. A cluster analysis showed that ECP treatment led to a sustained immunological improvement. Conclusions: We developed an immune monitoring assay for ECP treatment after heart transplantation by analyzing changes in tolerance-inducing immune cells. This assay allowed differentiation of patients who did and did not show immunological improvement. Based on these results, we propose classification criteria that may allow optimization of the duration of ECP treatment.

Maintaining balance in the human immune system is critical for the body’s ability to discriminate between foreign and self-antigens. This balance is achieved, in part, by a subpopulation of T cells known as induced regulatory T cells (iTregs). Dysregulation of this population may contribute to the onset and progression of cancer, chronic inflammation and autoimmune diseases. Therefore, manipulation of iTreg development holds promising therapeutic potential; however, studying this vital population has proven difficult due to low numbers, heterogeneous cell populations, substantial phenotypic differences between mouse and human cells, and the high plasticity seen in iTregs. These current limitations have prevented a full understanding of the molecular signaling events that govern their development and function. Our lab has established a novel cell culture system that mimics in vivo human iTreg development. This system allows for the discrimination and comparison of naïve, memory and iTreg T cell populations simultaneously within a single donor. These iTregs exhibit high levels of CD25, FoxP3, CTLA4, GITR, low levels of CD127 and display strong suppressor activity. Using this innovative system, we have demonstrated a rewiring of T cell receptor (TCR) signaling in iTregs compared to conventional T cells. We found that the voltage gated K+ ion channel-Kv1.3 is not active in response to TCR engagement in iTregs, even though Ca2+ influx remains intact. Kv1.3 and the linked Src-family kinase Lck were redistributed to the highly active IL2-Receptor (IL2-R) complex. Additionally, we have shown that there is increased AKT protein expression in iTregs versus conventional T cell populations that does not correlate with the TCR-induced increase in its active (phosphorylated) form. This blockage appears to be due to an imbalance of kinase to phosphatase activity in iTregs with a specific TCR-induced inhibition of mTOR activity. We have also demonstrated that AKT accumulation in iTregs leads to its physical association with SMAD3, suggesting a novel, non-enzymatic function of AKT through transcription factor inhibition. This study sheds light on the reciprocal cross talk between the IL-2R and TCR signaling pathways and uncovers the mechanism of AKT blockade in primary human iTregs, thus opening novel avenues for therapeutic manipulation

Regulatory T cells (Treg) play a key role in maintaining the immune tolerance. They suppress development of autoimmune diseases and contribute to maintaining the homeostasis of the immune system. Expansion and excessive ability of regulatory T cells to suppress the immune response is increasingly observed also at many types of cancer. Due to the active inhibition of the antitumor immune response Treg contribute to tumor progression. Specific phenotype based detection and analysis of Treg functional properties may contribute to the successful monitoring of Treg accounts and to the effective cancer immunotherapy itself. Tumor cells express high amounts of so-called tumor antigens, which may play a key role in the antitumor immune response. Expression level of the tumor antigens gives the evidence about relevancy of each antigen in the specific immune response and efficiency of cancer immunotherapy. These data are obviously important to be obtained from the tumor cell lines as well as primary tumor cells. In the first part of the thesis I was focusing on the quantitative analysis of regulatory T cells in tumor tissue and peripheral blood of patients with ovarian cancer. For this purpose I used the newly introduced methyl-sensitive quantitative PCR (MS-qPCR) method and compare the data with the widely...

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, CD25, CTLA-4, CD45RB, GITR e CD103 (fenótipo de células Tregs), por citometria de fluxo. Adicionalmente, as células obtidas dos linfonodos foram avaliadas quanto à expressão de mensagem para o fator de transcrição Foxp3 (característico da função regulatória), por PCR quantitativo. Paralelamente, saliva de R. sanguineus foi inoculada na orelha de animais da mesma linhagem, a fim de se comparar o infiltrado celular com o obtido na pele dos camundongos infestados com carrapatos. Os resultados mostraram que as infestações não alteraram a percentagem de células T CD4+CD25+ nem a expressão de moléculas associadas ao fenótipo de células Tregs nas células infiltradas na lesão de fixação dos carrapatos ou nos linfonodos em comparação a camundongos controles. Também não se verificou aumento da expressão do gene para Foxp3 nos linfonodos em nenhum dos grupos analisados. Por outro lado, a inoculação de saliva na orelha de camundongos induziu um aumento significativo da população de células T CD4+, porém estas também não apresentavam fenótipo regulatório, sugerindo que o mecanismo de imunomodulação exercido pelos carrapatos sobre seus hospedeiros não é mediado por essas células. Resultados adicionais mostraram que a saliva de carrapatos reduziu significativamente a percentagem de células dendríticas nas orelhas dos camundongos, sugerindo que carrapatos podem estar modulando a resposta imune de seus hospedeiros por diminuírem o repovoamento da pele com células dendríticas, as quais são essenciais na vigilância imune dos tecidos periféricos. / Ticks are bloodsucking arthropods that feed on vertebrates and are responsible for serious global economic losses both through the effects of blood sucking and as vectors of pathogens. A tick?s bloodmeal lasts for several days, during which it remains fixed to the host and avoids rejection by local inflammatory and immunological reactions. This status is achieved by the escape mechanisms ticks have evolved. In fact, many studies have demonstrated that ticks modulate the host immune response through salivary compounds inoculated during their bloodmeals. This study investigated if during bloodfeeding ticks can recruit regulatory T cells in an attempt to modulate the host immune response and to control inflammatory responses that could be harmful to tick feeding. BALB/c mice were infested with Rhipicephalus sanguineus, the skin at feeding sites and the regional lymph nodes were collected, and the cells forming the local infiltrates were analyzed by flow cytometry for simultaneous expression of CD4, CD25, CTLA-4, CD45RB, GITR and CD103 molecules. Additionally, expression of mRNA for Foxp3 was measured in the lymph node cells. Tick saliva was also inoculated into the ears of BALB/c mice in order to compare the local cellular infiltrate with that elicited by artificial infestation. Control animals were sham infested. The results show that, relative to sham-infested tissues, tick infestations did not alter the percentage of CD4+CD25+ T cells present at the site of their attachment or in draining lymph nodes. Infestations also did not increase the expression of Foxp3 in skin and lymph nodes. On the other hand, saliva inoculated into the ear induced a significant increase in the number of CD4+ T lymphocytes recruited to the site of inoculation, although these cells did not express a regulatory phenotype. These results suggest that the modulation of the host immune response by ticks does not involve CD4+CD25+ T cells. Additional results showed that tick saliva reduced the percentage of dendritic cells in the skin of infested mice. This finding indicates that ticks may modulate the host immune response by diminishing the repopulation of skin with dendritic cells, which are essential for maintaining surveillance of peripheral tissues for incoming antigens.