Background and Aim: CD4+CD25+ regulatory T (Treg) cells are important in the maintenance of self-tolerance, regulation of T cell homeostasis, prevention of allograft rejection and in the modulation of immune responses to pathogens. Several groups have recently reported an increased expression of fgl2 in Treg cells by microarray analysis. FGL2, a member of the fibrinogen-like family, was previously shown to act as an immunomodulator by inhibiting DC maturation and T cell proliferation. Based on these findings, the immunoregulatory role of FGL2 as a novel effector molecule of Treg cells was investigated. Results: In agreement with previous studies, high levels of fgl2 transcripts were detected in Treg cells by real-time PCR. In fgl2-/- mice, an increased number and percentage of Treg cells were found with a greater expression of Foxp3 compared with fgl2+/+ Treg cells; however, the suppressive activity of fgl2-/- Treg cells was significantly impaired. Antibody to FGL2 completely inhibited the activity of fgl2+/+ Treg cells in vitro. Consistent with FGL2 contribution to Treg cell activity, targeted deletion of the gene led to an increased immune reactivity of DC, T cells and B cells, and the manifestation of glomerular autoimmunity in aged fgl2-/- mice. The importance of FGL2 as an effector of Treg cells was also demonstrated in MHV-3-induced fulminant hepatitis. Uninfected susceptible BALB/cJ mice had increased numbers of Treg cells and expression of FGL2 compared to uninfected resistant A/J mice. Following MHV-3 infection, plasma levels of FGL2 in BALB/cJ mice were significantly increased, correlating with an increased percentage of Treg cells. Treatment with anti-FGL2 antibody completely inhibited Treg cell activity and protected susceptible BALB/cJ mice against MHV-3-induced liver injury and mortality. Adoptive transfer of fgl2+/+ Treg cells into resistant fgl2-/- mice increased their mortality following MHV-3 infection. Finally, FGL2 treatment led to prolonged skin graft survival in a murine allotransplant model. The suppressive activity of FGL2 was mediated through binding to the inhibitory FcγRIIB receptor expressed on APCs, resulting in inhibition of DC maturation and induction of B cell apoptosis. Conclusion: These studies indicate that FGL2 plays an important immunoregulatory role as an effector cytokine of Treg cells; targeting FGL2 may provide a novel therapeutic approach for the treatment of patients with viral hepatitis, autoimmunity and transplant rejection.

Background and Aim: CD4+CD25+ regulatory T (Treg) cells are important in the maintenance of self-tolerance, regulation of T cell homeostasis, prevention of allograft rejection and in the modulation of immune responses to pathogens. Several groups have recently reported an increased expression of fgl2 in Treg cells by microarray analysis. FGL2, a member of the fibrinogen-like family, was previously shown to act as an immunomodulator by inhibiting DC maturation and T cell proliferation. Based on these findings, the immunoregulatory role of FGL2 as a novel effector molecule of Treg cells was investigated. Results: In agreement with previous studies, high levels of fgl2 transcripts were detected in Treg cells by real-time PCR. In fgl2-/- mice, an increased number and percentage of Treg cells were found with a greater expression of Foxp3 compared with fgl2+/+ Treg cells; however, the suppressive activity of fgl2-/- Treg cells was significantly impaired. Antibody to FGL2 completely inhibited the activity of fgl2+/+ Treg cells in vitro. Consistent with FGL2 contribution to Treg cell activity, targeted deletion of the gene led to an increased immune reactivity of DC, T cells and B cells, and the manifestation of glomerular autoimmunity in aged fgl2-/- mice. The importance of FGL2 as an effector of Treg cells was also demonstrated in MHV-3-induced fulminant hepatitis. Uninfected susceptible BALB/cJ mice had increased numbers of Treg cells and expression of FGL2 compared to uninfected resistant A/J mice. Following MHV-3 infection, plasma levels of FGL2 in BALB/cJ mice were significantly increased, correlating with an increased percentage of Treg cells. Treatment with anti-FGL2 antibody completely inhibited Treg cell activity and protected susceptible BALB/cJ mice against MHV-3-induced liver injury and mortality. Adoptive transfer of fgl2+/+ Treg cells into resistant fgl2-/- mice increased their mortality following MHV-3 infection. Finally, FGL2 treatment led to prolonged skin graft survival in a murine allotransplant model. The suppressive activity of FGL2 was mediated through binding to the inhibitory FcγRIIB receptor expressed on APCs, resulting in inhibition of DC maturation and induction of B cell apoptosis. Conclusion: These studies indicate that FGL2 plays an important immunoregulatory role as an effector cytokine of Treg cells; targeting FGL2 may provide a novel therapeutic approach for the treatment of patients with viral hepatitis, autoimmunity and transplant rejection.

Tem sido demonstrada a grande importância dos linfócitos T reguladores (Treg) na manutenção da tolerância e no controle da resposta imune em infecções. Na paracoccidioidomicose, micose sistêmica causada pelo Paraccoccidioides brasiliensis (Pb), a atuação das células Treg tem sido estudada. Utilizando o modelo murino DEREG, foram realizadas depleções com toxina diftérica (DT) na doença estabelecida. A gravidade da doença foi avaliada 6 e 10 semanas pós infecção com Pb através da carga fúngica nos tecidos, desenvolvimento de imunidade TCD8+ e TCD4+ e histopatologia. Animais DEREG tratados com a DT apresentaram menor número de células Treg, menor carga fúngica no pulmão, fígado e baço e redução dos granulomas comparados ao controle (PBS). Observou-se também um maior influxo de células TCD4+ e TCD8+ efetoras nos pulmões dos camundongos tratados com DT e aumento na produção de citocinas dos tipos Th1 e Th17. Portanto, demonstramos efeitos benéficos da depleção de Treg na PCM já estabelecida, caracterizado pela menor carga fúngica e maior ativação da imunidade celular. / The great importance of regulatory T lymphocytes (Treg) has been demonstrated in maintaining tolerance and not controlling the immune response in infections. In paracoccidioidomycosis, systemic mycosis caused by Paraccoccidioides brasiliensis (Pb), the action of Treg cells has been studied. Using the DEREG murine model, depletions were performed with diphtheria toxin (DT) in the established disease. The severity of the disease was assessed 6 and 10 weeks post-infection with fungal load in tissues, development of TCD8+ and TCD4+ immunity and histopathology. DEREG animals treated with DT presented lower number of Treg cells, lower fungal load in the lung, liver and spleen and reduction of granulomas compared to control (PBS). There was also a greater influx of effector CD4+ and TCD8+ cells in the lungs of DT-treated mice and increased production of Th1 and Th17 cytokines. Therefore, we demonstrated beneficial effects of Treg depletion on PCM already established, characterized by lower fungal load and greater activation of cellular immunity.

Toxoplasmosis is an important cause of congenital disease, and it is one of the most common opportunistic infections in patients with acquired immunodeficiency syndrome. The need for a reliable experimental model of this infection is crucial not only for achieving a better understanding of the patho-physiology of the disease, but also for developing better methods for evaluating new therapeutic regimens. The purpose of the present study was to investigate the role of CD4+CD25+ T regulatory lymphocytes in mice infected with Toxoplasma gondii. T regulatory (Treg) cells have been shown to play an important role in our immune system in controlling the activity of other T lymphocytes. These cells are differentiated from other T lymphocyte populations based on the co-expression of CD4 and CD25 and expression of the Foxp3 gene. The results of several recent studies have suggested that certain pathogens may be able to increase their survival in the host by exploiting T reg cell activity. T regulatory cells have been shown to control the persistence of the protozoan parasite, Leishmania major, in mice; however, this population of cells plays only a limited role during murine infection with Trypanosoma cruzi. In the present study we have investigated the role of Treg cells during murine infection with the ME49 strain of T. gondii. In vivo depletion of Treg cells was accomplished by injecting mice with a monoclonal antibody (Mab) isolated from the 7D4 rat hybridoma cell line. This Mab is specific for the interleukin-2 receptor chain (also known as CD25). Female Swiss Webster mice of approximately 6-7 weeks of age were depleted of Treg cells by intraperitoneal injection of 400µg of Mab, mice were injected once 7days prior to infection, and a second time 1 day prior to infection, with 20 tissue cysts of T. gondii. Mouse weight and tissue cyst numbers were monitored to evaluate the impact of Treg depletion on the outcome of infection. Our results suggest that depletion of Treg cells has little measurable impact during the acute stage of infection with the ME49 strain of T. gondii. Further studies will be required to determine what role, if any, these cells play in the chronic stage of murine toxoplasmosis.

Une immunité concomitante à long terme est mise en place lors d’infections persistantes avec des parasites protozoaires intracellulaires responsables, par exemple, de la leishmaniose et du paludisme. Dans un modèle murin de leishmaniose, il a ainsi été démontré que les cellules T régulatrices CD4+CD25+ sont impliquées dans la persistance des leishmanies aux sites d’infection cutanés et protègent l’hôte contre une ré-infection.Le protozoaire Toxoplasma gondii est également à l’origine d’une infection chronique liée à l’installation du parasite dans le cerveau et les muscles de l’hôte dans des formes kystiques. Il était donc pertinent de s’intéresser à l’implication des cellules T régulatrices dans l’installation et la persistance du toxoplasme.Pour atteindre cet objectif, nous avons utilisé l’anticorps monoclonal anti-CD25 dans des expériences de déplétion pendant la phase aiguë de la toxoplasmose après infection de souris non consanguines avec une souche de toxoplasmes de type II. Aucune différence significative que ce soit en terme de mortalité ou de charge parasitaire cérébrale n’a été observée entre les souris infectées et déplétées et les souris infectées non déplétées. En complément de ces expériences, nous avons pu montrer que les cellules régulatrices CD4+CD25+Foxp3+ (Treg) sont une cible potentielle de l’anticorps anti-CD25 ainsi que les cellules T effectrices CD4+CD25+Foxp3- (Teff); cellules qui expriment le marqueur CD25 en phase aiguë de l’infection. / Long term concomitant immunity is developed in case of persistant infections with intracellular protozoan parasites like for example in leishmaniosis and malaria. In a murine model of leishmaniosis, it has been demonstrated that CD4+CD25+ regulatory T (Treg) cells are involved in the persistance of leishmania parasites at cutaneous sites of infection and protect the host against re-infection.The protozoan parasite Toxoplasma gondii is also responsible for a chronic infection associated with the settlement of parasite in the brain and the muscles of the host in the form of cysts. It was therefore pertinent to know about the implication of Treg cells in the development and the persistance of toxoplasma. To attain this objective, we have used a monoclonal antibody anti-CD25 in depletion experiments during the acute phase of toxoplasmosis after infection of outbred mice with a type II toxoplasma strain. No significant difference was found in terms of mortality or in brain cyst load between depleted mice and non-depleted mice. In addition to these experiments, we have shown that not only the CD4+CD25+Foxp3+ regulatory T (Treg) cells but also the CD4+CD25+Foxp3- T effector (Teff) cells are a potential target of anti-CD25 antibody-depletion. These cells are induced to express CD25 during acute phase of the infection.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13356号 / 論医博第2202号 / 新制||医||1044(附属図書館) / (主査)教授 生田 宏一, 教授 濵﨑 洋子, 教授 竹内 理 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

A Artrite Reumatóide (AR) é uma artropatia autoimune multifatorial com etiologia desconhecida que afeta aproximadamente 1% da população adulta. A estratégia padrão para o tratamento da AR consiste na administração de baixas doses de Metotrexato (MTX), cujo efeito anti-inflamatório está relacionado com a manutenção dos níveis elevados de adenosina (ADO) extracelular. No entanto, uma parte considerável dos pacientes com AR é refratária ao tratamento com MTX e o mecanismo pelo qual este fenômeno ocorre ainda não está totalmente esclarecido. Neste contexto, o presente estudo descreveu que a eficácia terapêutica ao MTX está associada com a expressão em células Tregs da ectoenzima CD39, cuja função biológica é a geração de ADO extracelular via metabolização do ATP. Especificamente, através da realização de um estudo longitudinal, observamos que pacientes respondedores ao MTX (R-MTX) apresentam uma expansão de células Tregs circulantes expressando CD39 após o tratamento com MTX. Por outro lado, identificamos que pacientes não respondedores ao MTX (UR-MTX) possuem uma redução da expressão de CD39 em células Tregs, o que culmina em um comprometimento das suas funções supressoras. Ainda, demonstramos que a expressão de CD39 em células Tregs é um biomarcador apto em predizer a resposta terapêutica ao MTX, visto que pacientes UR-MTX apresentam uma expressão reduzida de CD39 em Tregs mesmo antes do início do tratamento com MTX. Posteriormente, nós investigamos as bases moleculares que acarretam na expressão reduzida de CD39 observada em células Tregs de pacientes URMTX. Demonstramos que a estimulação com TGF-? tanto em células Tregs isoladas quanto diferenciadas in vitro aumenta a expressão de CD39 através da ativação sequencial da seguinte plataforma molecular: receptores de TGF-? (TGFBRII e TGFBRI), transdutor de sinal SMAD2, fator de transcrição CREB, de modo dependente da atividade de p38. Uma vez identificada a via envolvida com a indução da expressão de CD39, demonstramos que células Tregs diferenciadas de indivíduos que apresentam uma expressão reduzida de CD39 são incapazes de induzir a expressão desta ectoenzima através da estimulação com TGF-?. Por fim, transpondo nossos achados para pacientes com AR, observamos que pacientes UR-MTX apresentam uma redução nos níveis de RNAm para TGFBRII e CREB bem como também uma redução das proteínas fosforiladas SMAD2 e CREB em células CD4+ e Tregs, sugerindo que o comprometimento na cascata de sinalização de TGF-?, envolvida com a indução da expressão de CD39 em células Tregs, está associado com a resistência ao MTX. / Rheumatoid arthritis (RA) is an autoimmune multifactorial arthropathy with unknown etiology that affects approximately 1% of the adult population. The standard strategy for RA treatment comprises the administration of low doses of methotrexate (MTX), whose antiinflammatory effects are associated with maintenance of high levels of extracellular adenosine (ADO). However, a considerable proportion of RA patients is resistant to MTX treatment and the mechanisms underlying this phenomenon occurs is poorly understood. Within this context, the present study showed that therapeutic efficacy of MTX is associated with expression on Treg cells of the ectoenzyme CD39, whose function is related to the generation of extracellular ADO by ATP metabolism. Specifically, we conducted a longitudinal study and observed that responsive patients to MTX (R-MTX) exhibit an increase in the frequency of circulating Treg cells expressing CD39 after MTX treatment. On the other hand, we found that non-responsive patients to MTX (UR-MTX) have a reduction of CD39 expression on Treg cells, which culminates in an impairment of Treg function. Furthermore, these findings indicate that CD39 expression on Treg cells is a biomarker for therapeutic response to MTX, since UR-MTX patients had a depressed CD39 expression on Treg cells even before MTX treatment. Subsequently, the present study investigated the molecular mechanisms that would cause the reduction of CD39 expression on Treg cells from UR-MTX patients. For this, we demonstrated that TGF-? stimulation increases CD39 expression in isolated and in vitro differentiated Treg cells through participation/activation of the following molecules: receptors of TGF-?, TGFBRII and TGFBRI, signal transducer SMAD2 and transcription factor CREB, through p38 activity dependent-manner. Once identified these molecules involved with CD39 induction, we demonstrated that differentiated Treg cells from healthy individuals with an intrinsic reduction of CD39 expression on circulating Treg cells are unable to increase CD39 expression by TGF-? stimulation. Transposing our findings to RA patients, we found that UR-MTX patients exhibit a reduction of mRNA for TGFBRII and CREB as well as reduction on levels of phospho-SMAD2 and phospho-CREB in CD4+ and Treg cells, suggesting that an impairment in TGF-? signaling pathway, related to induction of CD39 expression on Treg cells, is associated with MTX resistance.

Psoriasiform diseases are a group of cutaneous disorders that are characterized by impaired keratinocyte maturation leading to epidermal hyperplasia and thickening of skin. This group of disorders includes psoriasis, seborrheic dermatitis (SD) and mycosis fungoides (MF). Psoriasis has been recently shown to be mediated by the pro-inflammatory T helper cell subset, namely Th17 cells, whereas the pathogenesis of SD and MF are still poorly understood. SD is characterized by inflamed skin that primarily manifests on areas populated with sebaceous glands. Interestingly, SD is very common amongst immunosuppressed patients such as those with HIV-AIDS, suggesting the importance of an immune response in the development of SD. Because SD shares common clinical and histopathological features with psoriasis, a disease in which Th17 axis cytokines is known to be involved, and given that Th17 cells and their related cytokines have been implicated in the pathogenesis of a wide range of autoimmune and inflammatory disorders, it is possible that Th17 axis cytokines play a role in the pathogenesis of SD. We explored the involvement of Th17 axis cytokines in a D2C mouse model of psoriasiform disease that shows a high degree homology to the clinicopathological characteristics of human seborrheic dermatitis. IL-6 and IL-23, which are important for the differentiation of Th17 cells, and IL-17 and IL-22, which are the Th17 effector molecules, were measured at both protein and mRNA levels in sera and lesional skin from D2C mice. An immunohistochemical analysis was also performed to detect the presence of IL-17 in D2C lesional skin relative to normal skin from DBA/2 controls. Our data demonstrated significantly elevated levels of IL-6, IL-17 and IL-22 in sera from diseased D2C mice compared to controls and/or convalescent mice. There were no significant differences in IL-23 protein levels in sera from D2C mice compared to those from wild type mice or convalescent D2C mice. RT-PCR revealed a significant increase in IL-23 and IL-17 gene expression in D2C lesional skin relative to normal skin. Gene expression levels of IL-22, but not IL-6, were statistically significant elevated in D2C skin lesions compared to controls, by real time PCR. Our IHC study of IL-17 expression showed an abundance of positively stained mononuclear cells in D2C lesional skin relative to DBA/2 normal skin. Altogether, our data demonstrate that Th17 axis cytokines are elevated locally at mRNA levels for IL-23, IL-17, and IL-22 and systematically at protein levels for IL-6, IL-17, and IL-22. This data lay the foundation for further studies investigating a role for Th17 axis cytokines in the cutaneous inflammatory disease seen in our mouse model of SD and, ultimately, in the development of human SD. Mycosis fungoides (MF) is the most common type of cutaneous T cell lymphoma (CTCL). The etiology of MF is unknown, but there is substantial evidence suggesting a potential role for a yet unidentified infectious agent in the pathogenesis of MF. Many studies have claimed that there is an association between MF and the Human T cell Lymphotorpic Virus Type 1 (HTLV-I); however, the involvement of this virus in the etiology of MF is a controversial topic. In our study, we used nested PCR to explore the association between HTLV-I infection and MF by screening genomic DNA from 114 skin biopsies for the presence of HTLV-I provirus. We also utilized a ViroChip and high-throughput sequencing (HTS), as a case study, to attempt to detect novel virus-specific oligonucleotides that may be associated with CTCL. Our data showed no evidence for HTLV-I proviral integration in the 114 MF samples that were screened using nested-PCR. The ViroChip and HTS results also did not reveal any signature sequence for known or unknown infectious agent in the CTCL case study. Collectively, this data argue against the involvement of HTLV-I provirus in the pathogenesis of MF.

A Artrite Reumatóide (AR) é uma artropatia autoimune multifatorial com etiologia desconhecida que afeta aproximadamente 1% da população adulta. A estratégia padrão para o tratamento da AR consiste na administração de baixas doses de Metotrexato (MTX), cujo efeito anti-inflamatório está relacionado com a manutenção dos níveis elevados de adenosina (ADO) extracelular. No entanto, uma parte considerável dos pacientes com AR é refratária ao tratamento com MTX e o mecanismo pelo qual este fenômeno ocorre ainda não está totalmente esclarecido. Neste contexto, o presente estudo descreveu que a eficácia terapêutica ao MTX está associada com a expressão em células Tregs da ectoenzima CD39, cuja função biológica é a geração de ADO extracelular via metabolização do ATP. Especificamente, através da realização de um estudo longitudinal, observamos que pacientes respondedores ao MTX (R-MTX) apresentam uma expansão de células Tregs circulantes expressando CD39 após o tratamento com MTX. Por outro lado, identificamos que pacientes não respondedores ao MTX (UR-MTX) possuem uma redução da expressão de CD39 em células Tregs, o que culmina em um comprometimento das suas funções supressoras. Ainda, demonstramos que a expressão de CD39 em células Tregs é um biomarcador apto em predizer a resposta terapêutica ao MTX, visto que pacientes UR-MTX apresentam uma expressão reduzida de CD39 em Tregs mesmo antes do início do tratamento com MTX. Posteriormente, nós investigamos as bases moleculares que acarretam na expressão reduzida de CD39 observada em células Tregs de pacientes URMTX. Demonstramos que a estimulação com TGF-? tanto em células Tregs isoladas quanto diferenciadas in vitro aumenta a expressão de CD39 através da ativação sequencial da seguinte plataforma molecular: receptores de TGF-? (TGFBRII e TGFBRI), transdutor de sinal SMAD2, fator de transcrição CREB, de modo dependente da atividade de p38. Uma vez identificada a via envolvida com a indução da expressão de CD39, demonstramos que células Tregs diferenciadas de indivíduos que apresentam uma expressão reduzida de CD39 são incapazes de induzir a expressão desta ectoenzima através da estimulação com TGF-?. Por fim, transpondo nossos achados para pacientes com AR, observamos que pacientes UR-MTX apresentam uma redução nos níveis de RNAm para TGFBRII e CREB bem como também uma redução das proteínas fosforiladas SMAD2 e CREB em células CD4+ e Tregs, sugerindo que o comprometimento na cascata de sinalização de TGF-?, envolvida com a indução da expressão de CD39 em células Tregs, está associado com a resistência ao MTX. / Rheumatoid arthritis (RA) is an autoimmune multifactorial arthropathy with unknown etiology that affects approximately 1% of the adult population. The standard strategy for RA treatment comprises the administration of low doses of methotrexate (MTX), whose antiinflammatory effects are associated with maintenance of high levels of extracellular adenosine (ADO). However, a considerable proportion of RA patients is resistant to MTX treatment and the mechanisms underlying this phenomenon occurs is poorly understood. Within this context, the present study showed that therapeutic efficacy of MTX is associated with expression on Treg cells of the ectoenzyme CD39, whose function is related to the generation of extracellular ADO by ATP metabolism. Specifically, we conducted a longitudinal study and observed that responsive patients to MTX (R-MTX) exhibit an increase in the frequency of circulating Treg cells expressing CD39 after MTX treatment. On the other hand, we found that non-responsive patients to MTX (UR-MTX) have a reduction of CD39 expression on Treg cells, which culminates in an impairment of Treg function. Furthermore, these findings indicate that CD39 expression on Treg cells is a biomarker for therapeutic response to MTX, since UR-MTX patients had a depressed CD39 expression on Treg cells even before MTX treatment. Subsequently, the present study investigated the molecular mechanisms that would cause the reduction of CD39 expression on Treg cells from UR-MTX patients. For this, we demonstrated that TGF-? stimulation increases CD39 expression in isolated and in vitro differentiated Treg cells through participation/activation of the following molecules: receptors of TGF-?, TGFBRII and TGFBRI, signal transducer SMAD2 and transcription factor CREB, through p38 activity dependent-manner. Once identified these molecules involved with CD39 induction, we demonstrated that differentiated Treg cells from healthy individuals with an intrinsic reduction of CD39 expression on circulating Treg cells are unable to increase CD39 expression by TGF-? stimulation. Transposing our findings to RA patients, we found that UR-MTX patients exhibit a reduction of mRNA for TGFBRII and CREB as well as reduction on levels of phospho-SMAD2 and phospho-CREB in CD4+ and Treg cells, suggesting that an impairment in TGF-? signaling pathway, related to induction of CD39 expression on Treg cells, is associated with MTX resistance.