INTRODUÇÃO: O diabetes tipo 1 autoimune (DM1A) está associado a alterações na imunidade inata e adaptativa. A agressão autoimune, órgão específica, determina a destruição das células beta do pâncreas e a deficiência da produção de insulina. O infiltrado inflamatório do tipo linfomononuclear, configurando a insulite, e a escassez ou a ausência das células ?, definem o quadro histológico do DM1A. Os autoanticorpos contra antígenos das células beta, que geralmente se desenvolvem na fase pré-clínica, conferem predisposição para DM1A. No entanto, é difícil definir quando e quais indivíduos progredirão para o diabetes manifesto, justificando a busca de outros biomarcadores que auxiliem nas indicações de tratamentos preventivos. Nesse contexto, sabe-se que os microRNAs (miRNAs), pequenos RNAs que atuam pós transcrição, desempenham papel crucial na regulação de genes, integrando fatores genéticos e ambientais e influenciando o funcionamento de órgãos e tecidos de maneira pontual ou sistêmica. OBJETIVOS: avaliar o envolvimento biológico e a relevância da expressão de miRNAs na resposta imunológica e na função das células ? na patogênese do DM1A. MÉTODOS: analisamos o perfil dos miRNAs séricos em 4 grupos, a saber: pacientes portadores de DM1A, até 6 meses do diagnóstico (DM1A recente), (n=30); pacientes portadores de DM1A com duração de 2-5 anos (DM1A 2-5)(n=26) e indivíduos com autoanticorpos pancreáticos positivos sem diabetes (AcP) (n=25), os quais foram comparados aos indivíduos controles saudáveis(n= 29). A expressão dos microRNAs foi obtida com ensaios individuais TaqMan® MicroRNA Assays 5x primers e TaqMan MicroRNA Human Array Card A, (Applied Biosystems- Forster City CA, USA) constituído por 377 alvos e 4 endógenos. Os dados de expressão foram analisados no Software Cloud, (Thermo Fisher Scientific) e no programa Limma (Linear Models for Microarray and RNA-Seq Data). RESULTADOS: Não houve diferença nas características demográficas, como idade, cor auto referida e sexo entre os grupos (p > 0,05). Pacientes portadores de DM1A (recente e com duração de 2-5 anos), diferiram do grupo controle pelos valores elevados de glicose, hemoglobina glicada, títulos de autoanticorpos pancreáticos, e menores de peptídeo C (p < 0,05) e foram semelhantes entre si. Os portadores de autoanticorpos (AcP) tinham características intermediárias entre os grupos: menores valores de HbA1c e de anticorpo anti-tirosina-fosfatase (anti-IA2) e maiores de peptídeo C em relação aos dois grupos com diabetes. Diferiram dos controles apenas pelos maiores títulos de anticorpo anti-insulina (IAA) e anti-descarboxilase do ácido glutâmico 65 (anti-GAD65). A frequência dos alelos HLA de risco para diabetes (-DR3 ou -DR4 e -DQ2 ou DQ-8) decresceu dos grupos DM1A recente e DM 2-5 para AcP e controles. Foram avaliados 135 miRNAs que estavam expressos em 20% ou mais das amostras dos quatro grupos analisados. Maior expressão foi observada em 13, 4 e 33 miRNAs dos grupos AcP, DM1A recente e DM1A 2-5 respectivamente e menor em 11, 7 e 31 miRNAs destes grupos. Destes, 4 miRNAs foram diferencialmente expressos nos grupos AcP, DM1A recente e DM1A 2-5 em relação ao grupo controle. Os miRNAs: miR -16, miR-195 e miR-454, relacionados com regeneração endócrina do pâncreas, efeito anti-inflamatório e resposta à injúria da célula ? estavam diminuídos nestes 3 grupos. O miR-200a, implicado em apoptose das células beta, estava aumentado nos grupos AcP e DM1A recente e diminuído nos pacientes com maior duração do diabetes (DM1A 2-5), possivelmente devido à escassez destas células. Outros 8 miRNAs apresentaram expressão diferente da do grupo controle em dois dos grupos avaliados, e tendência semelhante no terceiro grupo, sendo 4 deles elevados (miR-193a-5p, miR- 323-3p, miR-423-5p, e miR-92a) e 4, diminuídos (miR-191, miR-19a, miR- 376a, miR-590-5p) ou neutralidade no 3º grupo (miR-15b, miR-100, miR-181a e miR-483-5p) Resposta antagônica foi observada para o miR-25 e miR-485- 3p, diminuídos no grupo AcP e aumentados no DM1A 2- 5. Tais miRNAs estão relacionados com resposta imunológica, secreção de insulina, lesão de células ? e glicotoxicidade, à semelhança do observado para o miR-101-3p, validado por ensaios individuais numa casuística maior. CONCLUSÃO: nossos dados sugerem que miRNAs circulantes podem estar envolvidos na patogênese do DM1A / INTRODUCTION: Autoimmune type 1 diabetes (T1D) is associated with changes in innate and adaptive immunity. The organ-specific autoimmune aggression determines the destruction of beta-cells in the pancreas and the deficient insulin production. The inflammatory infiltration of the lymphomononuclear type, configuring the insulite, and the scarcity or the absence of the beta cells, define the histological picture of T1D. Autoantibodies against beta-cell antigens, which usually develop in the preclinical phase, confer predisposition to T1D. However, it is difficult to define when and which individuals will progress to overt diabetes, justifying the search for other biomarkers that could be indicative of preventive treatments. In this context, it is known that the microRNAs (miRNAs) - small RNAs that act post transcription - play a crucial role in regulating genes and in integrating genetic and environmental factors, influencing the function of organs and tissues in a punctual or systemic way. OBJECTIVES: to evaluate the biological involvement and relevance of miRNA expression in the immune response and ?-cell function in the pathogenesis of T1D. METHODS: we analyzed the profile of serum miRNAs of 4 groups, namely: patients with T1D up to 6 months after diagnosis (recent T1D), (n = 30); patients with T1D lasting 2-5 years (T1D 2- 5) (n = 26) and individuals expressing pancreatic autoantibodies without diabetes (AbP) (n = 25), which were compared to healthy controls (n = 29). Expression of the microRNAs was obtained with individual assays TaqMan® MicroRNA Assays 5x primers and TaqMan MicroRNA Human Array Card A (Applied Biosystems-Forster City CA, USA), consisting of 377 targets and 4 endogenous. The expression data was analyzed in the Cloud Software (Thermo Fisher Scientific) and Limma (Linear Models for Microarray and RNASeq Data) program. RESULTS: There was no difference in demographic characteristics, such as age, self-reported color, and sex among groups (p > 0.05). Patients with T1D (both recent and 2-5 years), similar to each other, differed from the control group by high glucose, glycated hemoglobin levels, pancreatic autoantibody titers, and lower C peptide values (p < 0.05) . Pancreatic autoantibodies (AbP) carriers had intermediate characteristics among the groups: lower HbA1c and anti-tyrosine phosphatase antibody (anti- IA2) values and higher C-peptide levels than the two groups with diabetes. They differed from controls only by the higher titers of anti-insulin (IAA) and anti-decarboxylase of glutamic acid 65 (anti-GAD65) autoantibodies. The frequency of high risk HLA alleles for diabetes (-DR3 or -DR4 and -DQ2 or DQ- 8) decreased from the recent T1D and T1D 2-5 groups to the AbP and controls. We evaluated 135 miRNAs that were expressed in 20% or more of the samples from the four groups analyzed. Higher expression was observed in 13, 4 and 33 miRNAs of the Abp, recent T1D and T1D 2-5 groups respectively and lower in 11, 7 and 31 miRNAs of these groups. Of these, 4 miRNAs were differentially expressed in the AbP, recent T1D and T1D 2-5 groups in relation to the control group.The miRNAs: miR -16, miR-195 and miR-454, related to endocrine regeneration of the pancreas, anti-inflammatory effect and response to beta-cell injury were decreased in these 3 groups. miR-200a, implicated in beta-cell apoptosis, was increased in the recent and decreased AbP and T1D groups in patients with longer duration of diabetes (T1D 2-5y), possibly due to the shortage of these cells. Another eight miRNAs showed different expression of the control group in two of the evaluated groups, and a similar trend in the third group, four of them high (miR-193a-5p, miR-323-3p, miR-423-5p, and miR- 92a ) and four, decreased (miR-191, miR-19a, miR-376a, miR-590-5p) or neutrality in the 3rd group (miR-15b, miR-100, miR-181a and miR-483-5p) was observed for miR-25 and miR-485-3p, decreased in the AbP group and increased in T1D 2-5y. Such miRNAs are related to immune response, insulin secretion, ?-cell damage and glycotoxicity, similar to that observed for the miR- 101-3p, validated by individual trials in a larger cohort. CONCLUSION Our data suggests that circulating miRNAs may be involved in the pathogenesis of T1D

A Imunodeficiência Comum Variável (ICV) é a imunodeficiência primária sintomática mais comum em adultos. Pacientes ICV frequentemente apresentam diversas alterações de linfócitos B, número reduzido de células Treg e ativação imune crônica, bem como infecções recorrentes, alta incidência de doenças autoimunes e um risco aumentado de doenças malignas. Testamos a hipótese de que a frequência de células B10 estaria diminuída nos pacientes ICV, já que elas desempenham um importante papel no desenvolvimento de células Treg, no controle da ativação de células T e na autoimunidade. Para tanto, nós avaliamos a frequência de células B10 em pacientes ICV buscando correlacioná-la com as diferentes manifestações clínicas e imunológicas associadas à doença. Quarenta e dois (42) pacientes com diagnóstico de ICV e 17 indivíduos saudáveis foram convidados a participar do estudo. A partir das CMSP criopreservadas foram realizados testes de perfil de ativação celular, presença de células T reguladoras (Treg) e caracterização das células B10. Os níveis de sCD14 no plasma foram determinados por ELISA. A produção de IL-10 foi determinada por ELISA em sobrenadante de cultura de células B. Pacientes ICV apresentam frequência diminuída de células B CD24hiCD38hi produtoras de IL-10 em diferentes condições de cultura celular e frequência diminuída de células B CD24hiCD27+ em cultura celular estimulada por CpG+PIB. No entanto, a produção de IL-10 por células B não demonstrou diferença significativa entre pacientes ICV e controles. A frequência de células B10 não teve correlação com a presença de autoimunidade, ativação celular ou frequência de células Treg em pacientes ICV. Este trabalho sugere que pacientes ICV têm um comprometimento na subpopulação de células B reguladoras, mas que não está correlacionado com as características clínicas e imunológicas apresentadas por esses indivíduos / Common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency in adults. CVID patients often present changes in the frequency and function of B lymphocytes, reduced number of Treg cells, chronic immune activation, recurrent infections, high incidence of autoimmunity and increased risk for malignancies. We hypothesized that the frequency of B10 cells would be diminished in CVID patients because these cells play an important role in the development of Treg cells and in the control of T cell activation and autoimmunity. Therefore, we evaluated the frequency of B10 cells in CVID patients and correlated it with the different clinical and immunological characteristics of this disease. Forty-two CVID patients and 17 healthy controls were recruited for this study. Cryopreserved PBMCs were used for analysis of T cell activation, frequency of Treg cells and characterization of B10 cells by flow cytometry. Plasma sCD14 levels were determined by ELISA. IL-10 production was determined in supernatant by ELISA after culture of B cells. We found that CVID patients presented a decreased frequency of IL-10-producing CD24hiCD38hi B cells in different cell culture conditions and decreased frequency of IL-10-producing CD24hiCD27+ B cells stimulated with CpG+PIB. However, the B cells secretion of IL-10 was similar between CVID patients and healthy controls. The frequency of B10 cells had no correlation with autoimmunity, immune activation and Treg cells in CVID patients. This work suggests that CVID patients have a compromised regulatory B cell compartment which is not correlated with clinical and immunological characteristics presented by these individuals

In der Pathogenese der Multiplen Sklerose (MS) wird autoantigenspezifischen proinflammatorischen T-Helferzellen eine entscheidende Rolle zugeschrieben. Das am meisten untersuchte Autoantigen ist das Myelin Basic Protein (MBP). Bisher waren zum Nachweis autoantigenspezifischer T-Zellen deren Kultur über Tage bis Monate unumgänglich. In dieser Arbeit wurden Methoden zum direkten ex vivo-Nachweis autoreaktiver T-Helferzellen etabliert, die die reaktive Sekretion der proinflammatorischen Zytokine Interferon gamma und Tumor Nekrose Faktor alpha nach sechsstündiger Stimulation nachweisen. Die durchflusszytometrische Analyse antigenreaktiver Zytokinexpression in fixierten Zellen wies eine Sensitivität von 1/10.000 in mononukleären Zellen des peripheren Blutes (PBMC) auf. Es konnten damit bei 34 untersuchten MS-Patienten und 25 gesunden Kontrollpersonen keine MBP-reaktiven T-Helferzellen detektiert werden, während sich die Reaktion auf die beiden Kontrollantigene Tetanus Toxoid und Cytomegalie Virus-Antigen in den beiden Gruppen nicht relevant unterschied. Deshalb wurde in einer anderen Methode reaktiv sezerniertes Zytokin extrazellulär auf lebenden Zellen gebunden und durch einen anschließenden magnetischen Anreicherungsschritt die Sensitivität auf 2/100.000 erhöht. Bei einem von acht MS-Patienten wurde so eine Population MBP-spezifischer Zellen mit einer Ausgangsfrequenz von 2,15/100.000 nachgewiesen. Im Liquor von drei MS-Patienten ließen sich keine MBP-reaktiven proinflammatorischen T-Helferzellen detektieren. Diese Ergebnisse implizieren, dass die Frequenz MBP-spezifischer T-Helferzellen im peripheren Blut und im Liquor der meisten MS-Patienten und Kontrollpersonen geringer ist als die Sensitivität der etablierten Methoden, diese Zellen jedoch bei einigen Patienten in höheren Frequenzen nachgewiesen werden können. / Autoantigen-specific proinflammatory T-helper cells are assumed to play an important role in the pathogenesis of Multiple Sclerosis (MS). The most extensively studied autoantigen is Myelin Basic Protein (MBP). To detect autoantigen-specific T-cells, so far these had to be cultured for several days or months. In this work methods for the direct ex vivo detection of autoreactive T-helper cells have been established by detecting the reactive secretion of the proinflammatory cytokines Interferon gamma and Tumor Necrosis Factor alpha after six hour stimulation. The flow cytometric analysis of antigen-reactive cytokine expression in fixed cells showed a sensitivity of 1/10.000 in peripheral blood mononuclear cells (PBMC). With this method there could not be detected any MBP-reactive T-helper cells in 34 MS-patients and 25 healthy controls, whereas the reaction after stimulation with the two control antigens Tetanus Toxoid and Cytomegalovirus antigen did not differ relevantly between the two groups. Therefore in another method the reactively secreted cytokine was bound on the surface of living cells and the sensitivity was then increased to 2/100.000 by following magnetic enrichment. With that, there could be detected a population of MBP-specific cells in one of eight MS-patients with a frequency of 2,15/100.000 in PBMC. There could not be found any MBP-reactive proinflammatory T-helper cells in the cerebrospinal fluid of three MS-patients. Our results suggest that the frequency of MBP-specific T-helper cells in peripheral blood and cerebrospinal fluid is below the employed methods' detection limit in most MS-patients, but seldom these cells can be detected in higher frequencies.

In Europa stellt die Multiple Sklerose (MS) die häufigste neuroimmunologische Erkrankung des zentralen Nervensystems (ZNS) dar. Relevante Daten zum Krankheitsverlauf der MS wurden durch Untersuchungen im Tiermodell der experimentellen autoimmunen Enzephalomyelits (EAE) gewonnen. Nach gegenwärtigem Kenntnisstand wandern autoreaktive T-Zellen vom T-Helfer-1 (Th-1) Typ in das ZNS ein und lösen eine gegen Bestandteile der Markscheide gerichtete Entzündung aus. Diese Zellen exprimieren große Mengen des für sie typischen Zytokin Interferon-gamma (IFN-gamma), was in zahlreichen Zelltypen die Expression des Tryptophan degradierenden Enzyms Indolamin 2,3-Dioxygenase (IDO) induziert. Seit 1998 ist bekannt, dass die Inhibition der IDO zu einer durch T-Zellen vermittelten Abstoßung allogener Feten führt. Diese immunregulatorische Funktion von IDO konnte auf den schnellen Abbau der essentiellen Aminosäure Tryptophan und der im folgenden Abbauweg synthetisierten T-Zell-toxischen Metabolite zurückgeführt werden. Da im entzündeten ZNS-Gewebe während der MS und der EAE das IDO induzierende Zytokin IFN-gamma ebenfalls exprimiert wird, sollte in der vorliegenden Arbeit untersucht werden in wie weit die IDO an der Immunregulation des ZNS unter autoimmuner Neuroinflammation während EAE, beteiligt ist. Mittels HPLC konnte gezeigt werden, dass die relative IDO Aktivität im ZNS während der akuten und der Erholungsphase der Erkrankung signifikant gesteigert ist. Erfolgte ab dem Ausbruch der Erkrankung die systemische Inhibition der IDO Aktivität mit dem spezifischen IDO-Inhibitor 1-Methyl-Tryptophan, so führte dies zu einem signifikant schwereren Krankheitsverlauf im Vergleich zu Kontrolltieren. Mittels Immunzytochemie wurde gezeigt, dass aktivierte Mikroglia IDO im entzündeten ZNS und in Zellkultur nach IFN-gamma Stimulation exprimieren. Aufgrund von RT-PCR Analysen weiterer Enzyme des Kynureninweges konnte dessen Regulation während EAE nachgewiesen werden Entlang dieses Stoffwechselweges werden T-zell-toxische Tryptophanmetabolite gebildet die an der Eliminierung autoreaktiver T-Zellen im ZNS während der Erholungsphase der EAE beteiligt sein könnten und somit die aktivierten Th1 Zellen im ZNS einen antiinflammatorischen Rückkopplungsmechanismus auslösen. / Multiple sclerosis (MS) is the most widespread neuroimmunological disease of the central nervous system (CNS) in Europe. By applying the animal model of MS, experimental autoimmune encephalomyelitis (EAE), important insights into the disease course of MS have been gained. At present it is accepted that at the onset of the disease myelin-reactive T helper type 1 (Th1) cells infiltrate the CNS and induce autoimmune neuroinflammation. Activated Th1 cells express high amounts of the cytokine interferon-gamma (IFN-gamma). This pro-inflammatory signaling molecule is known to induce the expression of the tryptophan-degrading enzyme Indolmaine 2,3- Dioxygenase (IDO) in several cell types. Since 1998 it is known that inhibition of IDO induces the T cell-mediated rejection of allogeneic concepti in mice. The mechanism of this immunregulatory function of IDO was shown to be due to the degradation of the essential amino acid tryptophan and the subsequent synthesis of T cell toxic metabolites. Since the IDO-inducing cytokine IFN-gamma is also expressed within inflamed CNS tissue during MS and EAE, the present work investigated the role of IDO in immunregulation of the CNS during autoimmune neuroinflammation in the EAE model. A significant increase in relative IDO activity within the CNS during the acute and remission phases of EAE was identified by HPLC analysis. Systemic inhibition of IDO activity by the specific IDO inhibitor 1-methyl-tryptophan reduced the remission and exacerbated the progression of the disease in comparison to control animals. Activated microglia were identified by immunocytochemistry as IDO-expressing cells within the acute inflamed CNS and in cell culture after IFN-gamma stimulation. Enzymes following IDO in the kynurenine pathway were shown by RT-PCR to be up-regulated in the disease course. The analyzed enzymes are known to produce T cell toxic tryptophan metabolites and might therefore be involved in the elimination of autoreactive T cells from CNS tissue. In conclusion, the presented data support the view that autoreactive Th1 cells in the CNS induce a self-limiting negative feedback mechanism which limits the spread of inflammation, thereby reducing bystander damage in the CNS.

Bei der adaptiven Immunantwort wird ein breites Repertoire an Effektor-T-Zellen gebildet, das sich durch spezifische und vielfältige funktionelle Fähigkeiten auszeichnet. Neben der Aktivierung der Immunantwort werden Mechanismen benötigt, die Immunantworten regulieren und abschalten können, um unerwünschte Immunreaktion zu verhindern. Die Arbeit befasst sich mit der Rolle von CD152 (CTLA-4), einem Homolog zu CD28, bei der Begrenzung von T-Zellantworten. Die Inhibition der Proliferationsbegrenzung der T-Zellen durch CD152 war ursprünglich auf eine späte Abschaltung der T-Zellproliferation zurückgeführt worden. Wir konnten zeigen, dass CD152 bereits die T-Zellaktivierung abschalten kann und somit die Aktivierungsschwelle der T-Zelle heraufsetzt. Wir konnten auch zeigen, dass CD152 die frühe T-Zellproliferation auf zwei Ebenen inhibiert: Durch die Transkriptionsinitiierung des autologen, Proliferation-induzierenden Zytokins IL-2 und durch die Expression von G1-Kinasen, die für das voranschreiten des Zellzyklus unerläßlich sind. Bisher war es nicht möglich, individuelle, CD152-oberflächen-exprimierende T-Zellen zu detektieren. Um die Expression von CD152 auf der Oberfläche von T-Zellen zu analysieren, haben wir eine sensitive Färbemethode für Oberflächen-exprimiertes CD152 etabliert. Wir konnten damit zeigen, dass während einer antigen-spezifischen Stimulation Zellmembran-gebundenes CD152 lediglich auf einer Subpopulation von aktivierten Zellen (CD152+ T-Zellen) exprimiert wird. Isolierte, aktivierte CD152+ T-Zellen waren im Gegensatz zu aktivierten CD152- T-Zellen bei Restimulation in ihrer Proliferation inhibiert. Dies zeigt auch, dass CD152 in der Lage ist, bereits aktivierte T-Zellen zu inhibieren. Die heterogene Expression von CD152 auf der Oberfläche lässt vermuten, dass die CD152 exprimierenden Zellen, wenn sie ein CD152-Signal bekommen, eine andere Zelldifferenzierung einschlagen als Zellen ohne CD152. Wiederholte oder chronische Aktivierung von Th-Zellen führt zu einer Form von Apoptose, dem Aktivierungs-induzierter Zelltod, gegen den Th2-Zellen resistent sind. Aktivierte Th2-Zellen exprimieren, im Gegensatz zu Th1-Zellen, häufiger CD152 auf ihrer Oberfläche. Wir konnten zeigen, dass CD152-Kreuzvernetzung von aktivierten T-Zellen direkt Resistenz gegen Apoptose vermittelt. Dies geschieht, indem ein Signaltransduktionsmolekül, die PI3´Kinase, aktiviert wird. Dies führt zur Inaktivierung von Apoptose-unterstützenden Molekülen (Phosphorylierung von FKHRL1 und Herunterregulation von FasL) und zur Induktion des Apoptose-verhindernden Moleküls Bcl-2. Vermeidung von Apoptose ist eine zentrale Voraussetzung zur Induktion von Gedächtniszellen. CD152 exprimierende Zellen wären somit gute Kandidaten, um zu Gedächtniszellen zu differenzieren. Um die Rolle von CD152 bei der späten T-Zelldifferenzierung in vivo untersuchen zu können, wurde das CD152 Gen konditionell in der Maus mutagenisiert. / During adaptive immune responses a broad repertoire of effector T-cells is generated, characterized by diverse functional capabilities. Besides activation of the immune response other mechanisms are needed in order to regulate and terminate responses, thus preventing unwanted immune reactions. Here I focus on the role of CD152 (CTLA-4), a homologue of CD28, in the limitation of T-cell responses. Inhibition of T-cell-proliferation by CD152 was originally attributed to a late regulation of the T-cell proliferation. We now show that CD152 is already able to prevent the activation of T-cells and to set the threshold for their activation. We also show that CD152 inhibits T-cell activation in two ways: It inhibits the induction of the growth-factor IL-2 and it inhibits the expression of G1-kinases mandatory for the progression of the cell cycle. Until now, it has not been possible to detect individual T-cells expressing CD152 at their surface. To analyze the expression of CD152 at the surface of individual cells, we developed a sensitive staining method. Using this technique we could show that antigen-specific stimulation of T-cells leads to the expression of surface-bound CD152 only on a fraction of the activated T-cells. Isolated, activated CD152+ T-cells were inhibited in their proliferation whereas CD152- T cells were not. This also shows that CD152 is indeed able to inhibit already activated T-cells. The heterogenous expression of CD152 at the cell surface of already activated T-cells also suggests that CD152+ T-cells will differentiate differently compared to CD152-T-cells. Repeated or chronic activation of Th-cells leads to one form of apoptosis, activation-induced cell death (AICD), against which Th2-cells are resistant. Activated Th2-cells express surface CD152 at higher frequencies than Th1-cells. We show here, that CD152-crosslinking of activated T-cells directly induces resistance against AICD by a mechanism requiring PI3´kinase. This leads to the inactivation of pro-apoptotic molecules (phosphorylation of FKHRL1 and downregulation of FasL). It also leads to the induction of the survival molecule Bcl-2. Prevention of apoptosis is a central prerequisite for the generation of memory cells. Therefore, surface CD152+ T-cells might be good candidates to differentiate into memory cells. To investigate the role of CD152 during the differentiation of T-cells in vivo, the CD152 gene was conditional mutagenese of the CD152 gene was generated.

Die systemische Autoimmunerkrankung Systemischer Lupus Erythematodes (SLE) ist durch die Produktion von autoreaktiven Antikörpern charakterisiert. In wie weit veränderte B-Zellrezeptor (BZR) Signalwege oder Co-Rezeptoren in diesem Prozess involviert sind, ist noch nicht ausreichend im humanen SLE untersucht worden. Aus diesem Grund wurde in der vorliegenden Arbeit eine detaillierte Analyse des inhibitorischen Co-Rezeptors CD22, der Kinase Syk und Akt in B-Zellen des peripheren Blutes von SLE Patienten durchgeführt. SLE Patienten zeigten eine Dysbalance in BZR abhängigen Signalwegen, welche eine B-Zellsubpopulationen unabhängige Reduktion der p-Syk/p-Akt Ratio versursacht. Diese Verschiebung könnte zu einer defekten negativen Selektion und somit zur Bildung von autoreaktiven Zellen führen, die wiederum durch Überlebensvorteile persistieren könnten. Zusätzlich wurde im peripheren Blut von SLE Patienten eine bislang nicht bekannte CD27 Syk++ B-Zellpopulation nachgewiesen. Diese wies, trotz des fehlenden Gedächtnismarkers CD27, Gedächtnismerkmale auf und könnte für die bekannte erhöhte Plasmazell-induktion in SLE Patienten verantwortlich sein. Somit konnte Syk als intrazellulärer Marker einer Gedächtnispopulation identifiziert werden. Des Weiterem stellt die Wiederherstellung der Balance von Syk- und Akt Phosphorylierung nach BZR Aktivierung einen erfolgsversprechenden Therapieansatz bei SLE Patienten dar, um die Entstehung und das Überleben von autoreaktiven B- und Plasmazellen besser kontrollieren zu können. / Systemic lupus erythematosus (SLE) is a severe systemic autoimmune disease in which loss of tolerance to nucleic acids results into the production of autoreactive antibodies (Ab) Therefore, B cells might play a key role in the pathogenesis of this disease. However, abnormalities of BCR associated co receptors and downstream kinases with potential implications in selection processes are rare for human SLE. Thus, a comprehensive analysis of the inhibitory BCR co-receptor CD22, the spleen tyrosine kinase (Syk) and the pro-survival serine kinase Akt has been undertaken to gain new insights into potential BCR signaling disturbances in this autoimmune disease. This data indicate that B cells from SLE patients display an intrinsically disturbed balance of BCR related signaling pathways, resulting in a B cell subset independent reduced p-Syk/p-Akt ratio. This may lead to a diminished BCR dependent negative selection and enhanced survival of SLE B cells, permitting the emergence of autoreactive B and plasma cells. Furthermore, SLE patients exhibit an increased frequency of a novel CD27-Syk++ B cell subset with memory features, enhanced tonic BCR signaling and the capacity to differentiate in auto-Ab secreting cells. The current study provides evidence that the use of intracellular markers, such as Syk, could permit a more precise delineation of CD27- memory B cell subsets in autoimmune diseases since the conventional used memory marker CD27 has some limitations. In addition, the balance between the BCR associated kinases Syk and Akt might be a promising therapeutic target to reduce the occurrence of autoreactive B and plasma cells.

Nach aktuellem Kenntnisstand zur Pathogenese der Multiplen Sklerose (MS) wird der Entzündungsprozeß im Zentralnervensystem (ZNS) durch autoreaktive, Myelin-spezifische T-Zellen aufrechterhalten, wobei virale Infektionen bekannte Auslöser klinischer Schübe darstellen. Wir zeigten, dass eine Infektion mit dem Epstein-Barr Virus (EBV) eine notwendige aber nicht hinreichende Voraussetzung für die Entwicklung einer MS darstellt, deren potentielle Bedeutung sich aus der Korrelation zwischen Reaktivierung einer latenten EBV-Infektion mit Krankheitsaktivität bei MS-Patienten ergibt, die eine Aktivierung autoreaktiver T-Zellen im Rahmen der antiviralen Immunantwort nahe legt. Als biologische Marker der Krankheitsaktivität identifizierten wir die Hochregulation der ß2-Kette des Interleukin-12 Rezeptors sowie des Chemokinrezeptors CCR5 in peripheren Blutlymphozyten von MS-Patienten. Ferner konnten wir erstmals direkt nachweisen, dass die Aktivierung und klonale Expansion Myelin-spezifischer T-Zellen in peripherem Blut direkt mit Krankheitsaktivität korreliert. Der Wirkmechanismus der immunmodulatorischen Therapie der MS mit Interferon (IFN)-ß ist weitestgehend unverstanden. Unverstanden ist auch, warum die Therapie nur in einer Subpopulation von Patienten ihre Wirksamkeit entfaltet. Mit dem Molekül TRAIL gelang uns die Identifizierung des ersten klinischen Response Markers einer IFN-ß Therapie bei MS, der zugleich als prädiktiver Marker eine prognostische Aussage über den individuellen Behandlungserfolg erlaubt. Auf Grund seiner funktionellen Relevanz für den Therapieerfolg einer IFN-ß Behandlung stellt TRAIL zudem ein vielversprechendes Zielmolekül für neue therapeutische Strategien bei der MS dar. / Multiple sclerosis (MS) is considered a T-cell mediated demyelinating disorder of the central nervous system (CNS). Furthermore, it is well established that viral infections might trigger disease relapses. We demonstrate that infection with the Epstein-Barr virus (EBV) represents a necessary but not alone sufficient prerequisite for developing MS. The potential significance of EBV infection in MS arises from the correlation between viral reactivation and disease activity, indicating an activation of autoreactive T cells in the course of EBV reactivation and rendering EBV one important trigger of MS relapses. As biological markers for monitoring disease activity, we identified expression and upregulation of the chemokine receptor CCR5 and of the interleukin-12 receptor b2-subunit on potential effector cells in the peripheral blood of MS patients. In addition, we demonstrate for the first time that activation and clonal expansion of autoreactive T cells in the peripheral blood is clearly correlated with MS activity. Despite the well documented efficacy of interferon-b (IFN-b) therapy in a subgroup of patients, it remains unclear how IFN-b alters the clinical course of MS. We identified TRAIL as the first clinical response marker of IFN-b therapy in MS that might even be used as prognostic marker of therapy response in individual patients. Furthermore, our data imply that the immunoregulatory potential of TRAIL represents a novel and promising target for future therapeutic strategies in MS.

Die adaptive Immunantwort wird von T-und B-Lymphozyten realisiert. Nachdem der Antigenrezeptor auf T-Zellen durch die Interaktion mit einem MHC-Peptid Komplex auf APZ getriggert wurde, sind kostimulatorische Moleküle ein zweiter Kontrollpunkt für die Immunantworten interagierender Zellen. Für das Abschalten von ungewollten Immunantworten, z.B. Autoimmunantworten, sind Moleküle der kostimulatorischen Molekülfamilien auf den T- und B-Zellen von außerordentlicher Bedeutung. Ein zentrales kostimulatorisches Molekül ist das Molekül CTLA-4. Wir untersuchten den Einfluss von regulatorischen Zellen auf den Verlauf einer EAE, die durch einen Wechsel von Remission und Rezidiv gekennzeichnet ist. Neben den CD4+CD25+Foxp3+CTLA-4+ T-Zellen konnten wir ebenfalls CD4+CD25-Foxp3+CTLA-4+ T-Zellen in den das Gehirn infiltrierenden Lymphozyten sichtbar machen. Wir fanden gleiche Zahlen an CD4+CD25+ Zellen, die auch intrazellulär CTLA-4 exprimierten, während der akuten Phase und ersten Remission, wobei aber Oberflächen CTLA-4+CD4+ Zellen während der akuten Phase deutlich erhöht waren. Eine Depletion der natürlich vorkommenden CD4+CD25+ Treg Zellen vor dem Auslösen einer EAE führte zu einem schnelleren Krankheitsausbruch und schwererem sekundär progressivem Krankheitsverlauf. Obwohl die erste Remission von der CD4+CD25+ Treg Zelldepletion unbeeinflußt blieb, konnten bereits im ersten Krankheitsschub signifikant erhöhte Antigen spezifische proinflammatorische Zytokine der T-Zellen detektiert werden. Damit wird deutlich, dass der sekundär progressive Verlauf durch CD4+CD25+ Treg Zelldepletion bereits zeitig während des Krankheitsverlaufes eingeleitet wird. Wir konnten CTLA-4 ebenfalls in B-Zellen nachweisen. Die Expression von intrazellulärem und Oberflächen CTLA-4 in aktivierten B-Zellen ist strikt T-Zellen abhängig und hat ihr Maximum 48-72h nach Stimulation in vitro. Durch den Einsatz hochsensitiver Zellanreicherungsverfahren konnte der Nachweis der mRNA für CTLA-4 in den B-Zellen aus T-Zellabhängigen Zellkultursystemen erbracht werden. Die Induktion der mRNA für CTLA-4 kann unter bestimmten Umständen durch CD19 Kreuzvernetzung in B-Zellen erfolgen. Durch den Einsatz von Knochenmarkschimären, in denen CTLA-4 spezifisch nur in B-Zellen deletiert wird, konnte gezeigt werden, dass CTLA-4 in B-Zellen die primäre IgE und IgM und die sekundäre IgM Produktion in Thymus abhängigen Immunantworten steuert. Diese Daten implizieren für alle Thymus abhängigen Immunantworten eine noch komplexere Regulation, bei der CTLA4 in B-Zellen deren Effektorfunktion intrinsisch modulieren kann. / T and B lymphocytes carry out the adaptive immune response. After the antigen receptor on T cells is triggered through interaction with an MHC:peptide complex on APCs, costimulatory molecules are a second checkpoint for immune responses of interacting cells. To terminate unwanted immune responses, such as autoimmune responses, molecules of the costimulatory molecule family on T and B cells are of great importance. A central costimulatory molecule is CTLA-4 (CD152). We investigated the influence of regulatory cells on the course of an EAE, a disease marked through alterations of remission and relapses. Apart from the CD4+CD25+Foxp3+CTLA-4+ T cells we also detected CD4+CD25-Foxp3+CTLA-4+ T cells within the brain-infiltrating lymphocyte population. Furthermore we found similar numbers of CD4+CD25+ cells that also expressed intracellular CTLA-4, during the acute phase and first remission from EAE, whereas surface CTLA-4+CD4+ cells were clearly elevated during the acute phase. The depletion of the natural occurring CD4+CD25+ Treg cells before EAE induction leads to an accelerated disease onset and an increase in disease severity combined with a secondary progressive disease course. Even though the first remission was unaffected by the depletion of CD4+CD25+ Treg cells before disease induction, the antigen specific proinflammatory cytokine production of T cells during the acute phase was already significantly increased. The data show, that the secondary progressive disease course after CD4+CD25+ Treg cell depletion is already determined early during the course of EAE. On a second approach we found CTLA-4 expression as well on B lymphocytes. The expression of intracellular and surface CTLA-4 on activated B cells is strictly T cell dependent and the expression is maximal 48-72h after stimulation in vitro. Through the use of highly sensitive cell sorting strategies we were able to detect the mRNA for CTLA-4 in B cells cultured with activated T cells. CTLA-4 mRNA in B cells is inducible in isolated B cells via crosslinking of CD19 in vitro. We generated bone marrow chimeric mice, in which only B cells were CTLA-4 deficient. With these mice we could show, that CTLA-4 on B cells controls the primary IgE and IgM, as well as the secondary IgM production in thymus dependent immune responses. These data imply a more complex regulation of thymus dependent immune responses, in which CTLA-4 on B cells can modulate B cell effector functions.

L’autophagie est un processus catabolique lié aux lysosomes. L’autophagie joue un rôle dans la biologie des lymphocytes et dans la réponse immunitaire en générale. Nous avons montré une dérégulation de l’autophagie dans les lymphocytes provenant de souris développant un lupus et de patients atteints d’un lupus érythémateux disséminé. Nous avons ensuite cherché à définir le rôle potentiel de l’autophagie des lymphocytes dans l’activation et le maintien des réponses humorales normales et pathologiques. Ainsi, nous avons généré des souris déficientes en autophagie spécifiquement dans les lymphocytes B. Ces modèles de souris nous ont permis de montrer que l’autophagie ne jouait pas de rôle majeur dans la mise en place de la réponse immunitaire humorale à court terme. Cependant, l’étude du même modèle murin sur fond génétique prédisposant à une auto-immunité systémique a démontré un rôle de l’autophagie dans la production d’auto-anticorps anti-nucléaires et dans le maintien d’un fort nombre de plasmocytes. L’autophagie est donc importante pour l’initiation de l’activation des lymphocytes B et leur survie en contexte d’auto-immunité à long terme. Ce modèle nous a également permis de montrer que l’absence d’autophagie lors de la stimulation du BCR compromet sa polarisation, conjointe à celle des molécules complexe majeur d’histocompatibilité de classe II et des lysosomes. Ce phénomène est important dans la mise en place de la synapse immunologique, structure qui permet la dégradation et l’internalisation d’antigène particulaires. Nous avons pu mettre en évidence que l’inhibition de l’autophagie impacte effectivement la présentation d’antigènes particulaires internalisés via le BCR aux lymphocytes T. Ainsi, la modulation de l’autophagie dans les lymphocytes pourrait permettre à plusieurs niveaux de limiter l’activation et la survie des lymphocytes autoréactifs dans le contexte de maladies auto-immunes. / Macroautophagy, called autophagy, is a catabolic lysosomal process. Macroautophagy was recently shown to regulate the immune response especially by regulating lymphocyte biology. We demonstrated that autophagy is deregulated in T cells from lupus mouse models and patients suffering from systemic lupus erythematosus. We suggest that autophagy could regulate the survival of autoreactive lymphocytes during lupus. We then wanted to better understand the role of autophagy in normal and pathologic humoral responses. We have generated mouse models conditionally deficient for ATG5 in B cells. In accordance with previous studies, we show that autophagy is dispensable for B cell survival and activation under short-term B cell receptor (BCR) activation. We then investigated long-term immunity on a spontaneous model of autoimmunity. In autoimmune-prone mice deficient for autophagy in B cells, we demonstrate that autophagy is important to maintain high levels of anti-nuclear auto-antibodies, and high number of long-lived plasma cells in the bone marrow. With these same mouse models, we show that autophagy contributes to the polarization of internalized BCR after stimulation, together with the recruitment of lysosomes and MHCMII molecules-containing compartments. The polarization of B cells is particularly important for the acquisition of particulate antigens for B cells. We postulate that ATG5 and possibly the autophagic machinery could facilitate the formation of the immune synapse. We indeed demonstrate that presentation of immobilized antigens to T cells is compromised in the absence of ATG5 in B cells. Thus, modulating autophagy in lymphocytes, could limit at several levels the activation and/or survival of autoreactive lymphocytes during autoimmunity.

INTRODUÇÃO: A deficiência de IgA (DIgA) é a imunodeficiência primária mais comum e caracteriza-se pela presença de concentrações de IgA sérica abaixo de 7 mg/dL e níveis normais de IgM e IgG. A maioria dos indivíduos acometidos não apresenta doença aparente embora alguns possam apresentar infecções recorrentes ou crônicas de mucosas, atopia e/ou doenças autoimunes (DAIs). Presumivelmente, a doença resulta de um defeito na troca de isótipo para IgA ou de falha na maturação de linfócitos produtores de IgA. A imunodeficiência comum variável (ICV) constitui uma deficiência primária de anticorpos caracterizada por níveis séricos baixos de IgG, IgA e/ou IgM, ao lado de valores normais ou diminuídos de linfócitos B e/ou T, levando a infecções crônicas ou recorrentes principalmente dos tratos respiratório e gastrintestinal. Embora a fisiopatologia da ICV ainda não esteja esclarecida, em muitos pacientes ela pode ser decorrente de algum defeito intrínseco de linfócitos B. De modo especial, as células B de memória (CD27+) têm sido correlacionadas com alguns aspectos clínicos da doença. Números elevados de células B de memória com persistência de IgM (CD27+IgM+) parecem estar correlacionados com a presença de infecções, enquanto valores diminuídos de células B de memória clássicas ou class-switched (CD27+IgG-IgM-) parecem estar associados a baixos níveis de IgG e presença de autoimunidade. A progressão de DIgA para ICV tem sido descrita em alguns pacientes embora não constitua regra geral. Uma hipótese é a de que uma base genética comum e a associação com DAIs possam constituir fatores de risco para a progressão de DIgA para ICV. Há relato anterior de que a persistência de células B imaturas IgM+ IgD+ em alguns pacientes com DIgA estava associada à progressão para ICV. Adicionalmente, há evidências de que a diminuição de células B de memória em uma proporção de pacientes com ICV esteja associada à presença de autoimunidade. OBJETIVOS: comparar em pacientes com DIgA e ICV várias subpopulações de células B e analisar a relação entre estas populações celulares e a presença de DAIs em ambos grupos. MÉTODO: Este estudo incluiu 56 pacientes adultos de ambos sexos com DIgA e ICV, distribuídos em grupos de acordo com a associação com DAI: grupo DIgA sem DAI (14 pacientes), grupo DIgA com DAI (14 pacientes), grupo ICV sem DAI (14 pacientes) e grupo ICV com DAI (14 pacientes). As seguintes subpopulações de células B foram determinadas por citometria de fluxo de quatro-cores: células B naive (CD19+IgM+), células B de memória clássicas ou class-switched (CD27+IgM-IgD-) e células B de memória imaturas (CD27+IgM+ or CD27+IgD+). Na análise estatística foi aplicado o teste de ANOVA; valores significativos foram determinados pela correção de Bonferoni. RESULTADOS: os grupos analisados foram homogêneos quanto à idade e distribuição de gêneros. Os valores de linfócitos totais e de células B naive foram similares nos quatro grupos estudados. Os pacientes com deficiência de IgA e ICV com DAIs associadas apresentaram valores igualmente aumentados de células B de memória imaturas CD27+IgM+ e CD27+IgD+ quando comparados a pacientes sem doenças autoimunes. CONCLUSÕES: estes resultados sugerem que a persistência de células B de memória imaturas possa estar relacionada à presença de autoimunidade em pacientes com DIgA e ICV. Especula-se se a persistência destas células em pacientes com DIgA e DAI associada possa constituir fator preditivo da progressão de DIgA para ICV. / INTRODUCTION: IgA deficiency (IgAD) is the most common primary immunodeficiency disorder and is characterized by serum IgA concentration below 7 mg/dL and normal serum IgM and IgG levels. Most of the affected individuals have no apparent disease, whereas selected patients suffer from recurrent or chronic mucosal infections, atopy and/or autoimmune diseases (AIDs). This defect is presumed to result from impaired class-switching to IgA or from maturational failure of IgA-producing lymphocytes. Common variable immunodeficiency (CVID) is a primary antibody deficiency disease characterized by low serum levels of IgG, IgA and/or IgM, and normal or decreased B and/or T cell numbers, leading to chronic or recurrent infections, noted mostly in the respiratory and gastrointestinal tract. While the pathophysiology of CVID remains elusive, in many patients it may be due to an intrinsic B cell defect. Memory B cells (CD27+) in particular, have been noted to correlate with certain clinical aspects of the disease. High numbers of IgM+ memory B cells (CD27+IgM+) appear to correlate with the presence of infections, whereas decreased numbers of classic (class-switched) memory B cells (CD27+IgG-IgM- ) correlate with lower serum IgG levels and increased rates of autoimmune features. Progression from IgAD to common variable immunodeficiency (CVID) has been reported in some patients, but is not a general rule. It is postulated that a common genetic base and association with AIDs could be risk factors for progression from IgAD to CVID. OBJECTIVES: The aim of this study was to compare B cell subpopulations of patients with IgAD and with CVID, and to assess the relationship between these populations and the presence of autoimmune diseases in both group of patients: . METHOD: The study included 56 adult patients of both genders with IgAD or CVID. Patients were grouped, according to the association with autoimmune disease,as follows: group IgAD with AID (14 patients), group IgAD without AID (14 patients), group CVID with AID (14 patients) and group CVID without AID (14 patients). We determined by immunophenotyping of lymphocytes by four-colour cytometry the following subpopulations of B cells: naïve B cells (CD19+IgM+), class-switched memory B cells (CD27+IgM-IgD-) and immature B memory cells (CD27+IgM+ or CD27+IgD+). Statistical analysis was performed by the ANOVA test; significant P-values were determined by means of Bonferonis correction. RESULTS: there is no statistically significant difference between the average ages and the gender of patients between the groups. the distribution of the sample values seem to indicating that, there is no statistically significant difference in the CD19 levels between the groups. patients with AID represent greater values of CD27 IgM+ and CD27+ IgD+ than patients without AID, independent of the group studied. CONCLUSIONS: These results suggest that the persistence of immature memory B cells in patients with IgAD and CVID can be related to autoimmune diseases. We speculate if the persistence of immature B cells can constitute risk factor to progression of IgAD for CVID.