Dissertationen zum Thema „Systemic lupus erythematosus Immunological aspects“

Subsets of systemic lupus erythematosus (SLE) patients with distinct patterns of disease manifestations and autoantibody production have been reported, but seldom have these two phenomena been analysed together. Cluster analysis was performed on 1928 Chinese SLE patients based on autoantibody profile and the frequencies of various clinical manifestations were compared between each cluster. Separate association analyses between individual autoantibodies and clinical manifestations, as well as between clinical manifestations, were also performed. This study identifies three separate autoantibody clusters each with different clinical manifestations, and proposes that the phenomena of autoantibody clustering and clinical subsets may be inter-related. Patient clusters could also be stratified into a bipolar spectrum. On one end are patients with over-representation of anti-dsDNA and renal disorder; whilst on the other end are two distinct autoantibody clusters (anti-Sm/anti-RNP/aPL and aPL/anti-Ro/anti-La) with overlapping of other non-renal manifestations. Patient stratification could aid disease prediction and subsequent management. These findings may also elucidate disease pathogenesis and guide future study on potential common pathological processes within autoantibody clusters.
published_or_final_version
Paediatrics and Adolescent Medicine
Master
Master of Research in Medicine

Systemic lupus erythematosus (SLE) is a chronic multi-organ autoimmune disease that is characterised by diverse clinical manifestations. Immunologically, SLE features a prominent “interferon (IFN) signature” which is marked by an elevated expression of type I IFN-regulated genes in blood and tissue cells of patients with this condition. Plasmacytoid dendritic cells (pDCs), also known as the most potent type I IFN-producing cells, are therefore considered the major culprit in SLE pathogenesis. Previous studies from our group have demonstrated abnormalities in circulating and bone marrow (BM)-derived pDCs from SLE patients. In the light of this, the present study was undertaken to further evaluate the role of pDCs in SLE development and to seek for key mediator(s) that might lead to functional aberrations of pDCs in this condition. Recently, a growing attention has been drawn to microRNAs (miRNAs) for their critical role in regulating immune cell function and strong association with autoimmune diseases. Therefore, the current study hypothesised that microRNAs played an important role in modulating pDC response(s) to toll-like receptor (TLR) stimulation, and that dysregulated microRNA expression induction was responsible for pDC abnormalities in SLE pathogenesis. The spontaneous lupus mouse model, F1 hybrid of New Zealand Black and White strains (NZB/W F1), was used in this study. The disease profile of NZB/W F1 was characterised based on the development of serum antinuclear antibodies and proteinuria. Specifically, the development of lupus in these mice (symptomatic mice) was illustrated by high titres of serum antinuclear antibodies, persistent proteinuria, glomerular immune complex deposition and elevated expression of pro-inflammatory cytokine genes in the kidney. Young NZB/W F1 (pre-symptomatic) as well as age- and sex-matched non-lupus maternal NZW mice were used as controls. While the development of pDCs appeared to be unaffected by lupus, elevated upregulation of MHC class II and co-stimulatory molecules, and induction of IFN-stimulated gene Ifitm3 in TLR7-stimulated lupus pDCs suggested phenotypic and functional hypersensitivity of these cells. Furthermore, analysis of the expression profile of miRNAs in pDCs upon TLR7 activation identified six differentially regulated targets. Among these, miR-155 was the most highly induced and its induction was consistently higher in pDCs from symptomatic NZB/W F1 mice. Nevertheless, transfection of miR-155 mimics into pre-symptomatic pDCs resulted in a reduced expression of Ifitm3, suggesting that miR-155 has a negative regulatory role in IFN production in pDCs. The finding of upregulated induction of miR-155 in lupus pDCs reported in this thesis is in line with previous studies, which showed increased expression of miR-155 in splenic lymphocytes of lupus NZB/W F1 mice. Results obtained from the transfection experiments are also in accordance with other previous studies, which showed miR-155 functioned as a negative feedback regulator of IFN production in pDCs. However, the mechanism of the association between miR-155 expression and increased IFN response in SLE requires further investigations. It is hoped that findings from this study contribute to a better understanding of SLE pathogenesis and ignite future interests in evaluating the molecular layer of regulation in autoimmunity.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy

New Zealand Black/White F₁ hybrid (NZB/W) and MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop a Systemic Lupus Erythematosus-like autoimmune disease. While the primary immunologic defect in the NZB/W is due to B cells, in the MRL/lpr it is a result of T cell abnormalities. Diethyldithiocarbamate (DTC), an agent suggested to enhance T cell function, was used to treat both strains. Weekly treatment of NZB/W mice with 25 mg/kg DTC had no significant effect upon survival or autoantibody levels but did induce changes in cell surface antigen expression. MRL/lpr mice treated with DTC displayed normalization of cell surface antigen expression (particularly increased expression of Lyt-2, macrophage markers and Lyt-2⁺/L3T4⁺ thymocytes), decreased lymphoproliferation and thymic atrophy, decreased serum autoantibody levels and kidney deposition of C3 and IgM, restored responses to mitogens and significantly prolonged survival. To determine both the influence of MRL background and lpr genes and to better understand on what cell populations DTC effects, changes in cell surface antigen expression were examined in DTC treated MRL-+/+, Balb/c, and Balb/lpr strains. The only consistent similarities observed between all strains tested were DTC induced changes in Mac-1 splenocyte surface antigen expression. In vitro studies showed DTC to have variable effects upon the mitogenic responses of lymphoid cells to phytohemagluttinin, but DTC alone stimulated both MRL/lpr and Balb/lpr lymphocytes. DTC stimulated the null cell population that predominates in lpr gene-bearing mice, but all observed in vitro effects of DTC were dependent upon the adherent cell population included in culture. DTC had no apparent direct effects upon adherent cells alone however. These studies have shown that DTC is capable of positive effects upon one autoimmune murine strain, the MRL/lpr, but not the NZB/W. DTC appears to affect macrophages, but other cell populations are required to obtain full activity of this compound. The variable effects of DTC emphasize the need to define the immunopathology of individual patients with autoimmune disease before initiating treatment with immunomodulative therapy.

The purpose of the study was to determine the point prevalence of cognitive dysfunction in patients with systemic lupus erythematosus (SLE) and to investigate its association with corticosteroids and depression. The severity of dysfunction and the pattern of cognitive changes were examined. This study hypothesized that cognitive dysfunction is common in SLE and many previous studies have underestimated its prevalence, partially due to using limited neuropsychological batteries and insensitive test instruments. It was further hypothesized that the pattern of cognitive changes in SLE patients will resemble that observed in subcortical dementias.

SLE is a complicated autoimmune disease with a strong genetic basis. Owing to the successful application of genome-wide association study (GWAS) on complex traits, significant progresses have been made in identifying susceptible loci for SLE. So far, more than 30 loci have been shown to have robust association with the disease, significantly improving our understanding of this disease. On the other hand, these loci only confer relatively small increments in disease risk, leaving a large amount of disease heritability unexplained. Several limitations may lead to the issue of “missing heritability”, such as the insufficient statistical power of standard GWAS, the failure to explore independent signals with marginal effects, and other forms of genetic variations or interactions that can hardly be detected by the current GWAS approach. In the current study, efforts have been made on solving the issue of missing heritability. Firstly, in order to increase the statistical power of GWAS, we performed meta-analysis of two existing SLE GWASs on Chinese populations, and replicated the findings in additional samples from Hong Kong, Anhui, and Thailand. We identified ARID5B and CDKN1B as novel SLE susceptibility genes through this approach. Secondly, considering the limitation of previous GWASs that only focus on the most significant signal in an individual region, we revisited the established loci by in-depth analysis on the basis of meta-analysis and followed up the findings with large-scale replication efforts. We found three additional independent signals in 11q23.3 as being associated with SLE. Thirdly, given the importance of independent effects in predisposing disease susceptibility, we performed gene-based analysis to combine the marginal independent signals within a gene to identify novel susceptibility genes. Our results demonstrated the widespread existence of independent effects within known SLE susceptibility genes, and we also identified ANXA6 as a novel SLE susceptibility gene. Lastly, we also made some efforts in the characterization of the related genes, and we identified several regulatory SNPs (rSNPs) predisposing individuals to SLE via affecting expression of the corresponding genes. In addition, we also found that epistatic interaction of variants in a previously established susceptibility gene, ETS1,correlates with cytokine (e.g.IL-17) abnormality in SLE cases. The current work represents several practical approaches to improve the power of GWAS. The findings might enrich the list of SLE susceptibility genes as well as advance our knowledge on the etiology of this complicated disease. Importantly, the current study highlights the importance of independent effects and rSNPs in conferring disease susceptibility, which may shed light on further exploration on the genetic architecture of SLE.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

Systemic lupus erythematosus (SLE) is characterized as an autoimmune disorder with unclear etiology. To identify the genetic effect of SLE, a genome wide association study (GWAS) and its further replication were conducted on SLE patients in Asian populations and ethnically matched controls. Before this study, most of the confirmed association loci were identified by GWAS studies in European populations. Apart from the established associations, we identified a SLE susceptible single nucleotide polymorphisms (SNP) located in ETS1 (rs1128334, P=2.3E-11, OR=1.29) in four different cohorts. This locus is probably an Asian-specific susceptibility locus since no Caucasian study has reported further validation in the last years. A new susceptibility variant in UHRF1BP1 (rs13205210, P=4.4E-09, OR=1.49) independent from the previously confirmed SNPs in Caucasian study was also confirmed to be associated with SLE in the Hong Kong Chinese population. Meta-analysis was performed by introducing another Chinese Han GWAS data set from Anhui province, China. Three loci, TET3-DGUOK, CD80, DRAM1, were confirmed to be associated with SLE. Two loci with suggestive signals in Hong Kong GWAS and further replication were also confirmed by the meta-analysis: PTTG1-MiRNA146a, YDJC. In order to identify the genetic effect for females who have predominant chance to suffer from SLE, X chromosome specific meta-analysis based on the Hong Kong and Anhui GWAS data and further replication study were performed by considering the difference between females and males. A signal in PRPS2, and three independent signals in the Xq28 were confirmed with the replication in three different cohorts by considering both females and males. Gene-gene interactions were also investigated genome-widely in a hypothesis free manner based on the meta-analysis results. The further validation processes were preceeded based on each independent GWAS data set. Four pairswise interacting loci were found and cross validated by three methods including logistic regression and Multifactor Dimensionality Reduction (MDR) and information gain theory based on the Anhui GWAS data set. Further studies are still needed to better explain the real features of genetic epistasis and the potential biological roles. By incorporating two GWAS from the same population, the population difference is efficiently avoided. Together with the putative gene-gene interactions, this study presents a comprehensive analysis based on the GWAS data conducted on SLE. It may shed new light on the disease mechanisms of SLE.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

Background. Rheumatoid Arthritis (RA) is an autoimmune inflammatory disease that, without powerful treatment, may lead to irreversible joint damage. During the past decade, anti-cytokine therapy has become available, e.g., infliximab, a chimeric antibody targeting the pro-inflammatory cytokine TNF that has a central role in the inflammatory process in RA patients. Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease that may affect all organs and is characterized by a massive antibody production. Chemokines, chemokine receptors and lipoprotein receptor-related protein 1(CD91) are regulators of inflammation in autoimmune diseases and T-cell migration. Objectives. The aim of this study was to get a deeper understanding how TNF blocking treatment influences inflammatory mechanisms and autoantibody formation in RA with special reference to similarities and differences with SLE. Methods. In patients with RA treated with anti-TNF, and in SLE patients (ACR criteria) clinical evaluation was performed and blood samples analyzed. Autoantibodies were analyzed using indirect immunofluorescence, ELISA and multiplex flow cytometry in samples from anti-TNF treated RA patients (n=59) followed longitudinally for 54 weeks, in pre-diseased samples from SLE patients (n=38) and matched population-based controls (n=152). T-cell expression of chemokine receptors and CD91 was analyzed by flow cytometry, whilst serum levels of chemokines were determined using ELISA in anti-TNF treated RA-patients (n=24) followed longitudinally (30 weeks), and cross-sectionally in SLE-patients (n=23). Expression of mRNA for chemokines was analyzed in T-cells from SLE-patients (n=10) using PCR. Results. After treatment with infliximab, RA patients produced ANA, anti-dsDNA and anti-nucleosome antibodies, but not anti-ENA antibodies. Although these antibodies are considered typical for SLE only one patient developed a transient lupus-syndrome. Antibodies against cell nuclear antigens, including ENA, were detected several years before the first clinical symptom of SLE; anti-SSA was the earliest detectable antibody. In RA-patients before infliximab treatment, the T-cell expression of several chemokine receptors was elevated compared with healthy controls. In contrast, only one soluble chemokine, IP-10 was elevated. After treatment the levels of soluble MIP-1β, MCP-1 and IP-10, and the T-cell expression of CCR2 were decreased. In SLE-patients MIP-1β, MCP-1, SDF-1, IP-10 and RANTES in blood were elevated, whilst expression of CXCR5 and CCR6 on T-cells was lower than in healthy controls. T-cell expression of CXCR2 and CCR1 was elevated in active disease (measured as SLEDAI index), whereas the CXCR5 and CCR2 expression was lower in inactive SLE. In SLE patients with nephritis IP-10 was lower and T-cell expression of CXCR3 and CCR3 elevated compared with patients without nephritis. The expression of CD91 was higher on T-cells from patients not responsive to infliximab treatment compared with responders. Conclusion. These findings indicate that anti-TNF (infliximab) treatment in RA-patients has a major impact on the production of autoantibodies and chemokines. The autoantibody profile in infliximab-treated patients was similar to that predating disease onset in SLE patients with the exception of anti-ENA being detectable in SLE, but the development of lupus-syndromes was rare. The expression of CD91 on T-cells may predict responsiveness to infliximab. The expression of chemokine receptors in SLE- patients seemed to be related to disease activity. Anti-nuclear antibodies were detectable years before clinical disease onset in patients who developed SLE suggesting a gradual pathogenic process.

Systemic lupus erythematosus is an autoinflammatory disorder driven by the development of autoantibodies to self-nucleic acids, in particular to DNA and to chromatin. Loss-of-function mutations of the secreted deoxyribonuclease DNASE1L3 have been implicated in the development of aggressive familial lupus. In addition, recent genome-wide association studies have linked a hypomorphic variant of DNASE1L3 to sporadic lupus. Studies in the lab determined that Dnase1l3-deficient mice develop rapid autoantibody responses against dsDNA and chromatin, and at older ages this leads to a lupus-like inflammatory disease. These disease manifestations were completely independent of the intracellular DNA sensor STING, which has been implicated in other examples of self-DNA driven autoinflammatory diseases. My project focused on developing assays to track the activity of DNASE1L3, as well as identifying the endogenous source of self-DNA normally processed by DNASE1L3. Using mouse models that allow the depletion of specific cell populations, we found that circulating DNASE1L3 is produced by hematopoietic cells, in particular by CD11c+ dendritic cells and by tissue macrophages. Taking into account the unique properties of DNASE1L3, we discovered that this enzyme is uniquely able to digest chromatin contained within and on the surface of apoptotic microparticles. Loss of DNASE1L3 activity in circulation results in elevated levels of DNA in plasma, in particular within microparticles. Microparticles are extensively bound by anti-chromatin autoantibodies isolated from both murine models of lupus as well as prototypical human clones. In addition, Dnase1l3-deficient mice have high levels of circulating IgG that bind to microparticles from young ages, and these titers increased as disease progressed in aged animals. Pretreatment of microparticles with DNASE1L3 largely abrogated this binding, demonstrating that DNASE1L3 directly reduces the immunogenicity of microparticles. We also studied two human patients with null mutations in DNASE1L3, and observed increased DNA circulating in plasma and, in particular, in their microparticles, demonstrating a conserved role for DNASE1L3 in mice and humans. Finally, we obtained plasma samples from a cohort of patients with sporadic SLE, and found that roughly 80% had circulating IgG that avidly bound microparticles. Roughly half of this group failed to bind to microparticles that had been pretreated with DNASE1L3, and this DNASE1L3-sensitive group also presented with lower levels of DNASE1L3 activity. We conclude that extracellular chromatin associated with microparticles acts as a potential self-antigen capable of causing loss of tolerance to self-DNA and inflammatory disease in both mice and humans. The secretion of a DNA-processing enzyme thus represents a novel, conserved tolerogenic mechanism by which dendritic cells restrict autoimmunity.

Background Systemic lupus erythematosus (SLE) is the prototypic multisystem autoimmune disease, with a broad spectrum of clinical presentations encompassing almost all organs and a chronic course, which can vary from mild to life-threatening. There is a major unmet need for outcome prediction enabling tailoring management and therapeutic interventions for SLE patients. Objectives To improve outcome prediction in SLE, the specific aims of this thesis were: (1) to evaluate the performance of the ACR and the SLICC classification criteria sets for SLE; (2) to evaluate the effect of the classification criteria fulfilled at time of SLE diagnosis and other predictors on long-term outcomes of damage and mortality; (3) to identify clinical predictors for SLE flares of disease activity; (4) to increase knowledge about the relationships between immunological markers and SLE disease activity. Methods We conducted a cross-sectional observational study of 2055 patients with a clinical diagnosis of SLE followed at 17 centers and registered in the Portuguese and Spanish national registries; the sensitivity of the ACR and SLICC classification criteria was compared using the McNemar’s test; the sensitivity of the two classification sets was further examined in five subgroups defined according to disease duration. We conducted a prospective inception cohort study of 192 SLE patients from time of diagnosis and followed up to 10 years at the CHUC Lupus Clinic; we assessed with multivariate Cox models the 10-year outcomes of damage and mortality, according to SLE classification status (fulfilling the ACR or only the SLICC criteria) at inception, and adjusting for potential baseline confounders. We conducted a prospective cohort study of 202 SLE patients followed up to 24 months at the CHUC Lupus Clinic over 1083 visits; we evaluated potential clinical predictors for disease activity flares with multivariate Cox regression models adjusting for potential confounders factors and estimating hazard ratios. We conducted cross-sectional studies of two groups of SLE patients, one with clinically active and another with inactive disease recruited at the CHUC Lupus Clinic and a group of healthy subjects enrolled at the same site; one peripheral blood sample was collected from each participant and analyzed with flow cytometry multiparametric immunophenotyping protocols to define relationships between immunological markers in B cells, Th17 cells and NK cells and SLE classification and disease activity status. Results The cross-sectional study on performance of classification criteria showed that the sensitivity for SLE clinical diagnosis was higher with the SLICC than with the ACR classification criteria (93.2% versus 85.6%, p<0.0001). From patients not fulfilling the ACR criteria, 62.8% could be classified with the SLICC. Patients within 5 years since disease onset presented the largest difference in sensitivity between the SLICC and the ACR criteria (respectively 89.3% and 76.0%, p<0.0001). In the 10-year prospective inception cohort study, patients meeting the ACR criteria compared to those with only the SLICC criteria at inception presented during follow-up with more cases of lupus nephritis (35.1% versus 13.8%, p<0.01), but less thrombotic antiphospholipid syndrome (4.5% versus 17.2%, p<0.01). The Cox models showed no significant differences in risk for damage or death between groups. In the 24-month prospective cohort study, the multivariate Cox models demonstrated that the risk of flare was more than two-fold, four-fold and three-fold higher for patients with SLE diagnosis up to 25 years, previous lupus nephritis or baseline immunosuppressant treatment, respectively. In the cross-sectional immunophenotyping studies, analysis of B cell subsets showed that differential expression of BAFFR, CD81 and CD38 in the transitional B cells allowed identifying two major clusters: the cluster 1 integrated all healthy subjects and 79% of SLE patients with clinically inactive disease, while in the cluster 2 there was only patients with SLE and 82% of those with clinically active disease. The analysis of Th17 cells showed no significant differences in the frequency of Th17 among healthy subjects and SLE patients, as well as among patients with clinically inactive and active disease. The analysis of NK cells showed a lower number and frequency of NK cells in SLE patients as compared to healthy subjects, regardless of disease activity status, and a lower frequency of CD56dim NK cells expressing CXCR3 was a marker of clinically active SLE (12.5% versus 24.1% in the active and inactive SLE group, respectively, p<0.01). Conclusions The SLICC classification criteria are more sensitive and may allow a SLE classification earlier in the disease course than the previous ACR criteria. Patients fulfilling at inception either of the classification criteria present no differences in the major long-term outcomes of organ damage and mortality. Patients with a SLE diagnosis before age 25, lupus nephritis or immunosuppressant treatment/severe SLE present higher risk for flares of disease activity; patients fulfilling at inception only the SLICC classification criteria may present higher risk of thrombotic antiphospholipid syndrome: these clinical predictors thus provide a basis for tailoring management strategies of SLE patients. Immunophenotyping studies suggested that SLE patients present: an upregulation of B cells, with subset clusters able to differentiate SLE patients from healthy subjects and clinically active from inactive SLE; a downregulation of NK cells, and less clear changes of Th17 cells. We provide proof-of-concept that a panel of immunological markers may provide a basis for biologic validation of clinical definitions for SLE disease activity states.
Introdução O Lúpus Eritematoso sistémico (LES) representa o paradigma das doenças autoimunes multissistémicas, apresentando um largo espectro de manifestações clínicas que potencialmente envolvem quase todos os órgãos e sistemas, com um curso clínico crónico e gravidade clínica variada, entre ligeira a severa e com risco de vida. Existe uma necessidade fundamental por cumprir de identificar preditores de prognóstico que permitam a individualização da monitorização e intervenções terapêuticas para os doentes com LES. Objetivos Identificar preditores de prognóstico em doentes com LES, através dos objetivos específicos desta tese: (1) avaliar o desempenho dos critérios de classificação ACR e SLICC para a identificação de doentes com LES; (2) avaliar o efeito dos critérios de classificação preenchidos à data de diagnóstico de LES e outros preditores no prognóstico a longo prazo em termos de dano irreversível e mortalidade; (3) identificar preditores clínicos para agudizações da atividade clínica do LES; (4) contribuir para o conhecimento das relações entre marcadores imunológicos e a atividade clínica do LES. Métodos Efetuámos um estudo observacional transversal de 2055 doentes com diagnóstico clínico de LES, seguidos em 17 centros e integrados nos registos nacionais de Portugal e Espanha; a sensibilidade dos critérios de classificação ACR e SLICC foi comparada através do teste de McNemar; a sensibilidade dos dois sistemas de classificação foi ainda analisada em 5 subgrupos definidos de acordo com a duração da doença. Realizámos um estudo prospetivo de coorte, incluindo 192 doentes com LES avaliados desde a data de diagnóstico e seguidos até 10 anos na CHUC Lupus Clinic; analisámos através de regressão multivariada de Cox o prognóstico a 10 anos, em termos de dano irreversível e mortalidade, em grupos definidos de acordo com os critérios de classificação cumpridos à data de diagnóstico (critérios ACR ou apenas os critérios SLICC) e ajustando para potenciais confundidores definidos à data de diagnóstico. Conduzimos um estudo prospetivo de coorte incluindo 202 doentes com LES seguidos até 24 meses na CHUC Lupus Clinic ao longo de 1083 consultas; analisámos potenciais preditores clínicos de agudizações da atividade do LES, aplicando regressão multivariada de Cox ajustada a potenciais confundidores e com estimativa dos hazard ratios dos preditores. Efetuámos estudos transversais incluindo dois grupos de doentes com LES, um com doença clinicamente ativa e outro com doença inativa, recrutados na CHUC Lupus Clinic e um grupo de indivíduos saudáveis recrutados no mesmo local; colhemos uma amostra de sangue periférico de cada participante, que foi processada através de protocolos de imunofenotipagem e analisada com citometria de fluxo multiparamétrica, para identificar relações entre marcadores imunológicos em células B, Th17 e NK e a classificação de LES e seus estados de atividade clínica. Resultados O estudo transversal do desempenho dos critérios de classificação demonstrou que a sensibilidade para o diagnóstico clínico de LES é mais elevado com o sistema de classificação SLICC do que com o ACR (93,2% e 85,6%, respetivamente, p<0,0001). Entre os doentes que não cumpriam os critérios ACR, 62,8% preencheram os critérios SLICC. Os pacientes com duração de doença até 5 anos apresentaram a maior diferença em sensibilidade entre o sistema SLICC e ACR de classificação (respetivamente 89,3% e 76,0%, p<0001). O estudo prospetivo de coorte de LES inicial e seguimento até 10 anos, mostrou que os doentes que preenchiam à data de diagnóstico os critérios ACR de classificação apresentaram ao longo do seguimento mais casos de nefrite lúpica do que aqueles cumprindo apenas critérios SLICC (35,1% e 13,8%, respetivamente, p<0,01), mas menos casos de síndrome antifosfolípido trombótico (4,5% e 17,2%, respetivamente, p<0,01). Os modelos multivariados de Cox não mostraram diferenças entre grupos no risco de dano irreversível nem de mortalidade. No estudo prospetivo de coorte com seguimento a 24 meses, os modelos multivariados de Cox demonstraram que o risco de agudizações clínicas do LES é mais de 2 vezes, 4 vezes e três vezes mais elevado para os doentes com diagnóstico de LES até aos 25 anos, com nefrite lúpica prévia ou sob terapêutica com imunossupressores à data de inclusão, respetivamente. Nos estudos transversais de imunofenotipagem, a análise da linhagem de células B demonstrou que a expressão diferencial de BAFFR, CD81 e CD38 nas células B de transição permite a identificação de dois principais clusters: o cluster 1, que integrou todos os indivíduos saudáveis e 79% dos doentes com LES clinicamente inativo, enquanto o cluster 2 incluiu apenas doentes com LES e 82% daqueles com doença clinicamente ativa. A análise das células Th17 mão mostrou diferenças significativas na frequência de Th17 entre o grupo de controlos e o de doentes com LES, nem entre pacientes com doença clinicamente inativa ou ativa. A análise das células NK revelou menor número e frequência de células NK em doentes com LES comparativamente aos controlos, independentemente da atividade clínica da doença; uma frequência mais baixa de células NK CD56dim expressando CXCR3 revelou ser um marcador de LES clinicamente ativo (12,5% e 24,1% no grupo com doença ativa e inativa, respetivamente, p<0,01). Conclusões Os critérios de classificação SLICC apresentam maior sensibilidade e podem permitir estabelecer a classificação como LES mais precocemente no curso da doença do que o prévio sistema de classificação ACR. Os doentes preenchendo à data de diagnóstico qualquer dos dois sistemas de classificação não apresentam diferenças no prognóstico a longo prazo em termos de dano irreversível nem de mortalidade. Os doentes com diagnóstico de LES até aos 25 anos, com nefrite lúpica ou necessitando terapêutica imunossupressora, apresentam risco mais elevado de sofrer agudizações clínicas do LES; pacientes preenchendo à data de diagnóstico apenas os critérios de classificação SLICC podem apresentar maior risco de síndrome anti-fosfolípido trombótico: estes preditores clínicos fornecem uma base para individualizar estratégias de monitorização e tratamento de doentes com LES. Os estudos de imunofenotipagem sugerem que os doentes com LES apresentam: hiperatividade de células B, com clusters de marcadores imunológicos em subtipos de células B que permitem diferenciar doentes com LES de indivíduos saudáveis e LES clinicamente ativo de inativo; hipoatividade das células NK e anomalias menos consistentes das células Th17. Fizemos prova do conceito que um painel de marcadores imunológicos pode providenciar uma base de validação biológica para definições clínicas de estados de atividade do LES.