The second meeting for the International Consensus on Antinuclear antibody (ANA) Pattern (ICAP) was held on 22 September 2015, one day prior to the opening of the 12th Dresden Symposium on Autoantibodies in Dresden, Germany. The ultimate goal of ICAP is to promote harmonization and understanding of autoantibody nomenclature, and thereby optimizing ANA usage in patient care. The newly developed ICAP website www.ANApatterns.org was introduced to the more than 50 participants. This was followed by several presentations and discussions focusing on key issues including the two-tier classification of ANA patterns into competent-level versus expert-level, the consideration of how to report composite versus mixed ANA patterns, and the necessity for developing a consensus on how ANA results should be reported. The need to establish on-line training modules to help users gain competency in identifying ANA patterns was discussed as a future addition to the website. To advance the ICAP goal of promoting wider international participation, it was agreed that there should be a consolidated plan to translate consensus documents into other languages by recruiting help from members of the respective communities.

Preclinical models of lupus indicate that T cell-B cell collaboration drives antinuclear antibody (ANA) production and sustains T cell activation. Autoreactive B lymphocytes are present in the normal repertoire but persist as ignorant or anergic cells. Mechanisms that normally limit T cell activation of autoreactive B cells remain incompletely resolved, but potentially include the absence of autoreactive effector T cell subsets and/or the presence of autoAgspecific regulatory T cells (Tregs). Several studies have addressed this issue by using experimental systems dependent on transgenic autoreactive B cells, but much less is known about the activation of autoreactive B cells present in a polyclonal repertoire. In the second chapter of this thesis, I have explored the role of effector T cells and Tregs using mice that express an inducible pseudoautoAg expressed on B cells and other antigen presenting cells (APCs). In this system, activated Th2 cells, but not naïve T cells, elicit the production of ANAs, but ANA production is severely limited by autoAg-specific Tregs. Bone marrow chimera experiments further demonstrated that this B cell activation is constrained by radioresistant autoantigen-expressing APCs (rAPC) present in the thymus as well as by non-hematopoietic stromal cells located in peripheral lymphoid tissue. Importantly, peripheral rAPC expression of autoAg induced the expansion of a highly effective subset of CD62L+CD69+ Tregs. The third chapter of this thesis focuses on the contribution of CD8+ T cells to fibrosis resulting from sterile lung injury. Type 2 effector production of IL-13 is v a demonstrated requirement in several models of fibrosis, and is routinely ascribed to CD4+ Th2 cells. However, we now demonstrate a major role for pulmonary CD8+ T cells, which mediate an exaggerated wound healing response and fibrosis through robust differentiation into IL-13-producing pro-fibrotic type 2 effectors (Tc2). Remarkably, differentiation of these Tc2 cells in the lung requires IL-21. We further show that the combination of IL-4 and IL-21 skews naïve CD8+ T cells to produce IL-21, which in turn acts in an autocrine manner to support robust IL-13 production. TGF-β negatively regulates production of IL-13 by suppressing CD8+ T cell responsiveness to IL-21. Our data illuminate a novel pathway involved in the onset and regulation of pulmonary fibrosis, and identify Tc2 cells as key mediators of fibrogenesis.

Trotz aller Fortschritte bei der Etablierung spezifischer Autoantikörperassays ist das Screening auf antinukleäre Antikörper (ANA) mittels indirekter Immunfluoreszenz an HEp-2-Zellen für eine qualitätsgerechte Labordiagnostik von ANA-assoziierten rheumatischen Erkrankungen (AARE) weiterhin unabdingbar. Mit den Erkenntnissen zur Relevanz von DFS-Mustern und DFS70-Antikörpern eröffnen sich neue Möglichkeiten zur Optimierung der serologischen Stufendiagnostik bei Verdacht auf AARE. Das dicht-feingranuläre („dense fine speckled“, DFS) ANA-Muster ist relativ gut von den klassischen, mit dsDNAAntikörpern assoziierten „homogenen“ ANA-Mustern differenzierbar. Die wichtigste bei diesem Muster nachweisbare ANA-Spezifität ist der DFS70-Antikörper (Synonym: LEDGFAntikörper). Dieser Antikörper ist auch die häufigste bei ANA-positiven gesunden Personen nachweisbare ANA-Spezifität. Die Prävalenz von DFS70-Antikörpern in AARE-Patienten ist signifikant niedriger im Vergleich zur Prävalenz bei ANA gesunden Personen. Es besteht eine negative Assoziation der DFS70-Antikörper mit AARE, insbesondere wenn der Antikörper nicht in Begleitung von klinisch relevanten Autoantikörpern vorliegt. Isolierte DFS70-Antikörper findet man in weniger als 1% der AARE, aber in 5%–11% bei gesunden Personen. Beim Vorliegen eines isolierten DFS70-Antikörpers verringert sich die post-Test-Wahrscheinlichkeit für eine AARE deutlich. DFS70-Antikörper sind daher wertvolle neue Biomarker zur besseren Interpretation positiver ANA bei Negativität für AARE-assoziierte Autoantikörper und sollten in modifizierte Testalgorithmen zur Vermeidung unnötiger Überweisungen und Folgeuntersuchung von ANA positiven Personen integriert werden.

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Das Sjögren-Syndrom (SjS) stellt eine mögliche autoimmune Ursache einer interstitiellen Lungenerkrankung dar. Die Abklärung in Richtung SjS ist aber im Vergleich zu anderen systemischen Autoimmunerkrankungen bisher kaum standardisiert. Die subjektive Sicca-Symptomatik, die Anti-SS-A/Ro-Antikörper und selbst die ANA-Diagnostik als Suchtest haben alle relevante Einschränkungen in ihrer Sensitivität und/oder Spezifität. Vor diesem Hintergrund haben wir in einer interdisziplinären Diskussion einen Konsens für die SjS-Abklärung entwickelt, den wir hier für die breitere Diskussion vorstellen. Neben ANA sollten sowohl Anti-SS-A/Ro-Antikörper als auch Antikörper gegen α‑Fodrin bestimmt werden. Wichtig ist die Objektivierung der Trockenheit mittels Schirmer- und Saxon-Test und bei fehlenden typischen Autoantikörpern die Speicheldrüsenbiopsie. / Sjögren’s syndrome (SjS) is a possible autoimmune cause of interstitial lung disease. The diagnostic pathway for SjS, however, is largely undefined in comparison to other systemic autoimmune diseases. Subjective sicca symptoms, anti-SS-A/Ro antibodies and even ANA as screening tests all have relevant limitations in sensitivity and/or specificity. Against this background, in an interdisciplinary discussion we have developed a consensus for the clarification of SjS, which is presented here for broader discussion. In addition to ANA and anti-SS-A/Ro antibodies, antibodies against alpha-fodrin should be included. Objective measures of dryness, such a Schirmer and Saxon tests are important, as is a salivary gland biopsy in the absence of typical autoantibodies.

Objetivos: Os objetivos deste estudo foram: avaliar a frequência de imunodeficiências primárias de anticorpos e Complemento em pacientes com lupus eritematoso sistêmico juvenil (LESJ); avaliar possíveis associações entre a presença de imunodeficiência primária (IDP) e dados demográficos, ocorrência de infecções, manifestações clínicas, atividade da doença, dano cumulativo e terapêutica direcionada ao LESJ; e determinar a frequência do anticorpo anti-C1q, estabelecendo a sua especificidade, sensibilidade e valores preditivos para o diagnóstico de LESJ. Métodos: Setenta e dois pacientes com LESJ foram avaliados para a determinação dos níveis séricos de imunoglobulinas (IgG, IgA, IgM e IgE) e subclasses de IgG, e dos componentes iniciais da via clássica do sistema Complemento (C1q, C1r, C1s, C4, C2, C3). Sessenta e sete pacientes e 26 controles saudáveis foram avaliados para a presença do anticorpo anti-C1q. O número de cópias do gene C4 foi determinado por PCR (reação de polimerase em cadeia) em tempo real nos pacientes com deficiência de C4. Setenta pacientes foram avaliados para a presença de deficiência de C2 tipo I. Resultados: Evidência de IDP foi identificada em 16 pacientes (22%): 3 com deficiência (D) de C2, 3 com C4D, 2 com C1qD, 4 com IgG2D (<20mg/dL), 3 com IgAD (<7mg/dL), e 3 com IgMD (<35mg/dL); um destes pacientes apresentou deficiência concomitante de IgA, C4 e C2. Quatro dos 13 pacientes do sexo masculino (30%) e 12 das 59 pacientes do sexo feminino (20%) apresentaram diagnóstico de IDP. As características clínicas de LES não diferiram entre os pacientes com e sem IDP. A mediana do SLICC/ACR-DI foi maior entre os pacientes com IDP (p=0,0033), assim como a frequência de SLICC/ACR-DI>1 (p=0,023). Os grupos também foram semelhantes quanto à ocorrência de infecção e terapêutica utilizada para o LESJ. Os únicos dois casos de LESJ com idade de início antes dos 2 anos apresentaram C1qD e IgMD, respectivamente. Para o diagnóstico de LESJ, o anticorpo anti-C1q apresentou especificidade de 100% (IC 86.7-100%), sensibilidade de 19.4% (IC 10.7-30.8%), valor preditivo positivo de 100% (IC 75.3-100%) e valor preditivo negativo de 32,5% (IC 22,4-43,9%). Conclusões: Foi observada uma elevada frequência de imunodeficiências de anticorpos e Complemento nos pacientes com LESJ, sugerindo que esses defeitos podem contribuir para a patogênese do lúpus. Esses achados indicam que os dois grupos de IDPs devem ser investigados em pacientes com LES de início precoce e de maior gravidade / Objectives. The objectives of this study were: to establish the frequency of primary immunoglobulin and Complement deficiency in Juvenile SLE (JSLE); to evaluate possible associations between the presence of primary immunodeficiency and demographic data, occurrence of infections, JSLE clinical manifestations, disease activity, cumulative damage and therapy; and to determine the frequency of anti-C1q antibody, establishing its sensitivity, specificity and predictive values for JSLE diagnosis. Methods. Seventy-two JSLE patients were analyzed for serum levels of immunoglobulin classes (IgG, IgA, IgM e IgE) and IgG subclasses and early components of the classical Complement pathway (C1q, C1r, C1s, C4, C2, C3). Sixty-seven patients and 26 healthy controls were evaluated for the presence of anti-C1q antibody. C4 gene copy number was determined by real time PCR (polymerase chain reaction) in C4 deficient patients. Seventy patients were analyzed by PCR for the presence of type I C2 deficiency. Results. Evidence of PID was identified in 16 patients (22%): 3 with C2 deficiency (D), 3 with C4D, 2 with C1qD, 4 with IgG2D (<20mg/dL), 3 with IgAD (<7mg/dL), and 3 with IgMD (<35mg/dL); one of these patients presented concomitant IgA, C2 and C4 deficiency. Four out of the 13 boys (30%) and 12 out of 59 girls (20%) had PID diagnosis. SLE features did not differ between patients with and without PID. The median SLICC/ACR-DI was higher among PID subjects (p=0.0033), as was the frequency of SLICC/ACR-DI>1 (p=0.023). Both groups did not differ regarding the occurrence of infections and therapeutic for JSLE. The only 2 cases with age of onset below 2 years presented C1qD and IgMD, respectively. For JSLE diagnosis, the anti-C1q antibodies presented a specificity of 100% (CI 86.7-100%), sensitivity of 19.4% (CI 10.7-30.8%), positive predictive value of 100% (CI 75.3-100%) and negative predictive value of 32,5% (CI 22,4-43,9%). Conclusions. A high frequency of immunoglobulin and Complement deficiency was observed in this JSLE series, suggesting that these defects may contribute to lupus development. Our findings indicate that these two groups of PID should be investigated in early-onset and severe lupus

Objetivos: Os objetivos deste estudo foram: avaliar a frequência de imunodeficiências primárias de anticorpos e Complemento em pacientes com lupus eritematoso sistêmico juvenil (LESJ); avaliar possíveis associações entre a presença de imunodeficiência primária (IDP) e dados demográficos, ocorrência de infecções, manifestações clínicas, atividade da doença, dano cumulativo e terapêutica direcionada ao LESJ; e determinar a frequência do anticorpo anti-C1q, estabelecendo a sua especificidade, sensibilidade e valores preditivos para o diagnóstico de LESJ. Métodos: Setenta e dois pacientes com LESJ foram avaliados para a determinação dos níveis séricos de imunoglobulinas (IgG, IgA, IgM e IgE) e subclasses de IgG, e dos componentes iniciais da via clássica do sistema Complemento (C1q, C1r, C1s, C4, C2, C3). Sessenta e sete pacientes e 26 controles saudáveis foram avaliados para a presença do anticorpo anti-C1q. O número de cópias do gene C4 foi determinado por PCR (reação de polimerase em cadeia) em tempo real nos pacientes com deficiência de C4. Setenta pacientes foram avaliados para a presença de deficiência de C2 tipo I. Resultados: Evidência de IDP foi identificada em 16 pacientes (22%): 3 com deficiência (D) de C2, 3 com C4D, 2 com C1qD, 4 com IgG2D (<20mg/dL), 3 com IgAD (<7mg/dL), e 3 com IgMD (<35mg/dL); um destes pacientes apresentou deficiência concomitante de IgA, C4 e C2. Quatro dos 13 pacientes do sexo masculino (30%) e 12 das 59 pacientes do sexo feminino (20%) apresentaram diagnóstico de IDP. As características clínicas de LES não diferiram entre os pacientes com e sem IDP. A mediana do SLICC/ACR-DI foi maior entre os pacientes com IDP (p=0,0033), assim como a frequência de SLICC/ACR-DI>1 (p=0,023). Os grupos também foram semelhantes quanto à ocorrência de infecção e terapêutica utilizada para o LESJ. Os únicos dois casos de LESJ com idade de início antes dos 2 anos apresentaram C1qD e IgMD, respectivamente. Para o diagnóstico de LESJ, o anticorpo anti-C1q apresentou especificidade de 100% (IC 86.7-100%), sensibilidade de 19.4% (IC 10.7-30.8%), valor preditivo positivo de 100% (IC 75.3-100%) e valor preditivo negativo de 32,5% (IC 22,4-43,9%). Conclusões: Foi observada uma elevada frequência de imunodeficiências de anticorpos e Complemento nos pacientes com LESJ, sugerindo que esses defeitos podem contribuir para a patogênese do lúpus. Esses achados indicam que os dois grupos de IDPs devem ser investigados em pacientes com LES de início precoce e de maior gravidade / Objectives. The objectives of this study were: to establish the frequency of primary immunoglobulin and Complement deficiency in Juvenile SLE (JSLE); to evaluate possible associations between the presence of primary immunodeficiency and demographic data, occurrence of infections, JSLE clinical manifestations, disease activity, cumulative damage and therapy; and to determine the frequency of anti-C1q antibody, establishing its sensitivity, specificity and predictive values for JSLE diagnosis. Methods. Seventy-two JSLE patients were analyzed for serum levels of immunoglobulin classes (IgG, IgA, IgM e IgE) and IgG subclasses and early components of the classical Complement pathway (C1q, C1r, C1s, C4, C2, C3). Sixty-seven patients and 26 healthy controls were evaluated for the presence of anti-C1q antibody. C4 gene copy number was determined by real time PCR (polymerase chain reaction) in C4 deficient patients. Seventy patients were analyzed by PCR for the presence of type I C2 deficiency. Results. Evidence of PID was identified in 16 patients (22%): 3 with C2 deficiency (D), 3 with C4D, 2 with C1qD, 4 with IgG2D (<20mg/dL), 3 with IgAD (<7mg/dL), and 3 with IgMD (<35mg/dL); one of these patients presented concomitant IgA, C2 and C4 deficiency. Four out of the 13 boys (30%) and 12 out of 59 girls (20%) had PID diagnosis. SLE features did not differ between patients with and without PID. The median SLICC/ACR-DI was higher among PID subjects (p=0.0033), as was the frequency of SLICC/ACR-DI>1 (p=0.023). Both groups did not differ regarding the occurrence of infections and therapeutic for JSLE. The only 2 cases with age of onset below 2 years presented C1qD and IgMD, respectively. For JSLE diagnosis, the anti-C1q antibodies presented a specificity of 100% (CI 86.7-100%), sensitivity of 19.4% (CI 10.7-30.8%), positive predictive value of 100% (CI 75.3-100%) and negative predictive value of 32,5% (CI 22,4-43,9%). Conclusions. A high frequency of immunoglobulin and Complement deficiency was observed in this JSLE series, suggesting that these defects may contribute to lupus development. Our findings indicate that these two groups of PID should be investigated in early-onset and severe lupus

The oldest-old (people aged 80 or older) constituted 5 % of the population in Sweden in 2000, an increase from 1.5 % fifty years earlier. The immune system undergoes dramatic changes at high age, sometimes referred to as “immunosenescence”. However, the natures of these changes, and in particular, their clinical consequences are incompletely understood. In a previous longitudinal study, a set of immune parameters were identified and termed immune risk phenotype (IRP) because of an association with increased mortality. The IRP consists of changes in the T lymphocyte compartment, in particular an inverted CD4/CD8 ratio. The IRP was found to be associated with cytomegalovirus (CMV) infection, which through expansions of cytolytic anti-viral CD8 cell responses was ascribed a role in the development of IRP. The general aim of this thesis was to increase the knowledge of changes in the immune system and their clinical consequences in the oldest-old. The population-based random sample of the longitudinal NONA-Immune Study (n = 138, mean age 90 years at baseline) was used for all investigations. In paper I, the effects on sample size of various exclusion protocols for immune studies of the elderly was examined. The commonly used SENIEUR protocol, selecting individuals representing ‘normal ageing’, excluded 90 % of nonagenarians. Based on different protocol criteria, individuals were grouped into ‘very healthy’, ‘moderately healthy’ or ‘frail’. The prevalence of CMV was similar across the groups. Further, differentiated CD8 populations associated with CMV, i.e. those expressing CD56, CD57 and CD45RA while lacking expression of CD27 and CD28, were equally distributed across the groups of the oldest-old, but were, as expected, significantly increased in the elderly compared to a middle aged control group. The findings showed that lymphocyte subsets associated with IRP might serve as significant biomarkers of ageing independent of the overall health status, also supporting the notion that immunological studies of the oldest-old should be done in population-based non-selected populations. The IRP and the presence of low-grade inflammation, for example increase of   IL-6 in plasma, constitute major predictors of 2-year mortality in the oldest-old. In paper II, the CD4/CD8 ratio and IL-6 were found to predict 97 % of observed survival and 57 % of deaths over 2 years. The impact of IRP and IL-6 on 2-year survival was independent of age, sex and several diseases. The longitudinal design allowed temporal evaluations, suggesting a sequence of events starting with IRP and leading to inflammation in the decline state.                                      Four-year mortality in the oldest-old (paper III) was found to be mainly related to markers of inflammation and IRP. Individuals with both inverted CD4/CD8 ratio and high IL-6 level had significantly higher 4 year mortality (82 %) compared to individuals with CD4/CD8 ratio ³ 1 and low IL-6 level (29 %) at baseline. The presence of IRP and increased IL-6 level showed some associations with presence of diseases; in particular, IL6 was associated with the presence of cognitive impairment. However, despite being strong predictors of mortality, IRP and IL-6 could not be linked to any specific cause of death, probably due to the multi-factorial nature of these factors.                                                                                                                             The prevalence of antinuclear antibodies (ANA) in the oldest-old was higher compared to younger controls (paper IV). The difference across age was most pronounced in men, showing low levels at younger age, whereas the prevalence among the oldest-old men reached a similar level as in women. There was no association between the presence of ANA and IRP, CMV status or health status in the oldest-old.