RESUMEN
BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune blistering disease driven by pathogenic antibodies to desmoglein-1 and -3, levels of which correlate with disease activity. Anti-desmoglein-3 IgG4 isotype antibodies are said to predominate in active disease and anti-desmoglein-3 IgG1 in remission; however, these observations arose from vertical studies, with limited assessments of clinical activity. The objective of this study was to examine the relationship between desmoglein autoantibodies, subdivided by isotype and disease activity using the validated PV activity tool "Pemphigus Disease Area Index (PDAI)." METHODS: Forty PV patients with predominantly mucosal disease were studied prospectively, 24 serially, and PDAI and anti-desmoglein antibodies recorded at each visit over a period of up to 15 months. RESULTS: At enrolment, only anti-desmoglein-3 IgG4 levels were significantly associated with disease activity but the correlation was weak. During follow-up, within-patient changes in disease activity correlated with changes in anti-desmoglein-3 IgG levels, but correlations were similar for both anti-desmoglein-3 IgG1 and IgG4. These trends were not observed in anti-desmoglein-1 IgG levels, although the majority of patients were negative at baseline. CONCLUSIONS: Anti-desmoglein-3 IgG4 levels correlated only weakly with PDAI scores at a single time point. Reciprocity of IgG1 vs IgG4 anti-desmoglein-3 with changes in disease activity over time could not be confirmed, but rather, changes in levels of anti-desmoglein-3 IgG, irrespective of isotype, were useful in following individual patient responses.
Asunto(s)
Pénfigo , Autoanticuerpos , Desmogleínas , HumanosRESUMEN
Most genetic defects that arrest B-cell development in the bone marrow present early in life with agammaglobulinemia, whereas incomplete antibody deficiency is usually associated with circulating B cells. We report 3 related individuals with a novel form of severe B-cell deficiency associated with partial persistence of serum immunoglobulin arising from a missense mutation in NFKB2. Significantly, this point mutation results in a D865G substitution and causes a failure of p100 phosphorylation that blocks processing to p52. Severe B-cell deficiency affects mature and transitional cells, mimicking the action of rituximab. This phenotype appears to be due to disruption of canonical and noncanonical nuclear factor κB pathways by the mutant p100 molecule. These findings could be informative for therapeutics as well as immunodeficiency.
Asunto(s)
Alopecia/genética , Alopecia/inmunología , Linfocitos B/inmunología , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Subunidad p52 de NF-kappa B/genética , Adulto , Alopecia/patología , Linfocitos B/patología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Salud de la Familia , Femenino , Genes Dominantes , Células HEK293 , Humanos , Síndromes de Inmunodeficiencia/patología , Datos de Secuencia Molecular , FN-kappa B/inmunología , Subunidad p52 de NF-kappa B/metabolismo , Linaje , Fosforilación/inmunología , Mutación Puntual , Homología de Secuencia de Aminoácido , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. OBJECTIVE: We sought to determine the signaling pathways and cellular interactions required for the development and function of TFH cells in human subjects. METHODS: Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cTFH) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS, or BTK. RESULTS: Loss-of-function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS, or BTK reduced cTFH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cTFH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cTFH cell function in vitro and in vivo, as corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1, and IL12RB1 LOF mutations. CONCLUSION: Specific mutations affect the quantity and quality of cTFH cells, highlighting the need to assess TFH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain TFH cell-induced B-cell differentiation. These findings shed new light on TFH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.
Asunto(s)
Síndromes de Inmunodeficiencia/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Agammaglobulinemia Tirosina Quinasa , Linfocitos B/inmunología , Ligando de CD40/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Células Cultivadas , Humanos , Quinasa I-kappa B/genética , Inmunidad Humoral/genética , Síndromes de Inmunodeficiencia/genética , Memoria Inmunológica , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Interferón gamma/genética , Interferón gamma/metabolismo , Activación de Linfocitos , Mutación/genética , Proteínas Tirosina Quinasas/genética , Receptores de Citocinas/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genética , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
B-cell responses are guided by the integration of signals through the B-cell receptor (BCR), CD40, and cytokine receptors. The common γ chain (γc)-binding cytokine interleukin (IL)-21 drives humoral immune responses via STAT3-dependent induction of transcription factors required for plasma cell generation. We investigated additional mechanisms by which IL-21/STAT3 signaling modulates human B-cell responses by studying patients with STAT3 mutations. IL-21 strongly induced CD25 (IL-2Rα) in normal, but not STAT3-deficient, CD40L-stimulated naïve B cells. Chromatin immunoprecipitation confirmed IL2RA as a direct target of STAT3. IL-21-induced CD25 expression was also impaired on B cells from patients with IL2RG or IL21R mutations, confirming a requirement for intact IL-21R signaling in this process. IL-2 increased plasmablast generation and immunoglobulin secretion from normal, but not CD25-deficient, naïve B cells stimulated with CD40L/IL-21. IL-2 and IL-21 were produced by T follicular helper cells, and neutralizing both cytokines abolished the B-cell helper capacity of these cells. Our results demonstrate that IL-21, via STAT3, sensitizes B cells to the stimulatory effects of IL-2. Thus, IL-2 may play an adjunctive role in IL-21-induced B-cell differentiation. Lack of this secondary effect of IL-21 may amplify the humoral immunodeficiency in patients with mutations in STAT3, IL2RG, or IL21R due to impaired responsiveness to IL-21.
Asunto(s)
Linfocitos B/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Interleucina-2/farmacología , Interleucinas/farmacología , Células Plasmáticas/efectos de los fármacos , Factor de Transcripción STAT3/genética , Linfocitos B/citología , Linfocitos B/metabolismo , Ligando de CD40/farmacología , Diferenciación Celular/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Expresión Génica/efectos de los fármacos , Humanos , Interleucina-10/farmacología , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Plasmáticas/citología , Células Plasmáticas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcriptoma/efectos de los fármacosRESUMEN
T follicular helper (Tfh) cells are critical for providing the necessary signals to induce differentiation of B cells into memory and Ab-secreting cells. Accordingly, it is important to identify the molecular requirements for Tfh cell development and function. We previously found that IL-12 mediates the differentiation of human CD4(+) T cells to the Tfh lineage, because IL-12 induces naive human CD4(+) T cells to acquire expression of IL-21, BCL6, ICOS, and CXCR5, which typify Tfh cells. We have now examined CD4(+) T cells from patients deficient in IL-12Rß1, TYK2, STAT1, and STAT3 to further explore the pathways involved in human Tfh cell differentiation. Although STAT1 was dispensable, mutations in IL12RB1, TYK2, or STAT3 compromised IL-12-induced expression of IL-21 by human CD4(+) T cells. Defective expression of IL-21 by STAT3-deficient CD4(+) T cells resulted in diminished B-cell helper activity in vitro. Importantly, mutations in STAT3, but not IL12RB1 or TYK2, also reduced Tfh cell generation in vivo, evidenced by decreased circulating CD4(+)CXCR5(+) T cells. These results highlight the nonredundant role of STAT3 in human Tfh cell differentiation and suggest that defective Tfh cell development and/or function contributes to the humoral defects observed in STAT3-deficient patients.
Asunto(s)
Diferenciación Celular , Interleucina-12/farmacología , Mutación/genética , Receptores de Interleucina-12/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genética , Linfocitos T Colaboradores-Inductores/citología , TYK2 Quinasa/genética , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Humanos , Huésped Inmunocomprometido , Activación de Linfocitos , Ratones , Receptores de Interleucina-12/deficiencia , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/deficiencia , Transducción de Señal , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , TYK2 Quinasa/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: The 1858T allele of protein tyrosine phosphatase nonreceptor type 22 (PTPN22; R620W) exhibits one of the strongest and most consistent associations with sporadic autoimmune disease. Although autoimmunity is common in patients with primary antibody deficiency (PAD), it remains unknown whether its pathogenesis is similar when it arises in this context compared with in immunocompetent patients. OBJECTIVE: We set out to determine whether the 1858T allele of PTPN22 was associated with PAD or with autoimmunity in the context of PAD. METHODS: We genotyped rs2476601 (g.1858C>T), a single nucleotide polymorphism encoding substitution of arginine for tryptophan in PTPN22 (R620W), in 193 patients with PAD and 148 control subjects from an Australian cohort. We also performed a subgroup analysis according to the presence of autoimmunity and B-cell phenotypes. RESULTS: C/T and T/T PTPN22 genotypes were more common in patients with PAD than in the matched control subjects (C/T, 18.1% vs 9.5%; T/T, 1.04% vs 0.6%). The T allele was associated with an increased risk of PAD relative to control subjects (odds ratio, 2.10; 95% CI, 1.11-4.00). The distribution of genotypes in control subjects was similar to those reported previously and did not deviate significantly from Hardy-Weinberg equilibrium. We found a strong association between the 1858T allele and PAD with coexistent autoimmune diseases. In patients with PAD and autoimmunity, 16 (43.2%) of 37 had at least one T allele of PTPN22 compared with 27 (17.3%) of 156 with the C/C genotype (P=.0014; odds ratio, 3.64; 95% CI, 1.68-7.88). We found no evidence that this effect was mediated by enrichment of CD21low B cells. CONCLUSION: The 1858T PTPN22 allele is strongly associated with autoimmunity in patients with PAD.
Asunto(s)
Sustitución de Aminoácidos , Autoinmunidad/genética , Síndromes de Inmunodeficiencia/genética , Polimorfismo de Nucleótido Simple , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Adulto , Alelos , Australia , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Técnicas de Genotipaje , Humanos , Síndromes de Inmunodeficiencia/inmunología , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Proteína Tirosina Fosfatasa no Receptora Tipo 22/inmunología , Factores de RiesgoRESUMEN
BACKGROUND: The capacity of CD8(+) T cells to control infections and mediate antitumor immunity requires the development and survival of effector and memory cells. IL-21 has emerged as a potent inducer of CD8(+) T-cell effector function and memory development in mouse models of infectious disease. However, the role of IL-21 and associated signaling pathways in protective CD8(+) T-cell immunity in human subjects is unknown. OBJECTIVE: We sought to determine which signaling pathways mediate the effects of IL-21 on human CD8(+) T cells and whether defects in these pathways contribute to disease pathogenesis in patients with primary immunodeficiencies caused by mutations in components of the IL-21 signaling cascade. METHODS: Human primary immunodeficiencies resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Lymphocytes from patients with loss-of-function mutations in signal transducer and activator of transcription 1 (STAT1), STAT3, or IL-21 receptor (IL21R) were used to assess the respective roles of these genes in human CD8(+) T-cell differentiation in vivo and in vitro. RESULTS: Mutations in STAT3 and IL21R, but not STAT1, led to a decrease in multiple memory CD8(+) T-cell subsets in vivo, indicating that STAT3 signaling, possibly downstream of IL-21R, regulates the memory cell pool. Furthermore, STAT3 was important for inducing the lytic machinery in IL-21-stimulated naive CD8(+) T cells. However, this defect was overcome by T-cell receptor engagement. CONCLUSION: The IL-21R/STAT3 pathway is required for many aspects of human CD8(+) T-cell behavior but in some cases can be compensated by other signals. This helps explain the relatively mild susceptibility to viral disease observed in STAT3- and IL-21R-deficient subjects.
Asunto(s)
Linfocitos T CD8-positivos/citología , Diferenciación Celular , Memoria Inmunológica , Síndrome de Job/genética , Mutación , Factor de Transcripción STAT3/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Humanos , Interleucinas/genética , Interleucinas/inmunología , Interleucinas/metabolismo , Síndrome de Job/inmunología , Síndrome de Job/patología , Receptores de Interleucina-21/genética , Receptores de Interleucina-21/inmunología , Receptores de Interleucina-21/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them, SH2B3-a negative regulator of cytokine and growth factor receptor signaling-harbors rare coding variants in over 5% of SLE patients. Here, we show that unlike the variant found exclusively in healthy controls, SH2B3 rare variants found in lupus patients are predominantly hypomorphic alleles, failing to suppress IFNGR signaling via JAK2-STAT1. The generation of two mouse lines carrying patients' variants revealed that SH2B3 is important in limiting the number of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair the negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role for SH2B3 in human B cell tolerance and lupus risk.
Asunto(s)
Autoinmunidad , Lupus Eritematoso Sistémico , Animales , Humanos , Ratones , Autoinmunidad/genética , Factor Activador de Células B/metabolismo , Linfocitos B , Lupus Eritematoso Sistémico/genética , Células Precursoras de Linfocitos BRESUMEN
As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection.
Asunto(s)
Linfocitos B , Linfocitos T CD4-Positivos , Linfocitos B/metabolismo , Antígenos CD57/metabolismo , Diferenciación Celular , Antígeno CTLA-4 , HumanosRESUMEN
The transitional stage of B-cell development represents an important step where autoreactive cells are deleted, allowing the generation of a mature functional B-cell repertoire. In mice, 3 subsets of transitional B cells have been identified. In contrast, most studies of human transitional B cells have focused on a single subset defined as CD24(hi)CD38(hi) B cells. Here, we have identified 2 subsets of human transitional B cells based on the differential expression of CD21. CD21(hi) transitional cells displayed higher expression of CD23, CD44, and IgD, and exhibited greater proliferation and Ig secretion in vitro than CD21(lo) transitional B cells. In contrast, the CD21(lo) subset expressed elevated levels of LEF1, a transcription factor highly expressed by immature lymphocytes, and produced higher amounts of autoreactive Ab. These phenotypic, functional, and molecular features suggest that CD21(lo) transitional B cells are less mature than the CD21(hi) subset. This was confirmed by analyzing X-linked agammaglobulinemia patients and the kinetics of B-cell reconstitution after stem cell transplantation, which revealed that the development of CD21(lo) transitional B cells preceded that of CD21(hi) transitional cells. These findings provide important insights into the process of human B-cell development and have implications for understanding the processes underlying perturbed B-cell maturation in autoimmune and immunodeficient conditions.
Asunto(s)
Células Precursoras de Linfocitos B/fisiología , Receptores de Complemento 3d/genética , Animales , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Receptores de Complemento 3d/metabolismoRESUMEN
"Experiments of nature" due to single gene mutations resulting in human immunodeficiency states have revealed critical roles for several genes in regulating lymphocyte development and the generation of protective immunity. Recently, heterozygous mutations in STAT3 were found to cause autosomal dominant hyper-IgE syndrome, a condition affecting not only the immune system but also other mesenchymal and ectodermal tissues, including bones, cranium, teeth, and skin. STAT proteins operate to integrate signals from surface receptors, including cytokine receptors, that regulate growth and differentiation of multiple cell lineages. In this article, we will review how the study of STAT3 deficiency in humans and mice has highlighted nonredundant roles of STAT3, and of specific cytokines, in diverse cellular processes such as antimicrobial immunity and protection at epithelial barriers, the generation of functional humoral immune responses, bone formation, and keratinocyte biology.
Asunto(s)
Diferenciación Celular/inmunología , Modelos Animales de Enfermedad , Síndrome de Job/inmunología , Linfocitos/inmunología , Factor de Transcripción STAT3/fisiología , Animales , Formación de Anticuerpos/genética , Diferenciación Celular/genética , Citocinas/fisiología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Humanos , Inmunidad Celular/genética , Síndrome de Job/genética , Síndrome de Job/patología , Linfocitos/microbiología , Linfocitos/patología , Ratones , Factor de Transcripción STAT3/deficiencia , Factor de Transcripción STAT3/genéticaRESUMEN
The production of immunoglobulin E (IgE) is tightly regulated. This is evidenced by the fact that it comprises less than 0.0001% of serum Ig, and aberrant production causes atopic conditions, including allergy, rhinitis, and anaphylaxis. Interleukin-4 (IL-4) is a well-characterized inducer of IgE by human and murine B cells, whereas interferon-gamma can antagonize this effect. IL-21 has also been recognized for its ability to suppress IL-4-induced IgE production by murine B cells. Here, we identified IL-21 as an inducer of IgE production by CD40L-stimulated human naive B cells. Furthermore, there was a striking synergy between IL-4 and IL-21 on inducing IgE secretion by CD40L-stimulated human B cells, such that the levels detected under these conditions exceeded those induced by IL-4 or IL-21 alone by more than 10-fold. IL-21 induced activation of STAT3 and analysis of B cells from patients with loss-of-function STAT3 mutations revealed that the ability of IL-21 to induce IgE secretion, and augment that driven by IL-4, was STAT3-dependent. These findings highlight a fundamental difference between the regulation of IgE production by human and murine B cells and have implications for the dysregulated production of IgE in conditions characterized by extremely high levels of serum IgE.
Asunto(s)
Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Inmunoglobulina E/biosíntesis , Interleucinas/farmacología , Factor de Transcripción STAT3/fisiología , Animales , Linfocitos B/citología , Linfocitos B/fisiología , Linfocitos T CD4-Positivos/inmunología , Ligando de CD40/farmacología , Diferenciación Celular , División Celular , Células Cultivadas , Sangre Fetal/citología , Humanos , Técnicas In Vitro , Recién Nacido , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-4/fisiología , Interleucina-6/fisiología , Interleucinas/química , Interleucinas/fisiología , Síndrome de Job/genética , Síndrome de Job/inmunología , Ratones , Mutación , Fosforilación , Receptores de IgE/metabolismo , Factor de Transcripción STAT3/deficiencia , Factor de Transcripción STAT3/genética , Especificidad de la EspecieRESUMEN
Genetic primary immunodeficiency diseases are increasingly recognized, with pathogenic mutations changing the composition of circulating leukocyte subsets measured by flow cytometry (FCM). Discerning changes in multiple subpopulations is challenging, and subtle trends might be missed if traditional reference ranges derived from a control population are applied. We developed an algorithm where centiles were allocated using non-parametric comparison to controls, generating multiparameter heat maps to simultaneously represent all leukocyte subpopulations for inspection of trends within a cohort or segregation with a putative genetic mutation. To illustrate this method, we analyzed patients with Primary Antibody Deficiency (PAD) and kindreds harboring mutations in TNFRSF13B (encoding TACI), CTLA4, and CARD11. In PAD, loss of switched memory B cells (B-SM) was readily demonstrated, but as a continuous, not dichotomous, variable. Expansion of CXCR5+/CD45RA- CD4+ T cells (X5-Th cells) was a prominent feature in PAD, particularly in TACI mutants, and patients with expansion in CD21-lo B cells or transitional B cells were readily apparent. We observed differences between unaffected and affected TACI mutants (increased B cells and CD8+ T-effector memory cells, loss of B-SM cells and non-classical monocytes), cellular signatures that distinguished CTLA4 haploinsufficiency itself (expansion of plasmablasts, activated CD4+ T cells, regulatory T cells, and X5-Th cells) from its clinical expression (B-cell depletion), and those that were associated with CARD11 gain-of-function mutation (decreased CD8+ T effector memory cells, B cells, CD21-lo B cells, B-SM cells, and NK cells). Co-efficients of variation exceeded 30% for 36/54 FCM parameters, but by comparing inter-assay variation with disease-related variation, we ranked each parameter in terms of laboratory precision vs. disease variability, identifying X5-Th cells (and derivatives), naïve, activated, and central memory CD8+ T cells, transitional B cells, memory and SM-B cells, plasmablasts, activated CD4 cells, and total T cells as the 10 most useful cellular parameters. Applying these to cluster analysis of our PAD cohort, we could detect subgroups with the potential to reflect underlying genotypes. Heat mapping of normalized FCM data reveals cellular trends missed by standard reference ranges, identifies changes associating with a phenotype or genotype, and could inform hypotheses regarding pathogenesis of genetic immunodeficiency.
Asunto(s)
Citometría de Flujo , Enfermedades Genéticas Congénitas/inmunología , Calor , Síndromes de Inmunodeficiencia/inmunología , Mutación , Adulto , Anciano , Linfocitos B/inmunología , Linfocitos B/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Femenino , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/patología , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Memoria Inmunológica , Masculino , Persona de Mediana Edad , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/patologíaRESUMEN
Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease. It is thought that many common variant gene loci of weak effect act additively to predispose to common autoimmune diseases, while the contribution of rare variants remains unclear. Here we describe that rare coding variants in lupus-risk genes are present in most SLE patients and healthy controls. We demonstrate the functional consequences of rare and low frequency missense variants in the interacting proteins BLK and BANK1, which are present alone, or in combination, in a substantial proportion of lupus patients. The rare variants found in patients, but not those found exclusively in controls, impair suppression of IRF5 and type-I IFN in human B cell lines and increase pathogenic lymphocytes in lupus-prone mice. Thus, rare gene variants are common in SLE and likely contribute to genetic risk.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Predisposición Genética a la Enfermedad , Lupus Eritematoso Sistémico/genética , Proteínas de la Membrana/genética , Familia-src Quinasas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adolescente , Adulto , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Estudios de Casos y Controles , Línea Celular , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Niño , Modelos Animales de Enfermedad , Femenino , Frecuencia de los Genes , Células HEK293 , Voluntarios Sanos , Humanos , Factores Reguladores del Interferón/inmunología , Factores Reguladores del Interferón/metabolismo , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Secuenciación del Exoma , Familia-src Quinasas/metabolismoRESUMEN
AIMS: Patients with common variable immunodeficiency (CVID) often have defects in post-antigenic B-cell differentiation with fewer memory B cells and impaired isotype switching. We aimed to classify CVID patients according to these defects and determine whether this predicted clinical manifestations. METHODS: We analysed the memory marker CD27, maturation marker CD21, and IgD on peripheral blood B cells from 31 CVID patients and 23 controls using a whole-blood lysis technique, allocated patients according to two classifications ('Freiburg' and 'Paris') and correlated results with clinical manifestations. RESULTS: CVID patients had fewer memory (CD27(+)) B cells and isotype-switched (IgD(-)) memory B cells in absolute number and proportion. Many CVID patients had increased immature (CD21(-)) B cells. Lymphoproliferation and autoimmune cytopenias were found almost exclusively in these patients, including Freiburg group Ia (decreased switched memory and increased immature B cells), but also those with normal switched memory and increased immature B cells. The Paris classification was less useful in predicting clinical manifestations. CONCLUSIONS: CVID is associated with defects in memory B-cell differentiation. Subclassification helps identify patients with clinical manifestations, particularly lymphoproliferation and autoimmune cytopenias in those with impaired B-cell maturation and isotype switching. Routine B-cell phenotyping may assist clinicians in predicting these clinical features.
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Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Inmunodeficiencia Variable Común/clasificación , Inmunodeficiencia Variable Común/inmunología , Adulto , Anciano , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Femenino , Humanos , Cambio de Clase de Inmunoglobulina , Inmunoglobulina D , Memoria Inmunológica , Masculino , Persona de Mediana Edad , Fenotipo , Receptores de Complemento 3d/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
A subset of human follicular helper T cells (TFH) cells expresses CD57 for which no distinct function has been identified. We show that CD57+ TFH cells are universally PD-1hi, but compared to their CD57- PD-1hi counterparts, express little IL-21 or IL-10 among others. Instead, CD57 expression on TFH cells marks cytotoxicity transcriptional signatures that translate into only a weak cytotoxic phenotype. Similarly, circulating PD-1+ CD57+ CD4+ T cells make less cytokine than their CD57- PD-1+ counterparts, but have a prominent cytotoxic phenotype. By analysis of responses to STAT3-dependent cytokines and cells from patients with gain- or loss-of-function STAT3 mutations, we show that CD4+ T cell cytotoxicity is STAT3-dependent. TFH formation also requires STAT3, but paradoxically, once formed, PD-1hi cells become unresponsive to STAT3. These findings suggest that changes in blood and germinal center cytotoxicity might be affected by changes in STAT3 signaling, or modulation of PD-1 by therapy.
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Antígenos CD57/inmunología , Regulación de la Expresión Génica/inmunología , Factor de Transcripción STAT3/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Tonsilitis/inmunología , Antígenos CD57/genética , Estudios de Casos y Controles , Proliferación Celular , Citotoxicidad Inmunológica , Humanos , Inmunofenotipificación , Interleucina-10/genética , Interleucina-10/inmunología , Interleucinas/genética , Interleucinas/inmunología , Tonsila Palatina/inmunología , Tonsila Palatina/patología , Tonsila Palatina/cirugía , Fenotipo , Cultivo Primario de Células , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Factor de Transcripción STAT3/genética , Transducción de Señal , Linfocitos T Citotóxicos/patología , Linfocitos T Colaboradores-Inductores/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Tonsilectomía , Tonsilitis/genética , Tonsilitis/patología , Tonsilitis/cirugíaRESUMEN
A commercial PLA2R Ab ELISA was validated by examining its ability to distinguish primary from secondary membranous nephropathy, correlating results with clinical markers of disease activity, and comparing its performance with an indirect immunofluorescence test (IIFT). PLA2R Ab levels were measured in 77 patients with biopsy proven membranous nephropathy, divided into either idiopathic (n = 61) or secondary groups (n = 6). In the idiopathic group, measures of contemporaneous disease activity (proteinuria, serum creatinine) were compared between seropositive and seronegative subjects. ELISA values were then compared with semi-quantitative results from an IIFT using PLA2R transfected HEK293 cells as substrate. The PLA2R Ab ELISA was positive in only 15 of 61 (25%) patients with idiopathic membranous nephropathy (IMN), but there was a significant negative relationship with time since diagnosis. Thus, in a subgroup of patients diagnosed within 6 months of analysis, the sensitivity was 6/15 (55%), rising to 6/8 (75%) in those recently-diagnosed patients who had not been treated. In the entire cohort, there was a significant positive correlation between ELISA values and degree of proteinuria, but our analysis did not control for variation of both variables with time. The PLA2R Ab ELISA also showed very high agreement with IIFT (96%). Therefore, the PLA2R Ab ELISA is a highly specific test for distinguishing primary from secondary membranous nephropathy that is most sensitive in newly diagnosed patients who have not received immunosuppression. Antibody levels correlated with degree of proteinuria, but this relationship was not shown to be independent of time. Both IIFT and ELISA platforms performed comparably.
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Anticuerpos/sangre , Ensayo de Inmunoadsorción Enzimática/normas , Glomerulonefritis Membranosa/diagnóstico , Receptores de Fosfolipasa A2/inmunología , Adulto , Anciano , Anticuerpos/inmunología , Australia , Biomarcadores/sangre , Biopsia , Estudios de Cohortes , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Glomerulonefritis Membranosa/inmunología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Naive CD4(+) T cells differentiate into specific effector subsets-Th1, Th2, Th17, and T follicular helper (Tfh)-that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4(+) T cell differentiation in vitro. IL12Rß1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10-secreting cells. IL12Rß1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4(+) T cell effector function in the settings of infection, vaccination, or immune dysregulation.
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Diferenciación Celular/inmunología , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunología , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Diferenciación Celular/genética , Femenino , Humanos , Interleucina-10/genética , Interleucina-10/inmunología , Masculino , Mutación , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Células TH1/citología , Células Th17/citologíaRESUMEN
Two patients with initial diagnoses of oral lichen planus and pemphigus vulgaris presented with refractory oral mucosal blistering. Subsequent positive serology results for paraneoplastic pemphigus led to the discovery of occult intra-abdominal malignancies in both, unicentric Castleman's and non-Hodgkin's lymphoma. The diagnosis of paraneoplastic pemphigus should be considered in patients with recalcitrant oral ulceration, even in the absence of clinical features of malignancy.
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Neoplasias Abdominales/complicaciones , Enfermedad de Castleman/complicaciones , Linfoma de Células B de la Zona Marginal/complicaciones , Úlceras Bucales/etiología , Síndromes Paraneoplásicos/etiología , Pénfigo/etiología , Neoplasias Abdominales/diagnóstico , Adulto , Enfermedad de Castleman/diagnóstico , Humanos , Linfoma de Células B de la Zona Marginal/diagnóstico , Masculino , Persona de Mediana Edad , Úlceras Bucales/tratamiento farmacológicoRESUMEN
Paraneoplastic pemphigus (PNP) is an autoimmune mucocutaneous blistering disease driven by autoantibodies against plakins expressed in mucosal epithelium. Diagnosis can be difficult as both clinical and biopsy features overlap with other blistering disorders, thus serology is important. Indirect immunofluorescence (IIF) on rat bladder substrate is the most widely used assay, but plakin-specific autoantibody assays have recently become available.The aim of this study was to compare the performance of five PNP assays in patients with mucosal blistering disease: IIF with rat bladder, monkey bladder and rat cardiac substrates, an envoplakin enzyme-linked immunosorbent assay (ELISA), and an envoplakin-transfected HEK cell based assay (CBA).Fifty-one patient serum samples, comprising three PNP patients and 48 disease controls, were collected along with 10 healthy control samples, and analysed using the five assays.IIF on rat and monkey bladder substrates both showed high specificity (97% and 95%, respectively), and correctly identified all three PNP sera. The envoplakin ELISA was equally specific (98%) but identified only one PNP patient. The CBA was difficult to interpret, and both this assay and IIF on rat cardiac substrate lacked specificity (82% and 83%, respectively).In this study IIF using either rat or monkey bladder substrates performed strongly, whilst the envoplakin ELISA seemed to lack sensitivity, and the CBA and IIF on rat cardiac substrate were inferior. Our findings suggest that traditional IIF-based assays remain the preferred approach in the serological diagnosis of PNP.