RESUMO
Sialic acid-binding Ig-like lectins, or Siglecs, vary in their specificity for sialic acid-containing ligands and are mainly expressed by cells of the immune system. Many Siglecs are inhibitory receptors expressed in innate immune cells that regulate inflammation mediated by damage-associated and pathogen-associated molecular patterns (DAMPs and PAMPs). This family also includes molecules involved in adhesion and phagocytosis and receptors that can associate with the ITAM-containing DAP12 adaptor. Siglecs contribute to the inhibition of immune cells both by binding to cis ligands (expressed in the same cells) and by responding to pathogen-derived sialoglycoconjugates. They can help maintain tolerance in B lymphocytes, modulate the activation of conventional and plasmacytoid dendritic cells, and contribute to the regulation of T cell function both directly and indirectly. Siglecs modulate immune responses, influencing almost every cell in the immune system, and are of relevance both in health and disease.
Assuntos
Sistema Imunitário/imunologia , Lectinas/metabolismo , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Imunidade Inata , Lectinas/classificação , Ativação Linfocitária/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
T follicular helper (Tfh) cells regulate humoral responses and present a marked phenotypic and functional diversity. Type 1 Tfh (Tfh1) cells were recently identified and associated with disease severity in infection and autoimmune diseases. The cellular and molecular requirements to induce human Tfh1 differentiation are not known. Here, using single-cell RNA sequencing (scRNAseq) and protein validation, we report that human blood CD1c+ dendritic cells (DCs) activated by GM-CSF (also known as CSF2) drive the differentiation of naive CD4+ T cells into Tfh1 cells. These Tfh1 cells displayed typical Tfh molecular features, including high levels of PD-1 (encoded by PDCD1), CXCR5 and ICOS. They co-expressed BCL6 and TBET (encoded by TBX21), and secreted large amounts of IL-21 and IFN-γ (encoded by IFNG). Mechanistically, GM-CSF triggered the emergence of two DC sub-populations defined by their expression of CD40 and ICOS ligand (ICOS-L), presenting distinct phenotypes, morphologies, transcriptomic signatures and functions. CD40High ICOS-LLow DCs efficiently induced Tfh1 differentiation in a CD40-dependent manner. In patients with mild COVID-19 or latent Mycobacterium tuberculosis infection, Tfh1 cells were positively correlated with a CD40High ICOS-LLow DC signature in scRNAseq of peripheral blood mononuclear cells or blood transcriptomics, respectively. Our study uncovered a novel CD40-dependent Tfh1 axis with potential physiopathological relevance to infection. This article has an associated First Person interview with the first author of the paper.
Assuntos
COVID-19 , Células T Auxiliares Foliculares , Humanos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Leucócitos Mononucleares , Células DendríticasRESUMO
Systemic sclerosis (SSc) is a progressive, multiorgan disease with limited treatment options. Although a recent proof-of-concept study using romilkimab or SAR156597, a bispecific IL-4/IL-13 antibody, suggests a direct role of these cytokines in the pathophysiology of SSc, their contributions to the balance between inflammation and fibrosis are unclear. Here, we determine the roles of type 2 inflammation in fibrogenesis using FRA2-Tg (Fos-related antigen 2-overexpressing transgenic) mice, which develop spontaneous, age-dependent progressive lung fibrosis. We defined the molecular signatures of inflammation and fibrosis at three key stages in disease progression, corresponding to preonset, inflammatory dominant, and fibrosis dominant biology, and revealed an early increase in cytokine-cytokine receptor interactions and antigen-processing and presentation pathways followed by enhanced Th2- and M2 macrophage-driven type 2 responses. This type 2 inflammation progressed to extensive fibrotic pathology by 14-18 weeks of age, with these gene signatures overlapping significantly with those seen in the lungs of patients with SSc with interstitial lung disease (ILD). These changes were also evident in the histopathology, which showed perivascular and peribronchiolar inflammation with prominent eosinophilia and accumulation of profibrotic M2-like macrophages followed by rapid progression to fibrosis with thickened alveolar walls with multifocal fibrotic bands and signs of interstitial pneumonia. Critically, treatment with a bispecific antibody targeting IL-4 and IL-13 during the inflammatory phase abrogated the Th2 and M2 responses and led to near-complete abrogation of lung fibrosis. These data recapitulate important features of fibrotic progression in the lungs of patients with SSc-ILD and enhance our understanding of the progressive pathobiology of SSc. This study also further establishes FRA2-Tg mice as a valuable tool for testing future therapeutic agents in SSc-ILD.
Assuntos
Doenças Pulmonares Intersticiais , Fibrose Pulmonar , Escleroderma Sistêmico , Humanos , Camundongos , Animais , Fibrose Pulmonar/patologia , Interleucina-13 , Interleucina-4 , Doenças Pulmonares Intersticiais/patologia , Fibrose , Pulmão/patologia , Inflamação/patologia , Camundongos Transgênicos , CitocinasRESUMO
Copy number heterogeneity is a prominent feature within tumors. The molecular basis for this heterogeneity remains poorly characterized. Here, we demonstrate that hypoxia induces transient site-specific copy gains (TSSGs) in primary, nontransformed, and transformed human cells. Hypoxia-driven copy gains are not dependent on HIF1α or HIF2α; however, they are dependent on the KDM4A histone demethylase and are blocked by inhibition of KDM4A with a small molecule or the natural metabolite succinate. Furthermore, this response is conserved at a syntenic region in zebrafish cells. Regions with site-specific copy gain are also enriched for amplifications in hypoxic primary tumors. These tumors exhibited amplification and overexpression of the drug resistance gene CKS1B, which we recapitulated in hypoxic breast cancer cells. Our results demonstrate that hypoxia provides a biological stimulus to create transient site-specific copy alterations that could result in heterogeneity within tumors and cell populations. These findings have major implications in our understanding of copy number heterogeneity and the emergence of drug resistance genes in cancer.
Assuntos
Hipóxia Celular/fisiologia , Variações do Número de Cópias de DNA/genética , Regulação da Expressão Gênica , Animais , Quinases relacionadas a CDC2 e CDC28/genética , Hipóxia Celular/genética , Linhagem Celular , Proliferação de Células , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Peixe-ZebraRESUMO
Fibrotic diseases take a very heavy toll in terms of morbidity and mortality equal to or even greater than that caused by metastatic cancer. In this review, we examine the pathogenesis of fibrotic diseases, mainly addressing triggers for induction, processes that lead to progression, therapies and therapeutic trials. For the most part, we have focused on two fibrotic diseases with lung involvement, idiopathic pulmonary fibrosis, in which the contribution of inflammatory mechanisms may be secondary to non-immune triggers, and systemic sclerosis in which the contribution of adaptive immunity may be predominant.
Assuntos
Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/terapia , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/terapia , Animais , HumanosRESUMO
BACKGROUND: IgG4-related disease (IgG4-RD) is an immune-mediated fibrotic disorder that has been linked to CD4+ cytotoxic T lymphocytes (CD4+CTLs). The effector phenotype of CD4+CTLs and the relevance of both CD8+ cytotoxic T lymphocytes (CD8+CTLs) and apoptotic cell death remain undefined in IgG4-RD. OBJECTIVE: We sought to define CD4+CTL heterogeneity, characterize the CD8+CTL response in the blood and in lesions, and determine whether enhanced apoptosis may contribute to the pathogenesis of IgG4-RD. METHODS: Blood analyses were undertaken using flow cytometry, cell sorting, transcriptomic analyses at the population and single-cell levels, and next-generation sequencing for the TCR repertoire. Tissues were interrogated using multicolor immunofluorescence. Results were correlated with clinical data. RESULTS: We establish that among circulating CD4+CTLs in IgG4-RD, CD27loCD28loCD57hi cells are the dominant effector subset, exhibit marked clonal expansion, and differentially express genes relevant to cytotoxicity, activation, and enhanced metabolism. We also observed prominent infiltration of granzyme A-expressing CD8+CTLs in disease tissues and clonal expansion in the blood of effector/memory CD8+ T cells with an activated and cytotoxic phenotype. Tissue studies revealed an abundance of cells undergoing apoptotic cell death disproportionately involving nonimmune, nonendothelial cells of mesenchymal origin. Apoptotic cells showed significant upregulation of HLA-DR. CONCLUSIONS: CD4+CTLs and CD8+CTLs may induce apoptotic cell death in tissues of patients with IgG4-RD with preferential targeting of nonendothelial, nonimmune cells of mesenchymal origin.
Assuntos
Antígenos CD/imunologia , Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Doença Relacionada a Imunoglobulina G4/imunologia , Células-Tronco Mesenquimais/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Linfócitos T CD4-Positivos/patologia , Feminino , Fibrose , Humanos , Doença Relacionada a Imunoglobulina G4/patologia , Masculino , Células-Tronco Mesenquimais/patologia , Linfócitos T Citotóxicos/patologiaRESUMO
BACKGROUND: IgG4-related disease (IgG4-RD) is a fibroinflammatory condition marked by rapid clinical improvement after selective depletion of B lymphocytes with rituximab. This feature suggests that B cells might participate in fibrogenesis and wound healing. OBJECTIVE: In the present work we aimed to demonstrate that B lymphocytes contribute directly to tissue fibrosis in patients with IgG4-RD. METHODS: Total circulating CD19+ B lymphocytes, naive B cells, memory B cells, or plasmablasts from patients with IgG4-RD were cultivated with human fibroblasts. Profibrotic soluble factors and collagen production in cocultures were assessed by using ELISAs and Luminex assays. RNA sequencing and quantitative RT-PCR were used to assess fibroblast activation in the presence of B cells, as well as induction of profibrotic pathways in B-cell subsets. Relevant profibrotic and inflammatory molecules were confirmed in vitro by using functional experiments and on IgG4-RD tissue sections by using multicolor immunofluorescence studies. RESULTS: B cells from patients with IgG4-RD (1) produced the profibrotic molecule platelet-derived growth factor B and stimulated collagen production by fibroblasts; (2) expressed enzymes implicated in extracellular matrix remodeling, such as lysyl oxidase homolog 2; (3) produced the chemotactic factors CCL4, CCL5, and CCL11; and (4) induced production of these same chemokines by activated fibroblasts. Plasmablasts expressed sets of genes implicated in fibroblast activation and proliferation and therefore represent cells with intrinsic profibrotic properties. CONCLUSION: We have demonstrated that B cells contribute directly to tissue fibrosis in patients with IgG4-RD. These unanticipated profibrotic properties of B lymphocytes, particularly plasmablasts, might be relevant for fibrogenesis in patients with other fibroinflammatory disorders and for wound-healing processes in physiologic conditions.
Assuntos
Linfócitos B/patologia , Fibroblastos/patologia , Doença Relacionada a Imunoglobulina G4/patologia , Pâncreas/patologia , Linfócitos B/imunologia , Células Cultivadas , Técnicas de Cocultura , Colágeno/biossíntese , Fibrose/imunologia , Fibrose/patologia , Humanos , Doença Relacionada a Imunoglobulina G4/imunologiaRESUMO
BACKGROUND: The antigenic trigger that drives expansion of circulating plasmablasts and CD4+ cytotoxic T cells in patients with IgG4-related disease (IgG4-RD) is presently unknown. OBJECTIVE: We sought to sequence immunoglobulin genes from single-cell clones of dominantly expanded plasmablasts and generate recombinant human mAbs to identify relevant antigens in patients with IgG4-RD by using mass spectrometry. METHODS: Paired heavy and light chain cDNAs from dominant plasmablast clones were expressed as mAbs and used to purify antigens by using immunoaffinity chromatography. Affinity-purified antigens were identified by using mass spectrometry and validated by means of ELISA. Plasma levels of the antigen of interest were also determined by using ELISA. RESULTS: mAbs expressed from the 2 dominant plasmablast clones of a patient with multiorgan IgG4-RD stained human pancreatic tissue sections. Galectin-3 was identified as the antigen specifically recognized by both mAbs. Anti-galectin-3 autoantibody responses were predominantly of the IgG4 isotype (28% of the IgG4-RD cohort, P = .0001) and IgE isotype (11% of the IgG4-RD cohort, P = .009). No significant responses were seen from the IgG1, IgG2, or IgG3 isotypes. IgG4 anti-galectin-3 autoantibodies correlated with increased plasma galectin-3 levels (P = .001), lymphadenopathy (P = .04), total IgG level increase (P = .05), and IgG4 level increase (P = .03). CONCLUSION: Affinity chromatography using patient-derived mAbs identifies relevant autoantigens in patients with IgG4-RD. IgG4 galectin-3 autoantibodies are present in a subset of patients with IgG4-RD and correlate with galectin-3 plasma levels. The marked increases in levels of circulating IgG4 and IgE observed clinically are, at least in part, caused by the development of IgG4- and IgE-specific autoantibody responses.
Assuntos
Autoantígenos/isolamento & purificação , Linfócitos T CD4-Positivos/imunologia , Galectina 3/isolamento & purificação , Doença Relacionada a Imunoglobulina G4/imunologia , Plasmócitos/imunologia , Autoanticorpos/metabolismo , Autoantígenos/imunologia , Proliferação de Células , Feminino , Galectina 3/imunologia , Humanos , Imunoglobulina E/metabolismo , Imunoglobulina G/metabolismo , Imunoglobulinas/genética , Técnicas de Imunoadsorção , Ativação Linfocitária , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteínas Recombinantes/genéticaRESUMO
We used Casd1-deficient mice to confirm that this enzyme is responsible for 9-O-acetylation of sialic acids in vivo. We observed a complete loss of 9-O-acetylation of sialic acid on the surface of myeloid, erythroid and CD4+ T cells in Casd1-deficient mice. Although 9-O-acetylation of sialic acids on multiple hematopoietic lineages was lost, there were no obvious defects in hematopoiesis. Interestingly, erythrocytes from Casd1-deficient mice also lost reactivity to TER-119, a rat monoclonal antibody that is widely used to mark the murine erythroid lineage. The sialic acid glyco-epitope recognized by TER-119 on erythrocytes was sensitive to the sialic acid O-acetyl esterase activity of the hemagglutinin-esterase from bovine coronavirus but not to the corresponding enzyme from the influenza C virus. During erythrocyte development, TER-119+ Ery-A and Ery-B cells could be stained by catalytically inactive bovine coronavirus hemagglutinin-esterase but not by the inactive influenza C hemagglutinin-esterase, while TER-119+ Ery-C cells and mature erythrocytes were recognized by both virolectins. Although the structure of the sialoglycoconjugate recognized by TER-119 was not chemically demonstrated, its selective binding to virolectins suggests that it may be comprised of a 7,9-di-O-acetyl form of sialic acid. As erythrocytes mature, the surfaces of Ery-C cells and mature erythrocytes also acquire an additional distinct CASD1-dependent 9-O-acetyl sialic acid moiety that can be recognized by virolectins from both influenza C and bovine coronavirus.
Assuntos
Eritrócitos/química , Gammainfluenzavirus/imunologia , Influenza Humana/imunologia , Ácido N-Acetilneuramínico/química , Acetilação , Animais , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/imunologia , Bovinos , Epitopos/química , Epitopos/imunologia , Eritrócitos/imunologia , Células Eritroides/química , Células Eritroides/imunologia , Hemaglutininas Virais/genética , Humanos , Influenza Humana/virologia , Gammainfluenzavirus/enzimologia , Gammainfluenzavirus/isolamento & purificação , Camundongos , Células Mieloides/química , Células Mieloides/imunologia , Ácido N-Acetilneuramínico/imunologia , Ratos , Proteínas Virais de Fusão/genéticaRESUMO
OBJECTIVES: IgG4-related disease (IgG4-RD) is a chronic, systemic, inflammatory condition of unknown aetiology. We have recently described clonally expanded circulating CD4+ cytotoxic T lymphocytes (CTLs) in IgG4-RD that infiltrate affected tissues where they secrete interleukin (IL)-1ß and transforming growth factor -ß1 (TGF-ß1). In this study, we sought to examine the role of CD4+ CTLs in the pathogenesis of IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS) and to determine whether these cells secrete interferon-gamma (IFN-γ) at lesional sites. METHODS: Salivary glands of 25 patients with IgG4-DS, 22 patients with Sjögren's syndrome (SS), 12 patients with chronic sialoadenitis (CS) and 12 healthy controls were analysed in this study. Gene expression analysis was performed on submandibular glands (SMGs) from five patients with IgG4-DS, three with CS and three healthy controls. Infiltrating CD4+ CTLs were examined by quantitative multicolour imaging in tissue samples from 20 patients with IgG4-DS, 22 patients with SS, 9 patients with CS and 9 healthy controls. RESULTS: In IgG4-DS tissues, nine genes associated with CD4+ CTLs were overexpressed. The expression of granzyme A (GZMA) mRNA was significantly higher in samples from patients with IgG4-RD compared with corresponding tissues from SS and healthy controls. Quantitative imaging showed that infiltrating CD4+ GZMA+ CTLs were more abundant in patients with IgG4-DS than in the other groups. The ratio of CD4+GZMA+ CTLs in SMGs from patients with IgG4-DS correlated with serum IgG4 concentrations and the number of affected organs. A large fraction of CD4+GZMA+ CTLs in SMGs from patients with IgG4-DS secreted IFN-γ. CONCLUSIONS: The pathogenesis of IgG4-DS is associated with tissue infiltration by CD4+GZMA+ CTLs that secrete IFN-γ.
Assuntos
Doenças Autoimunes/imunologia , Antígenos CD4/imunologia , Dacriocistite/imunologia , Imunoglobulina G/imunologia , Interferon gama/imunologia , RNA Mensageiro/metabolismo , Sialadenite/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Linfócitos T CD4-Positivos/imunologia , Estudos de Casos e Controles , Quimiocina CCL4/genética , Quimiocina CCL5/genética , Dacriocistite/genética , Dacriocistite/metabolismo , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Granzimas/genética , Humanos , Interferon gama/genética , Masculino , Pessoa de Meia-Idade , Perforina/genética , Sialadenite/genética , Sialadenite/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Síndrome de Sjogren/genética , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Glândula Submandibular/metabolismo , Proteínas com Domínio T/genética , Fator de Crescimento Transformador beta1/genética , Fator de Necrose Tumoral alfa/genética , Adulto JovemRESUMO
BACKGROUND: IgG4-related disease (IgG4-RD) is a systemic condition of unknown cause characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4(+) T cells constitute the major inflammatory cell population in IgG4-RD lesions. OBJECTIVE: We used an unbiased approach to characterize CD4(+) T-cell subsets in patients with IgG4-RD based on their clonal expansion and ability to infiltrate affected tissue sites. METHODS: We used flow cytometry to identify CD4(+) effector/memory T cells in a cohort of 101 patients with IgG4-RD. These expanded cells were characterized by means of gene expression analysis and flow cytometry. Next-generation sequencing of the T-cell receptor ß chain gene was performed on CD4(+)SLAMF7(+) cytotoxic T lymphocytes (CTLs) and CD4(+)GATA3(+) TH2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined by using quantitative multicolor imaging. RESULTS: CD4(+) effector/memory T cells with a cytolytic phenotype were expanded in patients with IgG4-RD. Next-generation sequencing revealed prominent clonal expansions of these CD4(+) CTLs but not CD4(+)GATA3(+) memory TH2 cells in patients with IgG4-RD. The dominant T cells infiltrating a range of inflamed IgG4-RD tissue sites were clonally expanded CD4(+) CTLs that expressed SLAMF7, granzyme A, IL-1ß, and TGF-ß1. Clinical remission induced by rituximab-mediated B-cell depletion was associated with a reduction in numbers of disease-associated CD4(+) CTLs. CONCLUSIONS: IgG4-RD is prominently linked to clonally expanded IL-1ß- and TGF-ß1-secreting CD4(+) CTLs in both peripheral blood and inflammatory tissue lesions. These active, terminally differentiated, cytokine-secreting effector CD4(+) T cells are now linked to a human disease characterized by chronic inflammation and fibrosis.
Assuntos
Doenças do Sistema Imunitário/imunologia , Imunoglobulina G/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Citocinas/imunologia , Feminino , Humanos , Doenças do Sistema Imunitário/sangue , Imunoglobulina G/sangue , Rim/citologia , Pulmão/citologia , Linfonodos/citologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Septo Nasal/citologia , Espaço Retroperitoneal , Glândula Submandibular/citologiaRESUMO
OBJECTIVE: IgG4-related disease (IgG4-RD) is a relapsing-remitting condition responsible for fibroinflammatory lesions that can lead to organ damage and life-threatening complications at nearly any anatomical site. The duration of remission following treatment varies and predictors of relapse are unclear. The objectives of this study were to review our experience with rituximab as remission induction in IgG4-RD, to clarify the duration of efficacy and to identify predictors of flare following treatment. METHODS: In this retrospective cohort study, all patients were treated with two doses of rituximab (1 g) separated by 15 days. Clinical, radiographic and laboratory data pertaining to rituximab response and disease relapse were collected from the electronic medical record. Kaplan-Meier curves were constructed to estimate the time to disease relapse. Log-rank analyses were performed to compare times to relapse among subgroups. Potential relapse predictors were evaluated with Cox regression analysis. RESULTS: Fifty-seven of 60 patients (95%) had clinical responses to rituximab. Forty-one patients (68%) were treated without glucocorticoids. Twenty-one patients (37%) experienced relapses following treatment at a median time from the first infusion of 244 days. Baseline concentrations of serum IgG4, IgE and circulating eosinophils predicted subsequent relapses, with hazard ratios of 6.2 (95% CI: 1.2, 32.0), 8.2 (95% CI: 1.4, 50.0) and 7.9 (95% CI: 1.8, 34.7), respectively. The higher the baseline values, the greater the risk of relapse and the shorter the time to relapse. Only 10% of the patients had elevations of all three major risk factors, underscoring the importance of measuring all three at baseline. CONCLUSION: Baseline elevations in serum IgG4, IgE and blood eosinophil concentrations all predict IgG4-RD relapses independently.
Assuntos
Doenças Autoimunes/sangue , Doenças Autoimunes/tratamento farmacológico , Imunoglobulina G/sangue , Fatores Imunológicos/administração & dosagem , Rituximab/administração & dosagem , Adulto , Idoso , Doenças Autoimunes/imunologia , Biomarcadores/sangue , Eosinófilos/metabolismo , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/imunologia , Estimativa de Kaplan-Meier , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Recidiva , Indução de Remissão/métodos , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
Somatic mutations altering lysine 171 of the IKBKB gene that encodes (IKKß), the critical activating kinase in canonical (NFκB) signaling, have been described in splenic marginal zone lymphomas and multiple myeloma. Lysine 171 forms part of a cationic pocket that interacts with the activation loop phosphate in the activated wild type kinase. We show here that K171E IKKß and K171T IKKß represent kinases that are constitutively active even in the absence of activation loop phosphorylation. Predictive modeling and biochemical studies establish why mutations in a positively charged residue in the cationic pocket of an activation loop phosphorylation-dependent kinase result in constitutive activation. Transcription activator-like effector nuclease-based knock-in mutagenesis provides evidence from a B lymphoid context that K171E IKKß contributes to lymphomagenesis.
Assuntos
Quinase I-kappa B , Linfoma , Mutação de Sentido Incorreto , NF-kappa B , Proteínas de Neoplasias , Transdução de Sinais/genética , Substituição de Aminoácidos , Células HeLa , Humanos , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Linfoma/genética , Linfoma/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação/genéticaRESUMO
OBJECTIVES: We examined the utility of circulating total and IgG4+ plasmablasts as biomarkers of diagnosis and disease activity in IgG4-related disease (IgG4-RD). MATERIALS METHODS: We evaluated patients with active, untreated, biopsy-proven IgG4-RD affecting various organs. Flow cytometry was used to measure total plasmablast and IgG4+ plasmablast counts by gating peripheral blood for CD19lowCD38+CD20-CD27+ cells and CD19lowCD38+CD20-CD27+IgG4+ cells. Serum IgG4 concentrations were measured by nephelometry. We compared 37 IgG4-RD patients to 35 controls, including healthy individuals (n=14) and patients with other inflammatory diseases before treatment (n=21). RESULTS: The IgG4-RD patients' mean age was 59, and 68% were male. Fourteen patients (38%) had three or more organs involved. The IgG4-RD patients had substantially elevated total plasmablast counts (median 4698/mL, range 610-79524/mL) compared to both untreated disease controls (median 592/mL, range 19-4294/mL; p < 0.001) and healthy controls (median 94/mL, range 1-653/mL; p < 0.001). Thirteen IgG4-RD patients (36%) had normal serum IgG4 concentrations (mean 60â mg/dL, range 5-123â mg/dL, normal <135â mg/dL). However, the median plasmablast count was not significantly lower in that subset with normal serum IgG4 concentrations (3784/mL) compared to those with elevated serum IgG4 (5155/mL) (p = 0.242). Among the 12 rituximab (RTX)-treated patients, the median plasmablast level during disease flare was 6356/mL (range 1123-41589/mL), declining to 1419/mL (range 386/mL-4150/mL) during remission (p < 0.01). CONCLUSIONS: Circulating plasmablasts are elevated in active IgG4-RD, even in patients with normal serum IgG4 concentrations. Plasmablast counts are a potentially useful biomarker for diagnosis, assessing response to treatment, and determining the appropriate time for re-treatment.
Assuntos
Doenças Autoimunes/diagnóstico , Imunoglobulina G/sangue , Plasmócitos/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Plasmócitos/imunologia , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: Fibrosis is a predominant feature of IgG4-related disease (IgG4-RD). B-cell depletion induces a prompt clinical and immunological response in patients with IgG4-RD, but the effects of this intervention on fibrosis in IgG4-RD are unknown. We used the enhanced liver fibrosis (ELF) score to address the impact of rituximab on fibroblast activation. The ELF score is an algorithm based on serum concentrations of procollagen-III aminoterminal propeptide, tissue inhibitor of matrix metalloproteinase-1 and hyaluronic acid. METHODS: Ten patients with active, untreated IgG4-RD were enrolled. ELF scores were measured and correlated with the IgG4-RD Responder Index, serum IgG4, circulating plasmablasts and imaging studies. Through immunohistochemical stains for CD3, CD20, IgG4 and α-smooth muscle actin, we assessed the extent of the lymphoplasmacytic infiltration and the degree of fibroblast activation in one patient with tissue biopsies before and after rituximab. RESULTS: The ELF score was increased in patients with IgG4-RD compared with healthy controls (8.3±1.4 vs 6.2±0.9; p=0.002) and correlated with the number of organs involved (R(2)=0.41; p=0.04). Rituximab induced significant reductions in the ELF score, the number of circulating plasmablasts and the IgG4-RD Responder Index (p<0.05 for all three parameters). Rituximab reduced both the lymphoplasmacytic infiltrate and myofibroblast activation. IgG4-RD relapse coincided with recurrent increases in the ELF score, indicating reactivation of collagen deposition. CONCLUSIONS: The ELF score may be a clinically useful indicator of active fibrosis and the extent of disease in IgG4-RD. B-cell depletion has the potential to halt continued collagen deposition by attenuating the secretory phenotype of myofibroblasts in IgG4-RD lesions.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Imunidade Celular , Imunoglobulina G/imunologia , Miofibroblastos/imunologia , Adulto , Doenças Autoimunes/sangue , Doenças Autoimunes/patologia , Biópsia , Progressão da Doença , Feminino , Fibrose , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Miofibroblastos/patologiaRESUMO
BACKGROUND: As individual naïve CD4 T lymphocytes circulate in the body after emerging from the thymus, they are likely to have individually varying microenvironmental interactions even in the absence of stimulation via specific target recognition. It is not clear if these interactions result in alterations in their activation, survival and effector programming. Naïve CD4 T cells show unimodal distribution for many phenotypic properties, suggesting that the variation is caused by intrinsic stochasticity, although underlying variation due to subsets created by different histories of microenvironmental interactions remains possible. To explore this possibility, we began examining the phenotype and functionality of naïve CD4 T cells differing in a basic unimodally distributed property, the CD4 levels, as well as the causal origin of these differences. RESULTS: We examined separated CD4hi and CD4lo subsets of mouse naïve CD4 cells. CD4lo cells were smaller with higher CD5 levels and lower levels of the dual-specific phosphatase (DUSP)6-suppressing micro-RNA miR181a, and responded poorly with more Th2-skewed outcomes. Human naïve CD4lo and CD4hi cells showed similar differences. Naïve CD4lo and CD4hi subsets of thymic single-positive CD4 T cells did not show differences whereas peripheral naïve CD4lo and CD4hi subsets of T cell receptor (TCR)-transgenic T cells did. Adoptive transfer-mediated parking of naïve CD4 cells in vivo lowered CD4 levels, increased CD5 and reactive oxygen species (ROS) levels and induced hyporesponsiveness in them, dependent, at least in part, on availability of major histocompatibility complex class II (MHCII) molecules. ROS scavenging or DUSP inhibition ameliorated hyporesponsiveness. Naïve CD4 cells from aged mice showed lower CD4 levels and cell sizes, higher CD5 levels, and hyporesponsiveness and Th2-skewing reversed by DUSP inhibition. CONCLUSIONS: Our data show that, underlying a unimodally distributed property, the CD4 level, there are subsets of naïve CD4 cells that vary in the time spent in the periphery receiving MHCII-mediated signals and show resultant alteration of phenotype and functionality via ROS and DUSP activity. Our findings also suggest the feasibility of potential pharmacological interventions for improved CD4 T cell responses during vaccination of older people via either anti-oxidant or DUSP inhibitor small molecules.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Fenótipo , Adulto , Animais , Células Cultivadas , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Estudos de Viabilidade , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Adulto JovemRESUMO
BACKGROUND: IgG4-related disease (IgG4-RD) is a poorly understood, multiorgan, chronic inflammatory disease characterized by tumefactive lesions, storiform fibrosis, obliterative phlebitis, and accumulation of IgG4-expressing plasma cells at disease sites. OBJECTIVE: The role of B cells and IgG4 antibodies in IgG4-RD pathogenesis is not well defined. We evaluated patients with IgG4-RD for activated B cells in both disease lesions and peripheral blood and investigated their role in disease pathogenesis. METHODS: B-cell populations from the peripheral blood of 84 patients with active IgG4-RD were analyzed by using flow cytometry. The repertoire of B-cell populations was analyzed in a subset of patients by using next-generation sequencing. Fourteen of these patients were longitudinally followed for 9 to 15 months after rituximab therapy. RESULTS: Numbers of CD19(+)CD27(+)CD20(-)CD38(hi) plasmablasts, which are largely IgG4(+), are increased in patients with active IgG4-RD. These expanded plasmablasts are oligoclonal and exhibit extensive somatic hypermutation, and their numbers decrease after rituximab-mediated B-cell depletion therapy; this loss correlates with disease remission. A subset of patients relapse after rituximab therapy, and circulating plasmablasts that re-emerge in these subjects are clonally distinct and exhibit enhanced somatic hypermutation. Cloning and expression of immunoglobulin heavy and light chain genes from expanded plasmablasts at the peak of disease reveals that disease-associated IgG4 antibodies are self-reactive. CONCLUSIONS: Clonally expanded CD19(+)CD27(+)CD20(-)CD38(hi) plasmablasts are a hallmark of active IgG4-RD. Enhanced somatic mutation in activated B cells and plasmablasts and emergence of distinct plasmablast clones on relapse indicate that the disease pathogenesis is linked to de novo recruitment of naive B cells into T cell-dependent responses by CD4(+) T cells, likely driving a self-reactive disease process.
Assuntos
Células Produtoras de Anticorpos/fisiologia , Linfócitos B/fisiologia , Doenças do Sistema Imunitário/imunologia , Imunoglobulina G/metabolismo , Linfocitose/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/efeitos adversos , Diversidade de Anticorpos/genética , Células Produtoras de Anticorpos/efeitos dos fármacos , Antígenos CD/metabolismo , Autoantígenos/imunologia , Linfócitos B/efeitos dos fármacos , Proliferação de Células , Células Clonais , Progressão da Doença , Feminino , Seguimentos , Humanos , Doenças do Sistema Imunitário/terapia , Ativação Linfocitária , Linfocitose/terapia , Masculino , Pessoa de Meia-Idade , Recidiva , Rituximab , Hipermutação Somática de Imunoglobulina/genética , Adulto JovemRESUMO
IgG4-related disease (IgG4-RD) is a fibroinflammatory disorder that can affect virtually every organ system. T-helper type 2 responses have been presumed to be pathogenic in this disease, and a high proportion of patients with IgG4-RD are reported to have longstanding allergies, peripheral blood eosinophilia, and serum IgE elevation. It has therefore been proposed that allergic mechanisms drive IgG4-RD. However, no epidemiological assessment of atopy, peripheral blood eosinophilia, and serum IgE concentrations has ever been undertaken in patients with IgG4-RD. In this study, we evaluated these parameters in a large cohort of patients with IgG4-RD in whom a wide range of organs were affected by disease. Our results demonstrate that the majority of patients with IgG4-RD are nonatopic. Nevertheless, a subset of nonatopic subjects exhibit peripheral blood eosinophilia and elevated IgE, suggesting that processes inherent to IgG4-RD itself rather than atopy per se contribute to the eosinophilia and IgE elevation observed in the absence of atopy.
Assuntos
Doenças Autoimunes/imunologia , Eosinofilia/epidemiologia , Hipersensibilidade Imediata/epidemiologia , Imunoglobulina E/sangue , Adulto , Idoso , Doenças Autoimunes/complicações , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Prevalência , Adulto JovemRESUMO
Time series RNASeq studies can enable understanding of the dynamics of disease progression and treatment response in patients. They also provide information on biomarkers, activated and repressed pathways, and more. While useful, data from multiple patients is challenging to integrate due to the heterogeneity in treatment response among patients, and the small number of timepoints that are usually profiled. Due to the heterogeneity among patients, relying on the sampled time points to integrate data across individuals is challenging and does not lead to correct reconstruction of the response patterns. To address these challenges, we developed a new constrained based pseudotime ordering method for analyzing transcriptomics data in clinical and response studies. Our method allows the assignment of samples to their correct placement on the response curve while respecting the individual patient order. We use polynomials to represent gene expression over the duration of the study and an EM algorithm to determine parameters and locations. Application to three treatment response datasets shows that our method improves on prior methods and leads to accurate orderings that provide new biological insight on the disease and response. Code for the method is available at https://github.com/Sanofi-Public/ RDCS-bulkRNASeq-pseudo ordering.
RESUMO
Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of cellular responses to perturbations such as therapeutic interventions and vaccines. Gene relevance to such perturbations is often assessed through differential expression analysis (DEA), which offers a one-dimensional view of the transcriptomic landscape. This method potentially overlooks genes with modest expression changes but profound downstream effects and is susceptible to false positives. We present GENIX (gene expression network importance examination), a computational framework that transcends DEA by constructing gene association networks and employing a network-based comparative model to identify topological signature genes. We benchmark GENIX using both synthetic and experimental datasets, including analysis of influenza vaccine-induced immune responses in peripheral blood mononuclear cells (PBMCs) from recovered COVID-19 patients. GENIX successfully emulates key characteristics of biological networks and reveals signature genes that are missed by classical DEA, thereby broadening the scope of target gene discovery in precision medicine.