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IgG 4 -related disease (IgG 4 -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 IgG 4 -RD. 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 IgG 4 -RD. 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. We establish that among circulating CD4+CTLs in IgG 4 -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. CD4+CTLs and CD8+CTLs may induce apoptotic cell death in tissues of patients with IgG 4 -RD with preferential targeting of nonendothelial, nonimmune cells of mesenchymal origin. [ABSTRACT FROM AUTHOR]

Background IgG 4 -related disease (IgG 4 -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 IgG 4 -RD lesions. Objective We used an unbiased approach to characterize CD4 + T-cell subsets in patients with IgG 4 -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 IgG 4 -RD. These expanded cells were characterized by means of gene expression analysis and flow cytometry. Next-generation sequencing of the T-cell receptor β chain gene was performed on CD4 + SLAMF7 + cytotoxic T lymphocytes (CTLs) and CD4 + GATA3 + T H 2 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 IgG 4 -RD. Next-generation sequencing revealed prominent clonal expansions of these CD4 + CTLs but not CD4 + GATA3 + memory T H 2 cells in patients with IgG 4 -RD. The dominant T cells infiltrating a range of inflamed IgG 4 -RD tissue sites were clonally expanded CD4 + CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B-cell depletion was associated with a reduction in numbers of disease-associated CD4 + CTLs. Conclusions IgG 4 -RD is prominently linked to clonally expanded IL-1β– and TGF-β1–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. [ABSTRACT FROM AUTHOR]

Background IgG 4 -related disease (IgG 4 -RD) is a poorly understood, multiorgan, chronic inflammatory disease characterized by tumefactive lesions, storiform fibrosis, obliterative phlebitis, and accumulation of IgG 4 -expressing plasma cells at disease sites. Objective The role of B cells and IgG 4 antibodies in IgG 4 -RD pathogenesis is not well defined. We evaluated patients with IgG 4 -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 IgG 4 -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 IgG 4 -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 IgG 4 antibodies are self-reactive. Conclusions Clonally expanded CD19 + CD27 + CD20 − CD38 hi plasmablasts are a hallmark of active IgG 4 -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. [ABSTRACT FROM AUTHOR]

How T follicular (Tfh) cells contribute to many different B-cell class-switching events during T-cell–dependent immune responses has been unclear. Diseases with polarized isotype switching offer a unique opportunity for the exploration of Tfh subsets. Secondary and tertiary lymphoid organs in patients with elevated tissue expression levels of IgE (Kimura disease, KD) and those of IgG 4 (IgG 4 -related disease, IgG 4 -RD) can provide important insights regarding cytokine expression by Tfh cells. We sought to identify disease-specific Tfh cell subsets in secondary and tertiary lymphoid organs expressing IL-10 or IL-13 and thus identify different cellular drivers of class switching in 2 distinct types of fibrotic disorders: allergic fibrosis (driven by type 2 immune cells) and inflammatory fibrosis (driven by cytotoxic T lymphocytes). Single-cell RNA sequencing, in situ sequencing, and multicolor immunofluorescence analysis were used to investigate B cells, Tfh cells, and infiltrating type 2 cells in lesion tissues from patients with KD or IgG 4 -RD. Infiltrating Tfh cells in tertiary lymphoid organs from IgG 4 -RD were divided into 6 main clusters. We encountered abundant infiltrating IL-10 –expressing LAG3 + Tfh cells in patients with IgG 4 -RD. Furthermore, we found that infiltrating AICDA + CD19 + B cells expressing IL-4 , IL-10 , and IL-21 receptors correlated with IgG 4 expression. In contrast, we found that infiltrating IL-13–expressing Tfh cells were abundant in affected tissues from patients with KD. Moreover, we observed few infiltrating IL-13–expressing Tfh cells in tissues from patients with IgG 4 -RD, despite high serum levels of IgE (but low IgE in the disease lesions). Cytotoxic T cells were abundant in IgG 4 -RD; in contrast, type 2 immune cells were abundant in KD. Our analysis revealed a novel subset of IL- 10 + LAG3 + Tfh cells infiltrating the affected organs of IgG 4 -RD patients. In contrast, IL-13+ Tfh cells and type 2 immune cells infiltrated those of KD patients. [Display omitted] [ABSTRACT FROM AUTHOR]

IgG 4 -related disease (IgG 4 -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. In the present work we aimed to demonstrate that B lymphocytes contribute directly to tissue fibrosis in patients with IgG 4 -RD. Total circulating CD19+ B lymphocytes, naive B cells, memory B cells, or plasmablasts from patients with IgG 4 -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 IgG 4 -RD tissue sections by using multicolor immunofluorescence studies. B cells from patients with IgG 4 -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. We have demonstrated that B cells contribute directly to tissue fibrosis in patients with IgG 4 -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. [ABSTRACT FROM AUTHOR]

Background The antigenic trigger that drives expansion of circulating plasmablasts and CD4+ cytotoxic T cells in patients with IgG 4 -related disease (IgG 4 -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 IgG 4 -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 IgG 4 -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 IgG 4 isotype (28% of the IgG 4 -RD cohort, P =.0001) and IgE isotype (11% of the IgG 4 -RD cohort, P =.009). No significant responses were seen from the IgG 1 , IgG 2 , or IgG 3 isotypes. IgG 4 anti–galectin-3 autoantibodies correlated with increased plasma galectin-3 levels (P =.001), lymphadenopathy (P =.04), total IgG level increase (P =.05), and IgG 4 level increase (P =.03). Conclusion Affinity chromatography using patient-derived mAbs identifies relevant autoantigens in patients with IgG 4 -RD. IgG 4 galectin-3 autoantibodies are present in a subset of patients with IgG 4 -RD and correlate with galectin-3 plasma levels. The marked increases in levels of circulating IgG 4 and IgE observed clinically are, at least in part, caused by the development of IgG 4 - and IgE-specific autoantibody responses. [ABSTRACT FROM AUTHOR]