Molecular Mechanisms Driving IL-10- Producing B Cells Functions: STAT3 and c-MAF as Underestimated Central Key Regulators?

Regulatory B cells (Bregs) have been highlighted in very different pathology settings including autoimmune diseases, allergy, graft rejection, and cancer. Improving tools for the characterization of Bregs has become the main objective especially in humans. Transitional, mature B cells and plasma cells can differentiate into IL-10 producing Bregs in both mice and humans, suggesting that Bregs are not derived from unique precursors but may arise from different competent progenitors at unrestricted development stages. Moreover, in addition to IL-10 production, regulatory B cells used a broad range of suppressing mechanisms to modulate the immune response. Although Bregs have been consistently described in the literature, only a few reports described the molecular aspects that control the acquisition of the regulatory function. In this manuscript, we detailed the latest reports describing the control of IL-10, TGFß, and GZMB production in different Breg subsets at the molecular level. We focused on the understanding of the role of the transcription factors STAT3 and c-MAF in controlling IL-10 production in murine and human B cells and how these factors may represent an important crossroad of several key drivers of the Breg response. Finally, we provided original data supporting the evidence that MAF is expressed in human IL-10- producing plasmablast and could be induced in vitro following different stimulation cocktails. At steady state, we reported that MAF is expressed in specific human B-cell tonsillar subsets including the IgD+ CD27+ unswitched population, germinal center cells and plasmablast.

Treatment of Sjögren's Syndrome with Mesenchymal Stem Cells: A Systematic Review.

Mesenchymal stem cells (MSCs) are ubiquitous in the human body. Mesenchymal stem cells were initially isolated from bone marrow and later from other organs such as fatty tissues, umbilical cords, and gingiva. Their secretory capacities give them interesting immunomodulatory properties in cell therapy. Some studies have explored the use of MSCs to treat Sjögren's syndrome (SS), a chronic inflammatory autoimmune disease that mainly affects exocrine glands, including salivary and lacrimal glands, although current treatments are only palliative. This systematic review summarizes the current data about the application of MSCs in SS. Reports show improvements in salivary secretions and a decrease in lymphocytic infiltration in salivary glands in patients and mice with SS after intravenous or infra-peritoneal injections of MSCs. MSC injections led to a decrease in inflammatory cytokines and an increase in anti-inflammatory cytokines. However, the intrinsic mechanism of action of these MSCs currently remains unknown.

A Proinflammatory Cytokine Network Profile in Th1/Type 1 Effector B Cells Delineates a Common Group of Patients in Four Systemic Autoimmune Diseases.

OBJECTIVE: The effector T cell and B cell cytokine networks have been implicated in the pathogenesis of systemic autoimmune diseases, but the association of these cytokine networks with the heterogeneity of clinical manifestations and immune profiles has not been carefully examined. This study was undertaken to examine whether cytokine profiles can delineate distinct groups of patients in 4 systemic autoimmune diseases (systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, and systemic sclerosis). METHODS: A total of 179 patients and 48 healthy volunteers were enrolled in the multicenter cross-sectional PRECISE Systemic Autoimmune Diseases (PRECISESADS) study. Multi-low-dimensional omics data (cytokines, autoantibodies, circulating immune cells) were examined. Coculture experiments were performed to test the impact of the cytokine microenvironment on T cell/B cell cross-talk. RESULTS: A proinflammatory cytokine profile defined by high levels of CXCL10, interleukin-6 (IL-6), IL-2, and tumor necrosis factor characterized a distinct group of patients in the 4 systemic autoimmune diseases. In each disease, this proinflammatory cluster was associated with a specific circulating immune cell signature, more severe disease, and higher levels of autoantibodies, suggesting an uncontrolled proinflammatory Th1 immune response. We observed in vitro that B cells reinforce Th1 differentiation and naive T cell proliferation, leading to the induction of type 1 effector B cells and IgG production. This process was associated with an increase in CXCL10, IL-6, IL-2, and interferon-γ production. CONCLUSION: This composite analysis brings new insights into human B cell functional heterogeneity based on T cell/B cell cross-talk, and proposes a better stratification of patients with systemic autoimmune diseases, suggesting that combined biomarkers would be of great value for the design of personalized treatments.

B cells in Sjögren's syndrome: from pathophysiology to therapeutic target.

Biological abnormalities associated with B lymphocytes are a hallmark of patients with primary Sjögren's syndrome. Those patients present abnormal distribution of B lymphocytes in peripheral blood and B cells in exocrine glands. B cells produce auto-antibodies, cytokines and present antigens but can also suppressive functions. In this review, we will summarize current knowledge on B cells in primary Sjögren's syndrome patients, demonstrate their critical role in the immunopathology of the disease and describe the past and current trials targeting B cells.

Innate B Cells: the Archetype of Protective Immune Cells.

The innate B cell (IBC) population is heterogeneous and involved in the primary immune response. IBC functions include a high ability to produce natural antibodies with IgM isotype, the elimination of apoptotic cells, and a capacity to be cognate help to T cells. Among IBC subsets, B-1 cells and marginal zone B cells are the main producers of IgM, act as rapid immune responders that may relocate to follicular lymphoid and differentiate to cytokine and antibody-secreting cells shortly after infection. IBCs functions are highly dependent on their localization site and the nature of their B cell receptor repertoire, suggesting a high plasticity range of different immune responses. In this review, we will describe the nature and functions of the different innate-like B cell subsets, first in mice and then in humans. Besides this, we will emphasize the strong ability of these cells to undertake different protective functions from the first line of defense against pathogens to the regulatory role of the broader immune response.

In-depth characterization of CD24(high)CD38(high) transitional human B cells reveals different regulatory profiles.

BACKGROUND: CD24(high)CD38(high) transitional B cells represent cells at a key stage in their developmental pathway. In addition, these B cells have been widely ascribed regulatory functions and involvement in the control of chronic inflammatory diseases. However, the phenotypic and functional overlap between these cells and regulatory B cells remains controversial. OBJECTIVE: In this study we wanted to explore the regulatory properties of CD24(high)CD38(high) human B cells. METHODS: We used multicolor flow cytometry in combination with bioinformatics and functional studies to show that CD24(high)CD38(high) B cells can be distinguished into multiple subsets with different regulatory functions. RESULTS: For the first time, the study reveals that human transitional B cells encompass not only transitional type 1 and type 2 B cells, as previously suggested, but also distinct anergic type 3 B cells, as well as IL-10-producing CD27(+) transitional B cells. Interestingly, the latter 2 subsets differentially regulate CD4(+) T-cell proliferation and polarization toward TH1 effector cells. Additional analyses reveal that the percentage of type 3 B cells is reduced and the frequency of CD27(+) transitional B cells is increased in patients with autoimmune diseases compared with those in matched healthy subjects. CONCLUSION: This study provides evidence for the existence of different transitional B-cell subsets, each displaying unique phenotypic and regulatory functional profiles. Furthermore, the study indicates that altered distribution of transitional B-cell subsets highlights different regulatory defects in patients with different autoimmune diseases.

Specific forms of BAFF favor BAFF receptor-mediated epithelial cell survival.

Although B cell activating factor (BAFF) and its receptor BR3 are produced and expressed by many cells, their role has been restricted to the lymphocyte lineage. Using various techniques (RT-PCR, indirect immunofluorescence, flow cytometry analysis), we observed the expression of BR3 and the production of BAFF by the human salivary gland cell line, by epithelial cells from biopsies of Sjögren's syndrome patients and their controls, but also by salivary gland epithelial cells in culture. To decipher the role of BAFF and BR3 on epithelial cells, BAFF and BR3 were neutralized by blocking antibodies or RNA specific inhibitor (siBR3) and epithelial cell survival was analyzed. Blocking BR3 promotes epithelial cell apoptosis in vitro. This apoptosis resulted in the nuclear translocation of PKCδ. BAFF neutralization by various anti-BAFF antibodies leads to different effects depending on the antibody used suggesting that only some forms of BAFF are required for epithelial cell survival. Our study demonstrates that BR3 is involved in the survival of cultured epithelial cells due to an autocrine effect of BAFF. It also suggests that epithelial cells produce different forms of BAFF and that only some of them are responsible for this effect.

Diagnostic criteria for autoimmune neutropenia.

Autoimmune neutropenia denotes that the number of circulating polymorphonuclear neutrophils is below 1.5×10(9)/L. This encompasses a wide range of disorders from primary conditions to complications of systemic autoimmune diseases or hematological neoplasms. Antineutrophil autoantibodies are particularly difficult to detect, and their amount does not correlate with the degree of neutropenia. Granulocyte colony-stimulating factor is the first-line therapy, but should be restricted to patients with total absence of neutrophils and/or severe infections.

The complexity of the BAFF TNF-family members: implications for autoimmunity.

The B-cell activating factor belonging to the tumor-necrosis factor family BAFF contributes to autoimmune disorders. As such, BAFF might become a therapeutic target. However, this molecule has pleiotropic effects that are as numerous as they are varied. The real effect of each form (spliced, glycosylated, membrane bound, soluble, homotrimerized, heterotrimerized, multimerized) has not been well characterized yet. Consequently, conflicting results, regarding the serum concentrations of BAFF or its functional effect, exist in literature. BAFF quantification based on ELISA commercial kits was indeed found to be inaccurate. The complexity of the various forms of BAFF will be reviewed by focusing on the different structural aspects of the molecule. These data have particular implications for autoimmunity, not only because of the role of these factors on B cell growth and survival, but also their influence on the onset and severity of several autoimmune diseases.

Role of Toll-like receptors in primary Sjögren's syndrome with a special emphasis on B-cell maturation within exocrine tissues.

Be they follicular cells within the germinal centers (GCs) or marginal zone (MZ), all naïve mature B lymphocytes need tonic signaling to stay alive. We reasoned that the same holds true for those B lymphocytes that proliferate in the salivary glands (SGs) of patients with primary Sjögren's syndrome. Based on B cell infiltration, 11 SGs and three tonsil samples were selected for further examination. Tissue sections were stained using CD20 combined with CD10, CD21, CD27, CD38 or IgD. They were also laser-microdissected for quantitative RT-PCR of transcription factors, GC-specific activation-induced cytidine deaminase (AID) and TLR9. Some B cell aggregates proved to be real GCs according to their membrane markers, whereas others were clusters of transitional type II B cells. These contained mRNAs for Notch-2 and Blimp-1, but not for Pax-5, Bcl-6 and AID. Unanticipated was the finding of mRNAs for TLR9 in these clusters of MZ B-cells, but not in the real GCs. Not only do TLR9 deliver sufficiency of tonic signaling to keep B cells alive, but they also confer autoreactive B cells with an MZ-like phenotype. Thus, TLRs might be targets for forthcoming biotherapies.

Colocalization of Porphyromonas gingivalis with CD4+ T cells in periodontal disease.

Porphyromonas gingivalis, an anaerobic, asaccharolytic gram-negative bacterium, is a causative agent in chronic periodontitis. It has many virulence factors that facilitate infection of the gingiva, but little is known about the local immune cells that respond to this bacterium. The aims of this study were to quantify P. gingivalis in gingival biopsies from patients with periodontitis using laser capture microdissection (LCM) plus qRT-PCR and to determine the phenotype of immune cells associated with the bacteria using immunofluorescence. The presence of P. gingivalis was confirmed in periodontitis gingival tissue from 10 patients, and differences in bacterial distribution in the epithelium and connective tissue with or without inflammatory infiltrates were observed. Immune cells found in the biopsy tissues, including CD20+ mature B cells and CD138+ plasma cells, were associated with the Th2-type immune response. Most P. gingivalis was in direct contact with CD4+ T cells. This study revealed for the first time the colocalization of P. gingivalis with immune cells. Use of LCM combined with qRT-PCR enabled quantitative analysis of bacteria in a selected area of a biopsy sample without any tissue degradation. Observation of the immune cells associated with these bacteria was also performed by immunofluorescence.

B-cell tolerance breakdown in Sjögren's syndrome: focus on BAFF.

Sjögren's syndrome (SS) is an autoimmune epithelitis hallmarked by a destruction of epithelial cells, the subsequent lymphocytic infiltration of exocrine glands and their ensuing dryness. Given the prominent role currently assigned to B cells in SS, it is not surprising that the B cell activating factor belonging to the Tumor Necrosis Factor family (BAFF) is involved in its pathogenesis. When overexpressed, this cytokine leads to self-reactive B cells emergence at the transitional B-cell stage. BAFF overexpression that has been observed in SS, is associated with B-cell tolerance breakdown and autoantibody production. Furthermore, BAFF is responsible for the abnormal distribution of B cells subsets and B-cell hyperactivity described in the blood. In the salivary glands, a minority of B-cell clusters represent ectopic germinal center cells, while the majority manifest features consistent with transitional type 2 (T2) and marginal-zone (MZ)-like B cells. Interestingly, both types of B-cell cluster include autoreactive B cells and BAFF is associated with expansion of T2 B cells and MZ-like B cells in the salivary glands. Finally, BAFF plays a major role in B-cell repopulation after their depletion by rituximab in SS.

New ELISA for B cell-activating factor.

BACKGROUND: The B cell-activating factor of the TNF family (BAFF) is upregulated in autoimmune diseases, but a number of conflicting results have cast doubts on the reliability of the ELISA protocols currently used for its quantification. This situation led us to develop a new ELISA for the measurement of BAFF. METHODS: BAFF was purified for use alongside nonglycosylated recombinant BAFF. Two monoclonal antibodies (mAbs) and two polyclonal antibodies (pAbs) to BAFF were used. RESULTS: The optimization process showed that the pAb format was preferable to the mAb format as capture antibody, because the pAbs recognized the glycosylated as well as the nonglycosylated forms of BAFF. The most efficient pair of Abs involved using the unconjugated form of a goat pAb to capture BAFF and the same biotinylated goat pAb to detect bound BAFF. This ELISA was not influenced by the presence of rheumatoid factor. CONCLUSIONS: This new ELISA helped provide insights into why serum concentrations of BAFF vary between studies for a given population of patients. It is a reliable tool for the management of the diseases in which BAFF is an indication of response to therapy.

Ectopic germinal centers are rare in Sjogren's syndrome salivary glands and do not exclude autoreactive B cells.

This study reports on the characterization of B cells of germinal center (GC)-like structures infiltrating the salivary glands (SGs) of patients with Sjögren's syndrome. Eight two-color combinations were devised to characterize the phenotype of these B cells in 11 SG specimens selected from biopsies obtained from 40 Sjögren's syndrome patients and three normal tonsils. The 9G4 mAb, which recognizes V4.34-encoded autoAbs, enabled us to identify autoreactive B cells. Quantitative RT-PCR was used to determine the level of mRNAs for activation-induced cytidine deaminase (AICDA), repressors and transcription factors. CD20(+)IgD(-)CD38(+)CD21(+)CD24(-) B cells, similar to those identified in tonsil GCs, were seen in the SGs of four patients and, and since they expressed AICDA, they were termed "real GCs". CD20(+)IgD(+)CD38(-)CD21(+)CD24(+) B cells, seen in aggregates from the remaining seven samples, were characteristically type 2 transitional B cells and marginal zone-type B cells. They lacked AICDA mRNAs and were termed "aggregates". Real GCs from SGs contained mRNAs for Pax-5 and Bcl-6, like tonsil GC cells, whereas aggregates contained mRNAs for Notch-2, Blimp-1, IRF-4, and BR3, similar to marginal zone B cells. Further experimental data in support of this dichotomy included the restriction of CXCR5 expression to real GC cells, while sphingosine 1-phosphate receptor 1 was expressed only in aggregates. In contrast, both types of B cell clusters expressed the idiotype recognized by the 9G4 mAb. Our data indicate that, in SGs, a minority of B cell clusters represent genuine GC cells, while the majority manifest features of being type 2 transitional B cells and marginal zone cells. Interestingly, both types of B cell aggregates include autoreactive B cells.

Does the BAFF dysregulation play a major role in the pathogenesis of systemic lupus erythematosus?

Given the prominent role currently assigned to B lymphocytes in systemic lupus erythematosus, it is not surprising that the B cell activity factor belonging to the tumor necrosis factor family (BAFF) is involved in its pathogenesis. This cytokine is produced in excess, and inserted into its receptors on the surface of circulating B cells. Up-regulation of BAFF is most likely to lead to breach of tolerance by aberrant survival of B cells directed to the self. Trials aimed at blocking BAFF have thus been set out. Yet the results are awaited.

Intravenous immunoglobulin and cytokines: focus on tumor necrosis factor family members BAFF and APRIL.

The presence of natural autoantibodies against cytokines has been reported in healthy individuals. Because circulating cytokines may be implicated in the clinical outcome of numerous diseases, the mode of action of intravenous immunoglobulin (IVIg) (pooled from sera over a thousand normal individuals) may involve immunomodulation of the cytokine network. We review the anti-cytokine effects of IVIg as well as the consequences of IVIg infusions on cytokine production. Furthermore, IVIg exerts therapeutic effects in autoimmune diseases and lymphoid malignancies. These two conditions have in common an overproduction of BAFF (for B-cell-activating factor of the TNF family). The presence of antibodies with BAFF and APRIL (a proliferation-inducing ligand) specificity was investigated. We found that IVIg recognizes BAFF and APRIL and that IVIg binding prevents BAFF from exerting its antiapoptotic effect on B cells. These anti-BAFF IgGs might prevent the deleterious effects of BAFF in B-cell-mediated autoimmune diseases.

BAFF, a new target for intravenous immunoglobulin in autoimmunity and cancer.

Intravenous immunoglobulin (IVIg) has been used to treat autoimmune diseases and lymphoid malignancies with some therapeutic effect. In both these pathological conditions, there is an overproduction of BAFF (for "B-cell-activating factor of the TNF family"), and APRIL (for "a proliferation-inducing ligand"). The presence of antibodies (Abs) with BAFF and APRIL specificities in IVIg preparations was investigated by enzyme-linked immunosorbent assay, and Western Blot analysis. Apoptosis was measured by the annexin-V binding method, and confirmed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique. Nonglycosylated recombinant BAFF, glycosylated affinity-purified BAFF, and recombinant APRIL (but not TNFalpha), were recognized by certain IgG in IVIg, and their F(ab')(2) fragments. Steric hindrance prevented the antiapoptotic effects of BAFF on B-lymphocytes. This work documents the presence of anti-BAFF and anti-APRIL Abs in IVIg. These can functionally neutralize the role of BAFF in B-cell survival. These anti-BAFF IgG might amend deleterious effects of BAFF in B-cell-mediated autoimmune diseases.

The mosaic of B-cell subsets (with special emphasis on primary Sjögren's syndrome).

Major breakthroughs have occurred with classification of B-cells into populations and subpopulations. With respect to their expression of CD5, they comprise the B1 and B2 populations, with the former further divided into B1a and B1b subpopulations. The oncologic process starts from transitional type 1 (T1) and T2 immature B-cells, through marginal zone or germinal center B-cells, ending up with memory B-cells and plasma cells (PCs). They may also be categorized based on their functional commitment with polarized B effector (Be)1 and Be2, with B-activating factor of the tumor-necrosis factor-producing B-cells, and with short-lived and long-lived PCs. Such a seemingly homogeneous family of cells has thus turned out to be a genuine mosaic of B-lymphocyte subsets.