Expansion of Activated Peripheral Blood Memory B Cells in Rheumatoid Arthritis,Impact of B Cell Depletion Therapy,and Biomarkers of Response

Although B cell depletion therapy (BCDT) is effective in a subset of rheumatoid arthritis (RA) patients, both mechanisms and biomarkers of response are poorly defined. Here we characterized abnormalities in B cell populations in RA and the impact of BCDT in order to elucidate B cell roles in the disease and response biomarkers. In active RA patients both CD27+IgD- switched memory (SM) and CD27-IgD- double negative memory (DN) peripheral blood B cells contained significantly higher fractions of CD95+ and CD21- activated cells compared to healthy controls. After BCD the predominant B cell populations were memory, and residual memory B cells displayed a high fraction of CD21- and CD95+ compared to pre-depletion indicating some resistance of these activated populations to anti-CD20. The residual memory populations also expressed more Ki-67 compared to pre-treatment, suggesting homeostatic proliferation in the B cell depleted state. Biomarkers of clinical response included lower CD95+ activated memory B cells at depletion time points and a higher ratio of transitional B cells to memory at reconstitution. B cell function in terms of cytokine secretion was dependent on B cell subset and changed with BCD. Thus, SM B cells produced pro-inflammatory (TNF) over regulatory (IL10) cytokines as compared to naïve/transitional. Notably, B cell TNF production decreased after BCDT and reconstitution compared to untreated RA. Our results support the hypothesis that the clinical and immunological outcome of BCDT depends on the relative balance of protective and pathogenic B cell subsets established after B cell depletion and repopulation.     

Expression of Toll-Like Receptor -7 and -9 in B Cell Subsets from Patients with Primary Sj?gren’s Syndrome

Introduction

Sjögren’s syndrome (SS) is a rheumatic autoimmune disease characterized by inflammation of exocrine glands. As autoantibodies are present in a majority of patients, B cells have been suggested to play an important role in onset and development of the disease. Toll-like receptors (TLRs) are pattern recognition receptors triggering innate immune responses. Since an increased expression of TLRs has been detected in other rheumatic diseases the purpose of this study was to explore TLRs in B cells of SS patients.

Methods

The expression of TLR-7 and -9 in B cell subsets of 25 patients with primary SS (pSS) and 25 healthy controls was analysed in peripheral blood using flow cytometry and real time quantitative PCR.

Results

We detected similar levels of CD19⁺ B cells in pSS patients and healthy controls. An increased number of naïve B cells, as well as fewer pre-switched memory B cells were found in pSS patients. No significant differences were observed in TLR-7 and -9 expression in B cells between pSS patients and healthy controls.

Conclusion

This study shows that pSS patients have an alteration in the B cell subpopulation composition compared to controls, with less pre-switched memory B cells and more naïve B cells. We did not detect any significant disparities in TLR-7 and -9 expression between the two groups.     

Germinal Center B Cell Depletion Diminishes CD4+ Follicular T Helper Cells in Autoimmune Mice

Background

Continuous support from follicular CD4⁺ T helper (Tfh) cells drives germinal center (GC) responses, which last for several weeks to produce high affinity memory B cells and plasma cells. In autoimmune Sle1 and NZB/W F1 mice, elevated numbers of Tfh cells persist, promoting the expansion of self-reactive B cells. Expansion of circulating Tfh like cells have also been described in several autoimmune diseases. Although, the signals required for Tfh differentiation have now been well described, the mechanisms that sustain the maintenance of fully differentiated Tfh are less understood. Recent data demonstrate a role for GC B cells for Tfh maintenance after protein immunization.

Methods and Finding

Given the pathogenic role Tfh play in autoimmune disease, we explored whether B cells are required for maintenance of autoreactive Tfh. Our data suggest that the number of mature autoreactive Tfh cells is controlled by GC B cells. Depletion of B cells in Sle1 autoimmune mice leads to a dramatic reduction in Tfh cells. In NZB/W F1 autoimmune mice, similar to the SRBC immunization model, GC B cells support the maintenance of mature Tfh, which is dependent mainly on ICOS. The CD28-associated pathway is dispensable for Tfh maintenance in SRBC immunized mice, but is required in the spontaneous NZB/W F1 model.

Conclusion

These data suggest that mature Tfh cells require signals from GC B cells to sustain their optimal numbers and function in both autoimmune and immunization models. Thus, immunotherapies targeting B cells in autoimmune disease may affect pathogenic Tfh cells.     

Current and Future Immunomodulation Strategies to Restore Tolerance in Autoimmune Diseases

Autoimmune diseases reflect a breakdown in self-tolerance that results from defects in thymic deletion of potentially autoreactive T cells (central tolerance) and in T-cell intrinsic and extrinsic mechanisms that normally control potentially autoreactive T cells in the periphery (peripheral tolerance). The mechanisms leading to autoimmune diseases are multifactorial and depend on a complex combination of genetic, epigenetic, molecular, and cellular elements that result in pathogenic inflammatory responses in peripheral tissues driven by self-antigen-specific T cells. In this article, we describe the different checkpoints of tolerance that are defective in autoimmune diseases as well as specific events in the autoimmune response which represent therapeutic opportunities to restore long-term tolerance in autoimmune diseases. We present evidence for the role of different pathways in animal models and the therapeutic strategies targeting these pathways in clinical trials in autoimmune diseases.Autoimmune diseases are debilitating conditions that affect a large and growing portion of the population (∼3%–5% in the United States) (Jacobson et al. 1997). Autoimmune diseases take a devastating toll on affected families and have a considerable economic impact. Thus, improving the understanding of autoimmune diseases and developing novel therapies have been significant goals in public health. The development of autoimmune diseases reflects a loss of tolerance of the immune system for self-antigens. With the exception of a few rare monogenic diseases such as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, the development of autoimmunity is a complex and multifactorial process. This process usually involves genetic predispositions and poorly defined environmental factors that result in slight alterations in many different checkpoints, which in turn tilts the balance toward autoreactivity and away from immunoregulation. Although clearly there are key roles for B cells, antigen-presenting cells (APCs), and the innate immune response in the development and progression of autoimmune diseases, this article will focus on autoreactive T cells and potential targets of tolerogenic treatments (Fig. 1). In addition, we will discuss selected strategies currently available or being developed in the clinic as well as future opportunities to prevent and treat these diseases. Finally, current clinical strategies available as the standard of care for autoimmune diseases rely on immunosuppressive and anti-inflammatory treatments that curtail the pathological events, alleviate symptoms, and provide short-term relief in some patients. Thus, we will focus for the most part on immunotherapies aimed at reestablishing long-term tolerance.Open in a separate windowFigure 1.Development of the pathogenic autoimmune response and targets for immunotherapy. Autoreactive T cells that escape thymic negative selection are usually controlled by intrinsic (inhibitory receptors) and extrinsic (regulatory cell populations) mechanisms of tolerance in the periphery. In individuals genetically prone to autoimmunity, one or several of these checkpoints are defective, resulting in expansion of autoreactive T cells that cannot be controlled by Tregs (red, autoreactive effector T cells; green, Tregs; gray, polyclonal conventional T cells). Autoreactive T cells migrate to their targeted tissue where cytotoxic mechanisms and uncontrolled inflammation mediated by soluble mediators released by T cells and innate cells result in tissue damage. Various immunotherapeutic strategies target different steps in this process. (A) The ultimate goal of immunotherapy is to alter the balance of pathogenic versus regulatory T cells to restore tolerance, as detailed in Figure 2. (B) Anti-CD3 mAbs, antigen-specific therapies, and costimulation blockade alter the interactions between autoreactive T cells and antigen-presenting cells (APCs) and/or the signaling pathways resulting from productive T-cell receptor (TCR) ligation after presentation of cognate self-peptide/MHC (major histocompatibility complexes) in the presence of costimulatory signals, leading to deletion, anergy, immune deviation, or induction of Tregs. (C) Many strategies aim at boosting Tregs, either by concomitantly deleting Teff and promoting Tregs, and thus resetting the immune system to various degrees, such as antithymocyte globulin (ATG), rapamycin plus IL-2, and autologous hematopoietic stem cell transplantation (HSCT), or directly providing Tregs through cellular therapy. (D,E) Some therapies target populations of APCs, such as depletion of B cells by rituximab or the promotion of self-antigen presentation specifically by tolerogenic dendritic cells (DCs). (F) The migration of autoreactive T cells to their target tissue is being altered by inhibitors of leukocyte trafficking such as natalizumab and fingolimod. These drugs may further promote tolerance by keeping autoreactive T cells in the lymph nodes (LN) during immunosuppression, a prerequisite for efficient immunomodulation in some cases. (G) Anti-inflammatory therapies such as tumor necrosis factor (TNF) antagonists reduce tissue damage but also create an immunological environment more favorable to the induction of Tregs and restoration of tolerance.     

Identification of a quantitative trait locus regulating B cell-dominant infiltration into autoimmune sialitis lesions of the IQI mouse model of primary Sjögren’s syndrome

Sjögren’s syndrome (SS) is caused by an autoimmune sialodacryoadenitis, and up to 5% of patients with SS develop malignant B cell growth. The IQI mouse is a spontaneous model of primary SS in which B cells are the dominant cellular subpopulation among mononuclear infiltrates in sialitis lesions. Understanding the genetic control of aberrant B cell growth in IQI mice may help elucidate the genetic mechanisms involved in B-lineage hyperplasia leading to malignant transformation in human SS. B cell-dominant infiltration in the submandibular glands of 6-month-old IQI and C57BL/6 (B6) mice and their F1 and F2 progenies was quantified as B-lymphocytic sialitis score, and a genome-wide scan of 179 (IQI x B6) F2 females was performed to identify a quantitative trait locus (QTL) controlling this phenotype. A QTL significantly associated with variance in B-lymphocytic sialitis score was mapped to the D6Mit138 marker (position of 0.68cM) on proximal chromosome 6, with a logarithm of odds score of 4.3 (p?=?0.00005). This QTL, named autoimmune sialitis in IQI mice, associated locus 1 (Asq1), colocalized with Islet cell autoantigen 1 (Ica1), which encodes a target protein of the immune processes that define the pathogenesis of primary SS in humans and in the nonobese diabetic mouse model.     

Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies

There is increasing interest in the role of antibodies targeting specific membrane proteins in neurological and other diseases. The target(s) of these pathogenic antibodies is known in a few diseases, usually when candidate cell surface proteins have been tested. Approaches for identifying new antigens have mainly resulted in the identification of antibodies to intracellular proteins, which are often very useful as diagnostic markers for disease but unlikely to be directly involved in disease pathogenesis because they are not accessible to circulating antibodies. To identify cell surface antigens, we developed a “conformational membrane antigen isolation and identification” strategy. First, a cell line is identified that reacts with patient sera but not with control sera. Second, intact cells are exposed to sera to allow the binding of presumptive autoantibodies to their cell surface targets. After washing off non-bound serum components, the cells are lysed, and immune complexes are precipitated. Third, the bound surface antigen is identified by mass spectrometry. As a model system we used a muscle cell line, TE671, that endogenously expresses muscle-specific tyrosine receptor kinase (MuSK) and sera or plasmas from patients with a subtype of the autoimmune disease myasthenia gravis in which patients have autoantibodies against MuSK. MuSK was robustly detected as the only membrane protein in immunoprecipitates from all three patient samples tested and not from the three MuSK antibody-negative control samples processed in parallel. Of note, however, there were many intracellular proteins found in the immunoprecipitates from both patients and controls, suggesting that these were nonspecifically immunoprecipitated from cell extracts. The conformational membrane antigen isolation and identification technique should be of value for the detection of highly relevant antigenic targets in the growing number of suspected antibody-mediated autoimmune disorders. The approach would also be very suitable for the analysis of human or experimental antitumor responses.Autoimmune diseases are conditions in which aberrant immune responses cause damage to and dysfunction of the body''s own tissue. They range from prevalent conditions, such as type 1 diabetes mellitus and rheumatoid arthritis, to various types of autoimmune thyroiditis (1), inflammatory bowel diseases (2), skin conditions such as bullous pemphigoid (3), and rarer neurological disorders such as myasthenia gravis (4).Understanding of most of these diseases is still highly incomplete. Fundamental knowledge includes the identity of the antigenic target of the immune response and whether the response is predominantly T cell- or antibody-mediated. In some of the above examples, “candidate” antigens have been proposed as a result of study of the pathophysiology of the disease (e.g. see Ref. 5). The detection of a disease-specific autoantibody allows the development of diagnostic tests, and if the target is a cell surface protein it usually implies that the disease will respond clinically to treatments that reduce the levels of the pathogenic antibodies.In recent years, there has also been increasing interest in natural (or experimental) immune responses to tumor cells that may slow the growth or spread of a tumor. In some cases, however, this immune response may result in pathogenic autoimmunity. For example, antibodies directed to voltage-gated calcium channels expressed on the surface of small cell lung cancer cells can cause neurological dysfunction by binding to similar calcium channels on the motor nerve endings (see Ref. 4). In other cancer-associated (paraneoplastic) disorders, however, there are antibodies to intracellular antigens, which are also shared between the tumor and neuronal tissue, that are highly useful as diagnostic markers for the disorders. In these patients, T cell immunity is thought to be responsible for the neurological disease (see Ref. 6), which generally does not improve with immunosuppressive treatments.Attempts to identify autoantigens and tumor antigens in many autoimmune and cancer-related syndromes have generally used techniques involving screening of mRNA expression libraries or, more recently, separation of soluble extracts of tissue or cell lines by one- or two-dimensional electrophoresis and blotting of the separated proteins onto membranes where they are probed with patient sera. Typically in any one experiment, a large number of protein bands or spots are bound by serum antibodies, and some of the corresponding bands or spots on the gel are then excised, digested, and analyzed by mass spectrometry (e.g. Refs. 7 and 8). The identified proteins have been claimed as novel antigens associated with the condition with sometimes a whole array of proteins identified from a single experiment and claimed to represent a disease-associated “autoimmune profile.” However, the identified proteins are often common intracellular proteins with the same or closely related proteins repeatedly implicated in seemingly unrelated autoimmune, allergic, and malignant diseases (see “Discussion”). The intracellular location of these proteins where they would be inaccessible to circulating antibodies and their lack of disease specificity cast doubt upon their relevance.The best understood example of an antibody-mediated disease is myasthenia gravis with acetylcholine receptor antibodies (for a review, see Ref. 9). Another subgroup of myasthenia gravis patients has antibodies to a muscle-specific tyrosine kinase (MuSK).¹ These antibodies are known to bind to the cell surface and to inhibit the clustering function of MuSK (10). Although the mechanisms of disease are not fully understood, the patients respond to immunotherapies, and the identification of this antigen by a candidate approach has revolutionized the diagnosis and treatment of this subtype of myasthenia (11). In many other conditions, however, no suitable candidate antigens have yet been proposed, limiting the diagnosis and treatment of the disorders.To develop a novel proteomics strategy for identifying cell membrane autoantigens, we used a model system involving antibodies from MuSK antibody-positive patients and from MuSK antibody-negative subjects. We first allowed the antibodies to bind to their target(s) on the intact cell surface, rather than after extraction and denaturation in detergents, so that the antibodies could recognize fully conformational epitopes. The cells, with antibodies already bound, were then solubilized, and the ready formed immune complexes were isolated and either visualized by SDS-PAGE and immunoblotting or identified by mass spectrometry. Although we show the current results as a “proof of principle,” the “conformational membrane antigen isolation and identification” (CMAII) technique could easily be adapted for use in studies of other diseases.     

Suppression of innate and adaptive B cell activation pathways by antibody coengagement of FcγRIIb and CD19

The Fc receptor (FcγRIIb) inhibits B cell responses when coengaged with B cell receptor (BCR), and has become a target for new autoimmune disease therapeutics. For example, BCR and FcγRIIb coengagement via the Fc-engineered anti-CD19 XmAb5871 suppresses humoral immune responses. We now assess effects of XmAb5871 on other activation pathways, including the pathogen-associated molecular pattern receptor, TLR9. Since TLR9 signaling is implicated in autoimmune diseases, we asked if XmAb5871 could inhibit TLR9 costimulation. We show that XmAb5871 decreases ERK and AKT activation, cell proliferation, cytokine, and IgG production induced by BCR and/or TLR9 signals. XmAb5871 also inhibited differentiation of citrullinated peptide-specific plasma cells from rheumatoid arthritis patients. XmAb5871 may therefore have potential to suppress pathogenic B cells in autoimmune diseases.     

Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Background

Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.

Methodology/Principal Findings

Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.

Conclusions/Significance

These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.     

Overexpression of activation-induced cytidine deaminase in B cells is associated with production of highly pathogenic autoantibodies

Defective receptor editing or defective B cell checkpoints have been associated with increased frequency of multireactive autoantibodies in autoimmune disease. However, Ig somatic hypermutation and/or class switch recombination may be mechanisms enabling the development of pathogenic multireactive autoantibodies. In this study, we report that, in the BXD2 mouse model of autoimmune disease, elevated expression of activation-induced cytidine deaminase (AID) in recirculating follicular CD86(+) subsets of B cells and increased germinal center B cell activity are associated with the production of pathogenic multireactive autoantibodies. CD4 T cells from BXD2 mice that expressed increased levels of CD28 and an increased proliferative response to anti-CD3 and anti-CD28 stimulation are required for this process. Inhibition of the CD28-CD86 interaction in BXD2 mice with AdCTLA4-Ig resulted in normalization of AID in the B cells and suppression of IgG autoantibodies. This treatment also prevented the development of germinal center autoantibody-producing B cells, suggesting that an optimal microenvironment enabling AID function is important for the formation of pathogenic autoantibodies. Taken together, our data indicate that AID expression in B cells is a promising therapeutic target for the treatment of autoimmune diseases and that suppression of this gene may be a molecular target of CTLA4-Ig therapy.     

Suppression of innate and adaptive B cell activation pathways by antibody coengagement of FcγRIIb and CD19

The Fc receptor (FcγRIIb) inhibits B cell responses when coengaged with B cell receptor (BCR), and has become a target for new autoimmune disease therapeutics. For example, BCR and FcγRIIb coengagement via the Fc-engineered anti-CD19 XmAb5871 suppresses humoral immune responses. We now assess effects of XmAb5871 on other activation pathways, including the pathogen-associated molecular pattern receptor, TLR9. Since TLR9 signaling is implicated in autoimmune diseases, we asked if XmAb5871 could inhibit TLR9 costimulation. We show that XmAb5871 decreases ERK and AKT activation, cell proliferation, cytokine, and IgG production induced by BCR and/or TLR9 signals. XmAb5871 also inhibited differentiation of citrullinated peptide-specific plasma cells from rheumatoid arthritis patients. XmAb5871 may therefore have potential to suppress pathogenic B cells in autoimmune diseases.     

In-cell infection: bringing uninvited guests

Cell-in-cell structures resulting from live cell engulfment were identified more than 100 years ago, but their physiological significance has remained largely obscure. Now Ni et al. identify a new role for cell-in-cell structure formation, called “in-cell infection” that spreads Epstein-Barr virus from infected B cells to epithelial cells, an activity that may predispose to cancer.Epstein-Barr virus (EBV) is a common herpesvirus infecting up to 90% or more of the human population that causes mononucleosis, is associated with autoimmune conditions, and predisposes to cancer¹. EBV persists as a latent infection within B cells and predisposes to cancers of B cell origin, including Hodgkin''s and Burkett''s lymphoma, due to expression of latency genes, which leads to B cell transformation. Infected individuals are also predisposed to developing nasopharyngeal and gastric carcinoma, as epithelial cells also harbor latent EBV. However, while the mechanism of EBV entry into B cells is well characterized, how EBV infects epithelium has remained obscure. In a recent paper published in Cell Research, Ni et al.² identify a novel mechanism for EBV infection of epithelial cells, which they term “in-cell infection”, an insidious mode of viral entry that takes advantage of whole cell ingestion.Viral infection is generally mediated by viral envelope glycoproteins that bind to specific receptors on target cells, leading to membrane fusion and viral entry. To infect B cells, the EBV envelope protein gp350 binds to the complement receptor 2 (CR2) on target cells, followed by interaction of gp42 with MHC class II molecules, and virus-to-target cell fusion is mediated by gp42, gH and gL proteins³. Unlike B cells, epithelial cells do not normally express complement receptors or MHC class II molecules, and are generally not infected by purified EBV. Previously described alternative modes of EBV infection of epithelial cells include “cell-to-cell” and “transfer” infection, where B cells have been found to act as carriers to mediate infection through cell adhesion protein-dependent conjugation^3,4,5,6.Ni et al.² now describe a different mode of epithelial cell infection, called “in-cell infection”, that occurs by ingestion of whole EBV-infected B cells, leading to the formation of “cell-in-cell” structures. B cell ingestion in this context resembles “entosis”, a mechanism previously found to mediate cell-in-cell structure formation in epithelial cultures and human tumors⁷. Entosis also promotes the uptake of hematopoietic cells into epithelial cells or cancer cells of various types^8,9. Incredibly, the authors find that entosis-like internalization of latent EBV-infected B cells (Akata) into cultured nasopharyngeal carcinoma cells (CNE-2) leads to the activation of EBV and the transfer of virus to host (CNE-2) cells. Cells infected in this manner express viral gene products, and produce virions upon stimulation that can infect naïve cells of either B cell or epithelial cell origin, indicating potent infection ability and altered tropism of EBV produced by this mechanism.Frequent cell-in-cell structure formation involving EBV-infected B cells is shown by the authors to occur in clinical nasopharyngeal carcinoma samples, suggesting that the in-cell infection mechanism is a likely contributor to viral spread in vivo, and may be linked to carcinoma development. Intriguingly, entosis itself may participate in tumorigenesis by promoting aneuploidy¹⁰, and by supplying cancer cells with nutrients¹¹. As the authors found that EBV infection promoted entosis-like cell uptake, this mode of viral spread could affect tumorigenesis by multiple mechanisms. For in-cell infection, it seems that the nutrients taken in upon the death of internalized cells come mixed with virus that is insidiously transferred to hosts, in a manner perhaps like a Trojan horse enterring with a hidden viral payload (Figure 1).Open in a separate windowFigure 1In-cell infection delivers virus to insusceptible host cells. The B cell infected by EBV resembles a Trojan horse that delivers a hidden viral payload to host epithelial cells.The identification of in-cell infection by Ni et al.² makes a significant contribution to cell-in-cell research by identifying a new pathophysiological role for an entosis-like process. Cell-in-cell structures were first reported over 100 years ago, but the mechanisms that control the formation of such structures and their significance are only now starting to emerge¹². As is often the case with groundbreaking research, the discovery of in-cell infection² raises many new interesting questions. What is the mechanism of B cell internalization into epithelial cells? Entosis is previously described to involve cell adhesion receptors, such as E-cadherin, and Rho-kinase that promotes the actomyosin contraction that drives cell uptake⁷. The molecular mechanism controlling the entry of EBV-infected B cells into epithelial cells will be important to uncover, as other mechanisms in addition to entosis can also mediate the uptake of live cells¹². How is EBV activated by the formation of cell-in-cell structures? How is EBV transferred from internalized cells to hosts? And importantly, can other viruses, such as HIV, spread by in-cell infection? The answers to these questions await further research.     

Rare incidence of methotrexate-specific lesions in liver biopsy of patients with arthritis and elevated liver enzymes

Introduction

The present study objective was to evaluate the incidence of methotrexate (MTX)-specific liver lesions from the analysis of a liver biopsy of inflammatory arthritis patients with elevated liver enzymes.

Methods

A case-control study was performed with 1,571 arthritis patients on long-term low-dose MTX therapy. Results of liver biopsy were analyzed in 41 patients with elevated liver enzymes. The expression of autoimmune markers was also assessed. This population was compared with 41 disease control subjects obtained from the same database, also on MTX but without elevated liver enzymes, matched for age, sex and rheumatic disease.

Results

Compared with the disease controls, patients with liver biopsy showed lower disease duration and lower MTX exposure, weekly and cumulative doses, reflecting shorter treatment duration due to liver abnormalities. Liver biopsies showed 17 autoimmune hepatitis-like (AIH-like) lesions, 13 nonalcoholic steatohepatitis-like lesions, seven limited liver lesions, and two primary biliary cirrhoses. However, MTX-specific lesions with dystrophic nuclei in hepatocytes were seen in only two cases. Liver biopsy lesions were associated with autoimmune markers (P = 0.007); notably, AIH-like lesions were associated with rheumatoid arthritis and with the presence of the HLA-DR shared epitope.

Conclusions

MTX-specific liver lesions are rarely observed in arthritis patients under long-term MTX therapy and elevated liver enzymes.     

IgG4-related disease: why high IgG4 and fibrosis?

The hallmarks of IgG4-related disease (IgG4-RD) are lymphoplasmacytic tissue infiltration with a predominance of IgG4-positive plasma cells, accompanied by fibrosis, obliterative phlebitis, dacryoadenitis, and elevated levels of IgG4. In a recent issue of Arthritis Research & Therapy, Tsuboi and colleagues demonstrated that regulatory T (Treg) cell-and T helper 2 (Th2) cell-derived cytokines contribute to the pathogenesis of Mikulicz''s disease, an activation pathway that appears to be common for IgG4-RD. Additional organ-specific factors may account for the different organ involvement of IgG4-RD.IgG4-related disease (IgG4-RD) is a newly categorized disease entity initially recognized in Japan but increasingly also in other parts of the world [1,2]. Most often the diagnosis is made in patients with autoimmune pancreatitis. Additional presentations include patients with lacrimal and salivary gland involvement, formerly Mikulicz''s disease (MD), which was once thought to be a subset of Sjögren''s syndrome (SS).The hallmarks of IgG4-RD are lymphoplasmacytic tissue infiltration with a dominance of IgG4-positive plasma cells, accompanied by fibrosis, obliterative phlebitis, dacryoadenitis, and elevated levels of IgG4. The pathogenesis of IgG4-RD is poorly understood; findings consistent with both an autoimmune disorder and an allergic disorder are present [1,2].IgG4 production is controlled primarily by T helper 2 (Th2) cells [3,4]. Th2 cytokines interleukin-4 (IL-4) and IL-13 enhance the production of IgG4 and IgE. In contrast, IL-10, IL-12, and IL-21 shift the balance between IgG4 and IgE, favoring IgG4. In the Th2 cytokine-driven immune reaction, IgG4 production is induced preferentially by the activation of IL-10 produced by regulatory T (Treg) cells [3]. Thus, selective IgG4 induction is referred to as the combined effect of Th2 and Treg cells.In a recent issue of Arthritis Research & Therapy, Tsuboi and colleagues [5] analyzed the expression of IgG4-specific class switch molecules such as Th2 cytokines (IL-4 and IL-13) and Treg cytokines (IL-10 and TGF-β), IgG4-nonspecific B cell regulatory molecules (CD40, CD154, BAFF, APRIL, and IRF4), and activation-induced cytidine deaminase (AID) in the labial salivary glands (LSGs) and peripheral blood mononuclear cells (PBMCs) from patients with IgG4-RD (MD) and SS. The authors provided evidence that IL-10, TGF-β, and AID were overexpressed in LSGs from IgG4-RD (MD) compared with those in patients with SS, suggesting that Treg cytokines (IL-10 and TGF-β) contribute to IgG4-specifc class switch recombination and fibrosis in patients with IgG4-RD (MD) in combination with the IgG4-unrelated molecule, AID (Figure ​(Figure11).Open in a separate windowFigure 1Molecular mechanism of IgG4-related disease. AID, activation-induced cytidine deaminase; IL, interleukin; TGF-β, transforming growth factor-beta; Th, T helper; Treg, regulatory T.Very recently, Tanaka and colleagues [6] examined the Th1, Th2, and Treg cytokine expression in LSGs from patients with IgG4-RD and SS. The authors showed that the levels of mRNA for both Th2 and Treg cytokines were significantly higher in LSGs from patients with IgG4-RD (MD) but that the expressions of Th1 and Th2 cytokines were higher in LSGs from patients with SS. The upregulation of Treg cytokines is identical to the findings reported by Tsuboi and colleagues [5], indicating that Treg cells play an important role in the pathogenesis of IgG4-RD (MD). In contrast, Tsuboi and colleagues showed that Th2 cytokines such as IL-4 and IL-13 were not significantly overexpressed in LSGs from patients with IgG4-RD (MD) but were increased if compared with those in healthy subjects. This finding supports the notion that Th2 cytokines such as IL-4 and IL-13 play a common B cell-activating role in both IgG4-RD (MD) and SS. Contrary to Th2 and Treg cytokines, Th1 cytokines were upregulated only in LSGs from patients with SS [6], suggesting that Th1 cells function as key players in the pathogenesis of SS.Consistent with the findings in MD, analyses of the expression of cytokines in inflammatory lesions from patients with IgG4-related sclerosing pancreatitis and cholangitis [7] or tubulointerstitial nephritis [8] showed that tissue mRNA expression of Th2 (IL-4) and Treg cytokines (IL-10 and TGF-β) was substantially higher than in other diseases. Many mononuclear cells expressing IL-4 or IL-10 were identifiable in affected organs by in situ hybridization [7]. Moreover, circulating CD4⁺CD25⁺Treg cells were significantly increased in PBMCs from patients with autoimmune pancreatitis [9].Further examinations should shed light on the molecular mechanisms controlling the activation of this pathway.     

Active systemic lupus erythematosus is associated with a reduced cytokine production by B cells in response to TLR9 stimulation

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with a break in self-tolerance reflected by a production of antinuclear autoantibodies. Since autoantibody production can be activated via nucleic acid Toll-like receptor 9 (TLR9), the respective pathway has been implicated in the development of SLE and pathogenic B cell responses. However, the response of B cells from SLE patients to TLR9 stimulation remains incompletely characterized.

Methods

In the current study, the response of B cells from SLE patients and healthy donors upon TLR9 stimulation was analyzed in terms of proliferation and cytokine production and correlated with the lupus disease activity and anti-dsDNA titers.

Results

B cells from SLE patients showed a reduced response to TLR9 agonist compared to B cells from healthy donors in terms of proliferation and activation. B cells from SLE patients with higher disease activity produced less interleukin (IL)-6, IL-10, vascular endothelial growth factor, and IL-1ra than B cells from healthy donors. Further analyses revealed an inverse correlation of cytokines produced by TLR9-stimulated B cells with lupus disease activity and anti-dsDNA titer, respectively.

Conclusion

The capacity of B cells from lupus patients to produce cytokines upon TLR9 engagement becomes less efficient with increasing disease activity, suggesting that they either enter an exhausted state or become tolerant to TLR stimulation for cytokine production when disease worsens.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0477-1) contains supplementary material, which is available to authorized users.     

GWASs and the age of human as the model organism for autoimmune genetic research

Genetic studies have identified more than 150 autoimmune loci, and next-generation sequencing will identify more. Is it time to make human the model organism for autoimmune research?Human genetics - linking inherited variation in DNA sequence with traits such as susceptibility to disease - provides prima facie evidence that a gene and a pathway are associated with a disease. The most recent wave of genomic technology has allowed human genomes to be scanned for variant DNA sequences (or alleles) in many people to determine which alleles are associated with a particular disease or phenotype of interest. Termed genome-wide association studies, or GWASs, this approach has identified hundreds of alleles that are associated with a variety of human traits [1,2]. By most accounts, the GWAS approach has been very successful at identifying new regions of the genome (or loci) that are important in disease, even though the effect sizes of most alleles are modest.The GWAS approach has been particularly successful at uncovering risk alleles for autoimmune diseases. Collectively, autoimmune diseases are common, affecting more than 5% of the adult population [3]. These diseases include rheumatoid arthritis (RA), type 1 diabetes (T1D), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), multiple sclerosis (MS), psoriasis and celiac disease (among others). RA is a chronic inflammatory disease that destroys free moving joints. T1D is a form of diabetes that results from the destruction of insulin-producing beta cells of the pancreas. IBD is a group of inflammatory conditions of the colon and small intestine; the two major types are Crohn''s disease and ulcerative colitis. In SLE, the immune system attacks a wide variety of organs, including the heart, joints, skin, lungs, blood vessels, liver, kidneys and nervous system. MS is an autoimmune disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to a broad spectrum of signs and symptoms. Psoriasis is a chronic disease in which the skin develops red, scaly patches, which is the result of areas of inflammation and excessive skin production. Celiac disease is an autoimmune disorder of the small intestine caused by a reaction to storage proteins (called glutens) found in cereal grains; the ensuing excessive immune reaction leads to an attack on the intestinal villi and tissue damage, resulting in malabsorption of nutrients.So far, approximately 150 loci have been identified that increase risk of these autoimmune diseases [4-14]. For each disease, the strongest genetic risk factors reside within the major histocompatibility complex (MHC) region on chromosome 6 [15]. Most associated alleles in other regions are common in the general population, but increase the disease risk by only 10 to 20% (corresponding to an odds ratio (OR) of 1.10 to 1.20 per copy of the risk allele). (The OR is a measure of the strength of association; it refers to the ratio of the odds of an event occurring in one group (such as cases) to the odds of it occurring in another group (such as controls).) For any given autoimmune disease, the known genetic risk alleles explain between 10 and 20% of variance in disease risk, whereas more than 50% of disease risk is estimated to be heritable. The remaining 30% or so of unexplained genetic disease risk is termed the missing heritability.The challenges now are, first, to find the causal mutation responsible for the signal of association; second, to understand which gene is disrupted by the causal mutation and how it is disrupted (that is, whether the mutation results in gain of function, loss of function, or a new function altogether); third, to understand which cell type and biological pathways are altered by these mutations; and finally to find additional mutations that explain the missing heritability [16]. The next wave of genomic technology - next-generation sequencing - will be a powerful ally in this effort. In particular, next-generation sequencing will help localize the causal mutation, as well as help identify rare alleles that confer risk of autoimmune disease.Thus, an important question remains: what is the most appropriate scientific approach to understand function of risk alleles discovered in human genetics research? Is the mouse the most appropriate model organism, or do these genetic discoveries provide new resources to enable functional studies directly in human immune cells?Here, I discuss the confluence of events that create a unique opportunity to use human subjects as the ''model organism'' for the study of autoimmune disease pathogenesis. In addition to GWASs and next-generation sequencing, registries of blood draws from healthy, consenting human volunteers enable functional studies of genetic variants in a wide range of primary human immune cells, and human stem cell technology has advanced to the point at which induced pluripotent stem (iPS) cells can be derived from patients with specific mutations and differentiated into diverse immune lineages. These resources should allow investigators to understand the altered cellular state in diseases that are uniquely human, which should ultimately lead to new therapeutics to treat or prevent the devastating consequences of autoimmune disease.     

Cholangiocytes: Cell transplantation

Background

Due to significant limitations to the access to orthotropic liver transplantation, cell therapies for liver diseases have gained large interest worldwide.

B cell subsets and dysfunction of regulatory B cells in IgG4-related diseases and primary Sj?gren’s syndrome: the similarities and differences

Introduction

IgG4-related disease (IgG4-RD) is a multisystem-involved autoimmune disease. Abnormally activated and differentiated B cells may play important roles. Regulatory B cells (Breg) are newly defined B cell subgroups with immunosuppressive functions. In this study, we investigated the differences of B cell subsets, the expressions of co-stimulatory molecules on B cells, and the function of Breg cells in patients with IgG4-RD, primary Sjögren’s syndrome (pSS) as well as in healthy controls (HC).

Methods

Newly diagnosed IgG4-RD patients (n = 48) were enrolled, 38 untreated pSS patients and 30 healthy volunteers were recruited as disease and healthy controls. To analyze B cell subsets and B cell activity, PBMCs were surface stained and detected by flow cytometry. The function of Breg cells was tested by coculturing isolated CD19 + CD24^hiCD38^hi Breg cells with purified CD4 + CD25- T cells. Serum cytokines were measured by ELISA and cytometric bead array. Relationship between clinical data and laboratory findings were analyzed as well.

Results

Compared with pSS patients and HC, IgG4-RD patients had a lower frequency of peripheral Breg cells. Interestingly, CD19 + CD24-CD38^hi B cell subsets were significantly higher in peripheral B cells from IgG4-RD patients than in pSS patients and HC, which correlated with serum IgG4 levels. The expression of BAFF-R and CD40 on B cells was significantly lower in IgG4-RD patients compared with those in pSS patients and HC. Unlike HC, Breg cells from pSS patients lacked suppressive functions.

Conclusions

B cells in patients with IgG4-RD and pSS display a variety of abnormalities, including disturbed B cell subpopulations, abnormal expression of key signaling molecules, co-stimulatory molecules, and inflammatory cytokines. In addition, a significantly increased B cell subset, CD19 + CD24-CD38^hi B cells, may play an important role in the pathogenesis of IgG4-RD.     

Prevalence and risk factors of vertebral compression fractures in female SLE patients

Introduction

The present study objective was to evaluate the incidence of methotrexate (MTX)-specific liver lesions from the analysis of a liver biopsy of inflammatory arthritis patients with elevated liver enzymes.

Methods

A case-control study was performed with 1,571 arthritis patients on long-term low-dose MTX therapy. Results of liver biopsy were analyzed in 41 patients with elevated liver enzymes. The expression of autoimmune markers was also assessed. This population was compared with 41 disease control subjects obtained from the same database, also on MTX but without elevated liver enzymes, matched for age, sex and rheumatic disease.

Results

Compared with the disease controls, patients with liver biopsy showed lower disease duration and lower MTX exposure, weekly and cumulative doses, reflecting shorter treatment duration due to liver abnormalities. Liver biopsies showed 17 autoimmune hepatitis-like (AIH-like) lesions, 13 nonalcoholic steatohepatitis-like lesions, seven limited liver lesions, and two primary biliary cirrhoses. However, MTX-specific lesions with dystrophic nuclei in hepatocytes were seen in only two cases. Liver biopsy lesions were associated with autoimmune markers (P = 0.007); notably, AIH-like lesions were associated with rheumatoid arthritis and with the presence of the HLA-DR shared epitope.

Conclusions

MTX-specific liver lesions are rarely observed in arthritis patients under long-term MTX therapy and elevated liver enzymes.     

Relapse Rate and Associated-Factor of Recurrence after Stopping NUCs Therapy with Different Prolonged Consolidation Therapy in HBeAg Positive CHB Patients

Background

Many chronic hepatitis B (CHB) patients recur after off-therapy and have to accept prolonged consolidation therapy with NUCs. We investigated the rate of HBV relapse after stopping NUCs therapy with different time period of prolonged consolidation therapy in HBeAg positive CHB patients, and analyzed the associated-factor of recurrence.

Methods

We recruited 162 HBeAg-positive CHB patients who met the standard of stopping NUCs therapy recommended by the 2005 APASL. Patients in group A, without the prolonged consolidation therapy, were as controls. Patients in group B were divided into 3 subgroups (group B1, 7 (range 3–11) months of the prolonged consolidation therapy; group B2, 17 (range 13–20) months of the prolonged consolidation therapy; group B3, 28 (range 25–34) months of the prolonged consolidation therapy). Virologic relapse was defined as an increase in serum HBV DNA to >10³copies/ml after off-therapy.

Results

One hundred and thirty-six patients (group A, 40 patients; group B1, 54 patients; group B2, 23 patients; group B3, 19 patients) were eligible for this study. The cumulative rates of relapse in group B at 6 months and 48 months were 29.2%, 41.7% after off-therapy, respectively. The cumulative rates of relapse in group B were statistically lower than that in group A at the same time periods. The cumulative rate of relapse in group B3 or group B2 was statistically lower than that in group B1, respectively. On multivariate analysis by Cox’s proportional hazard model, age at off-therapy, baseline ALT and the different time period of the prolonged consolidation therapy were associated with the relapse of HBV after off-therapy.

Conclusions

Consolidation therapy with NUCs after HBeAg seroconversion should be further prolonged. Age at off-therapy, ALT at baseline and the time period of the prolonged consolidation therapy could provide information to direct anti-viral therapy.