SummaryAnti-citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline-and arginine-containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline-peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline-peptides identify antibody-secreting cells in in vitro cultures of RA B cells. Recognition of citrulline-and arginine-containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide-specific microarray. B cells were purified from blood by negative selection; antibody-producing cells were enumerated by ELISPOT assay. The panel composed of citrulline-peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline-peptide panel including the new short epitope peptide of filaggrin, fil311-315, also identified nearly one-third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide-specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline-containing filaggrin peptides (fil311-315 and fil306-326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline-peptides of filaggrin and vimentin detect ACPA-producing cells, and so could also be applied to study the B cells of RA patients.

Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication. These tethers can form spontaneously between many cell types, including cells of the immune and nervous systems. Traffic of viral proteins, vesicles, calcium ions, mRNA, miRNA, mitochondria, lysosomes and membrane proteins/raft domains have all been reported so far via the open ended tunneling nanotubes (TNTs). Recently we reported on existence of plasma membrane derived GM/GM ganglioside enriched microvesicles and costimulatory proteins in nanotubes connecting B lymphocytes, the way they are formed and transported across TNTs, however, still remained unclear. Here, using live cell confocal and Structured Illumination (SR-SIM) superresolution imaging, we show that B cells respond to bacterial (Cholera) toxin challenge by their subsequent internalization followed by rapid formation of intracellular microvesicles (MVs). These MVs are then transported between adjacent B cells via nanotubes. Selective transport-inhibition analysis of two abundant motor proteins in these cell types demonstrated that actin-based non-muscle myosin 2A dominantly mediates intercellular MV-transport via TNTs, in contrast to the microtubule-based dynein, as shown by the unchanged transport after inhibition of the latter. As suggested by SR-SIM images of GFP-CD86 transfected macrophages, these costimulatory molecules may be transferred by unusually shaped MVs through thick TNTs connecting macrophages. In contrast, in B cell cultures the same GFP-CD86 is dominantly transported along the membrane wall of TNTs. Such intercellular molecule-exchange can consequently improve the efficiency of antigen-dependent T cell activation, especially in macrophages with weak costimulator expression and T cell activation capacity. Such improved T cell activating potential of these two cell types may result in a more efficient cellular immune response and formation of immunological memory. The results also highlight the power of superresolution microscopy to uncover so far hidden structural details of biological processes, such as microvesicle formation and transport.

IntroductionIn mice, marginal zone (MZ) B cells are mostly found in the outer white pulp of the spleen between the marginal sinus and the red pulp. They shuttle between MZ and follicles and transport antigens to follicles where they activate T cells and differentiate into plasma cells [1][2][3] self-renew and primarily express polyreactive BCRs, which recognize conserved molecular patterns that are often shared by foreign and autologous antigens [4][5][6]. Besides the BCRs mouse MZ B cells express high levels of TLRs including TLR9, which recognizes hypomethylated CpG motifs in bacterial DNA or in chromatin complexes expressed on the surface of apoptotic cells and bodies [7][8][9]. Collagen-induced arthritis (CIA) is an animal model of rheumatoid arthritis (RA) that has been used extensively for elucidating the role of individual cell types in disease onset and progression. The use of CIA as a model for RA is based on pathological similarities between the two diseases [10]. However, CIA differs from chronic RA in that the inflammatory phase is usually of limited . CIA is mostly considered as a Th1-mediated autoimmune disease. Differentiation of Th1 cells is crucially dependent on the expression of the transcription factor T-bet (T-box transcription factor), which is induced by the IFN-γ signaling pathways [13]. Beyond its well-known role in Th1 cells, T-bet has an important impact on B-cell functions. Experiments using T-bet-deficient, lupus-prone mice showed that T-bet is required for the induction of IgG2a germ-line transcripts by IFN-γ and suggested that it regulates IgG2a class-switching in a B-cell intrinsic way [14]. B-cell intrinsic T-bet expression was also found in ageassociated B cells, a recently discovered B-cell subset with a unique surface phenotype that arise prematurely in autoimmune-prone mouse strains or accumulates progressively with age and prone to IgG2a/c production [15]. Moreover, it was recently demonstrated that in mature B cells the signaling cascades driven by the BCR and TLR9 converged at the level of T-bet and acted synergistically on it [16]. Considered all these observations on T-bet and the functional plasticity of MZ B cells with their confirmed but not entirely clear role in arthritis prompted us to examine the involvement of MZ B cells in the regulation of CIA particularly focusing on T-bet involvement.Our results clearly demonstrate that MZ B cells express an elevated level of T-bet in the remission phase of CIA. We confirmed the existence of an IL-10 producing, T-bet positive subpopulation of MZ B cells. Furthermore showed that T-bet positive MZ B cells are able to migrate toward ligand interacting with T-bet regulated CXCR3 and robustly secrete IL-10 in response to inflammatory stimuli. These data suggest that T-bet might contribute to the downregulation of CIA by promoting the regulatory capacity of IL-10 producing MZ B cells. Results and discussionMZ B cells express elevated levels of T-bet in the remission phase of CIA Autoimmune diseases related to Th1 immunity require T...

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism.

The importance of the BCR and TLR9 in autoimmunity and in the production of autoantibodies is well established but the underlying molecular mechanism still needs to be determined. Here, we aim to characterize the BCR-TLR9 cross-talk by its effect on T-bet, as T-bet is activated and regulated by both receptors and has an important role in classswitching to pathological IgG2a in mice. Using primary mouse B cells, we demonstrate that T-bet expression is synergistically elevated by the cross-talk between the BCR and TLR9. To test the effect of this synergy on IgG2a-switching, the levels of switched B cells were checked by functional tests. We found that BCR costimulation had no additional effect on TLR9-induced IgG2a expression, however the expression of Rad51 was synergistically increased. To check the biological significance of the synergy, we compared T-bet expression in B cells from healthy and collagen-induced arthritis mice but no differences were found. Taken together, we demonstrate here that signaling cascades driven by the BCR and TLR9 have a newly identified meeting point at T-bet. The two cascades act synergistically on T-bet; however additional signals may be needed to induce prolonged functional responses such as class-switch recombination.Keywords: BCR r Signaling r Synergy r T-bet r TLR9 Additional supporting information may be found in the online version of this article at the publisher's web-site Introduction B cells recognize and respond to antigens through the surface BCR, a heterotrimeric complex consisting of a membrane Ig responsible for antigen binding and the signal transducing chains Igα and Igβ [1]. Antigen-mediated engagement of the BCR induces the activation of signaling cascades that lead to the activation of MAPKs and the transcription of a variety of genes associated with B cell activation, proliferation, and antibody production [2,3].In addition to the BCR, B cells also express germ-line encoded TLRs [4]. TLR9 is an endosomal TLR that recognizes hypomethylated CpG motifs derived from microbial DNA [5]. TLR9-initiated Correspondence: Dr. Dorottya Kövesdi e-mail: dkovesdi@elte.hu; sarmayg@elte.hu signaling ultimately results in the activation of the MAP kinases, p38 and JNK, and different transcription factors, such as NF-κB, although these signals are distinct from those initiated by the BCR [6]. Remarkably, simultaneous stimulation of B cells through the BCR and TLR9 triggers the recruitment of TLR9 from endosomes to autophagosome-like compartments by a Src-family kinase dependent, MyD88-independent mechanism, resulting in a synergistic response [7].Direct stimulation of TLR9 in B cells contributes to the development and pathogenesis of autoimmune diseases, such as systemic lupus erythematosus [8]. In line with this, it has been shown that autoreactive B cells can be activated after dual ligation of the BCR and TLR9 [9]. Moreover, TLR9 signaling pathways triggered by CpG-DNA in immune complexes synergize with the BCR to costimulate autoreactive B cells, leading to autoantibody produc...