Judit Pozsgay

Recognition of new citrulline-containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients

Anti‐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.

Antigen-specific immunotherapies in rheumatic diseases

The main goal of antigen-specific immunotherapy (ASI) in autoimmune and rheumatic diseases is to reprogramme or remove autoreactive cells and/or induce immune tolerance to self-antigens. Current therapies in these diseases either treat symptoms or slow down disease progression but are not yet curative or preventative — disease-specific treatments are urgently needed. In contrast to the nonspecific treatments in current use that induce generalized immune suppression, which is associated with several adverse effects including increased risk of infections, ASIs target a restricted subset of B cells or T cells, and thus do not compromise systemic immunity and host defence. This Review provides a summary of novel approaches for identifying autoepitopes and detecting and targeting autoreactive cells that might help in the development of ASIs. Promising approaches include the use of tolerizing peptides coupled to MHC constructs and/or nanocompounds, tolerizing dendritic cells and antigen-specific vaccines. Following studies in animal models of rheumatoid arthritis and systemic lupus erythematosus, several of these strategies have now entered clinical trials. However, to use these approaches in humans, several important limitations must first be addressed, such as; selecting the proper immunodominant autoantigen; identifying the optimal timing, dosing and route of administration; finding biomarkers for monitoring the therapy; and optimizing methodology.

Induction and Differentiation of IL-10-Producing Regulatory B Cells from Healthy Blood Donors and Rheumatoid Arthritis Patients.

The most important feature of B cells is the production of Abs upon activation; additionally, B cells produce pro- and anti-inflammatory cytokines in response to certain stimuli. IL-10–producing B cells represent a major subset of regulatory B cells (Bregs) that suppress autoimmune and inflammatory responses. B cells play a crucial role in the development and maintenance of the chronic inflammatory autoimmune disease rheumatoid arthritis (RA); however, controversial data are available on IL-10– producing Bregs in RA. Our aim was to identify the optimal conditions that induce IL-10+ Bregs and, furthermore, to shed light on the signaling pathways that are responsible for their expansion. The results show that dual stimulation by CpG and CD40L for 48 h is optimal for IL-10 induction, and this can be synergistically boosted by IL-21. We identified the CD19+CD27+ memory B cell population as the major source of IL-10+ Bregs. We detected significantly fewer CD19+CD27+IL-10+ cells in RA patients compared with healthy controls, and these were functionally defective in suppressing IFN-γ production by CD4+ T cells in coculture. IL-21 drastically increased the number of IL-10+ Bregs within the CD19+CD27+ and CD19+CD27− populations; furthermore, it induced the appearance of IL-10+Blimp-1+ plasmablasts. Monitoring the phosphorylation of key signaling molecules revealed that activation of ERK, p38, and CREB is indispensable for the induction of IL-10 production, whereas phosphorylation of STAT3 further enhances IL-10 expression in human Bregs. We conclude that CREB and STAT3 are the key transcription factors responsible for the expansion and differentiation of human IL-10–producing Bregs.

Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

Background: In rheumatoid arthritis (RA), anti-citrullinated protein/peptide antibodies (ACPAs) are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR) analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. Results: KD values of affinity-purified ACPA IgGs varied between 10−6 and 10−8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two–two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

A2.18 Induction and characterisation of the dominant IL-10 producing B cell subset in healthy blood donors and rheumatoid arthritis patients

Background and objectives The most important feature of B-cells is the production antibodies upon activation; additionally, B-cells produce both pro-inflammatory and anti-inflammatory cytokines in response to certain stimuli. IL-10 producing B10 cells represent a major subset of regulatory B-cells (Bregs). Bregs suppress autoimmune and inflammatory responses by multiple mechanisms. B-cells play crucial role in the development and maintenance of the chronic inflammatory autoimmune disease, Rheumatoid arthritis (RA); however, controversial data are available on B10 population in RA. Our aim was to identify the optimal conditions inducing B10 cells in samples from healthy controls and RA patients; furthermore, to shed light on signalling pathways resulting in the expansion of the B10 subset. Materials and methods Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood of healthy donors and RA patients. B cells were purified by magnetic separation, using negative selection. We assessed IL-10 and TNF expressing B-cells after intracellular staining by flow cytometry, and measured the secreted cytokines with multiplex bead array. Phosphorylation of key signalling molecules was monitored by phospho-flow method. Results The results show that dual stimulation by CpG and CD40L for 48h was optimal for IL-10 induction, which was synergistically boosted by IL-21. We identified CD19+ CD27+ memory B-cells as the major source of B10 cells. IL-21 increased the ratio of BLIMP1 and IL-10 double positive plasmablasts. In RA patients we detected significantly less CD27+ B10 cells as compared to the controls, while addition of IL-21 to the dual stimuli significantly elevated the number of B10 cells both in the CD19+CD27+ and in the CD19+ CD27- population. Different combinations of stimuli induced preferentially the secretion of pro-inflammatory cytokines (TNF, IFNγ, IL-17) and the suppressor cytokine, IL-10. We assumed that activation of ERK, p38 and CREB are indispensable to induce IL-10, while STAT3 appears to be a co-activator for IL-10 transcription in human Bregs. Conclusions CD19+CD27+ memory B-cells are the major source of human B10 cells, which may expand and differentiate to IL-10 producing plasmablasts in the presence of IL-21. CREB and the co-activator STAT3 are the key transcription factors responsible for the expansion of the B10 population. Support: National Research, Development and Innovation Office (OTKA104846), and ELTE TÁMOP 4.2.1./B-09/1/KMR-2010–0003

A1.15 Rheumatoid arthritis patients possess a reduced number of IL-10 producing CD27+ regulatory B cells

Background Regulatory B cells (Breg) are newly defined B cell subsets that downregulate the immune response. The pleiotrophic cytokine, IL-10 seemed to be responsible for this function. B cells play a crucial role in the development and maintenance of Rheumatoid arthritis (RA). Therefore this study was undertaken to identify the optimal stimuli (BCR, CpG, CD40L) that induce Breg cells, furthermore, our aim was to compare the number of Breg in RA patients and healthy controls. We also aimed to examine, which inflammatory cytokines are produced by B cells in response to the same stimuli. Materials and methods Blood samples were collected from healthy donors and RA patients. Intracellular IL-6, IL-10 and TNF were measured in purified B cells and in PBMC. Cytokines were detected in B cells prior or after stimulation by intracellular fluorescent staining using specific antibodies. IL-10, IL-6, TNF, IL-1b and INFg were measured in the supernatants of purified B cell with FlowCytomix multiplex bead array. Results At our experimental conditions, dual stimulation by CpG and CD40L for 48 h was found to be optimal for IL-10 induction in B cells. We identified CD19+ CD27+ memory B cells as the main source of IL-10. The dual stimuli induced significantly lower number of IL-10 producing B cells from RA patients as compared to healthy controls, while we didn’t find a difference in the inflammatory cytokine (IL-6 and TNF) production. However, in unstimulated samples the frequency of IL-6 producing B cells was lower in healthy controls, than in RA patients. Furthermore, we observed that in the supernatant of purified B cells, beside IL-10, other inflammatory cytokines (IL-6, TNF, IL-1b and INFg) were also present. Therefore we double stained B cells with anti-IL-10 and anti-TNF antibodies and found that these cytokines were produced by different subsets. Conclusion We detected a significant difference between the number of IL-10 producing CD27+Breg cells of RA patients and healthy controls. The lower frequency of activation-induced IL-10 producing Bregs and on the other hand, the increased ratio of spontaneously IL-6 secreting B cells in RA patients may contribute to the exacerbation of the disease. Support: OTKA NK 104846

A5.23 Multiparameter Phospho-Flow Analysis of B Cells from Patients with Rheumatoid Arthritis

Background and Objectives Rheumatoid arthritis (RA) is a common, relapsing autoimmune disease, which affects approximately 1% of the population worldwide. While the specific molecular events that lead to initiation and onset of RA are not known, an uncontrolled activation of the immune system is considered to be a critical component of the disease. B lymphocytes undoubtedly play a critical role in disease aetiology. Antigen binding to B-cell receptor (BCR) triggers B cell activation, although the threshold of activation can be influenced by other receptors, such as TLR9. TLR9 has received substantial attention as a pathogenic co-stimulator of autoreactive B cell responses. Our aim was to compare basal activity and induced phosphorylation of AKT, ERK, p38 MAPK and CREB after stimulation of B cells from RA patients and healthy individuals via BCR and/or TLR9. Materials and Methods Blood samples were collected from healthy donors and RA patients having moderate (DAS28 3.2 < 5.1) and active (DAS28 > 5.1) disease. B cells were stimulated with anti-Ig (Fab’)2 and/or CpG ODN. Naive and memory B cells were identified by anti-CD20-A647 and anti-CD27-PE Phosphorylation level of AKT, ERK, p38 and CREB was detected before and after the stimuli by specific phospho-antibodies using multiparameter phospho-flow analysis. Results were evaluated by the FlowJo software. Results The basal level of phosphorylation of signalling molecules was significantly higher in RA patients as compared to healthy donors. The induced level of phosphorylation was also higher in RA samples in most of cases, CpG stimulated memory B cells from patients with active disease have shown the highest values. In anti-Ig plus CpG ODN stimulated samples phosphorylation of all molecules was significantly higher in both naïve and memory RA B cells as compared to healthy controls. However, when compared to unstimulated cells, the increment of phosphorylation in the stimulated cells was the same or lower in RA samples. Conclusions We have shown differences in the activation state of AKT, ERK, p38 and CREB in B cells from healthy individuals and RA patients. The higher basal phosphorylation level indicates the activated state of RA B cells. The lower capability of activation-induced phosphorylation may be a result of lower responsiveness of RA B cells. The analysis of phosphorylation signature in RA B-cells may provide new information to a better understanding of the disease.

A5.11 Detection of ACPA Producing B-Cells by a Citrulline Peptide Panel

Background and Objectives Anti-citrullinated protein/peptide antibodies (ACPAs) are the most sensitive and specific serological markers of RA. To identify the optimal epitopes that detect different subgroups of RA patients with high sensitivity and specificity, we have investigated citrulline and arginine containing peptides derived from filaggrin, collagen or vimentin. We have identified a citrulline-containing peptide panel that was recognised by RA sera with high specificity. Our aim was to compare this peptide panel with the conventionally used serological assays and to detect peptide-specific ACPA producing B-cells in in vitro cultures. Materials and Methods Previously selected citrulline- and arginine-containing filaggrin, vimentin and collagen peptide epitopes were investigated. We compared the recognition of these peptides by RA and control sera using indirect ELISA. B-cells were purified from peripheral blood by negative selection, IgG production was stimulated by B-cell activators (R848 and recombinant human IL-2) provided with the human ELISPOT kit. Antibody producing cells were enumerated after 4 days culture by using peptide-specific ELISPOT assay. Results Sera samples from 247 RA and 148 age-matched (57 ± 14 years) healthy controls were collected. The citrulline peptide panel detected approximately 80% of RA patients, including 20% of seronegative/CCP negative patients as well. Individual peptides detected different subgroups of RA patients. The more peptides recognised by a particular RA serum sample, the more severe the disease of the patient was. In vitro cultured B-cells from selected RA patients synthesised multiple citrulline-containing peptide-specific antibodies after polyclonal stimulation, while B-cells from healthy blood donors did not. Conclusions The citrulline peptide panel can detect 20% of ACPA negative RA patients thus may have a prognostic value. Furthermore, the panel is suitable to detect citrulline peptidespecific antibody producing cells, thus enables us to study ACPA producing B-cells of RA patients.

Short citrullinated epitope of filaggrin is recognised by sera as well as antibodies produced in vitro by B cells of rheumatoid arthritis patients

Background and objectives Autoantibodies in about 70% of rheumatoid arthritis (RA) patients recognising citrullinated proteins (ACPA) are cross-reactive with citrullinated filaggrin epitopes. Although filaggrin is an epithelial protein and is not expressed in synovial tissues, so is probably not the in vivo target of ACPA, sysnthetic peptid epitopes of filaggrin are good tools for the early diagnosis of RA. Our main goals are to identify the optimal citrullinated epitope(s) applicable in highly sensitive diagnostic tests and also recognised by ACPA producing B cells, thus allowing phenotyping and functional studies of this population. Materials and methods The previously identified citrullinated epitopes were synthesised by SPPS, according to Fmoc/tBu strategy, biotinylated at either the C- or the N-terminal end respectively, and tested for their reactivity with sera of RA and other autoimmune patients by ELISA. B cells were isolated from peripheral blood by negative selection, cultured with TLR9 ligand, CpG oligodeoxynucleotide, and with B cell activating factor of TNF family (BAFF), then the citrullinated peptide specific antibody production was measured on day 7. Results 5-mer and 19-mer citrullinated filaggrin peptides were recognised by the RA sera, with a sensitivity and specificity that was comparable with the currently used tests. RA sera specifically recognised the C terminally, and not by the N-terminally biotinylated 5-mer peptide, while did not differentiate between C and N terminally biotinylated 19-mer peptide, suggesting that the N-terminal amino acids in the 5-mer peptide are crucial for recognition. The data obtained with both the 5-mer and the19-mer peptides showed significant correlation with the anti-CCP titers of commercial test. To see whether B cells from RA patients and normal individuals produce citrullinated peptide specific antibodies in vitro, purified B cells were stimulated via TLR9 by CpG in the presence of BAFF. B cells from RA patients synthesised a substantial amount of antibodies that specifically recognised the 5-mer citrullinated filaggrin peptide, while B cells from normal blood donors did not, indicating that autoreactive B cells that are specific for citrullinated proteins can be stimulated via TLR9 to produce anticitrullinated peptide antibodies. Such mechanism may contribute to the pathogenicity of ACPA. Conclusion The five-mer citrullinated filaggrin peptide as a well defined short epitope could be important for development of new diagnostic tools for RA, and also may facilitate to reveal phenotypic and functional differences of autoreactive and normal B cells.

Cooperation between signalling pathways leading to survival, proliferation or death of B cells

Background and objectives Antigen driven positive selection of B cells depends on signals from B cell receptor (BCR) and a plethora of positive and negative regulating receptors determining B cells’ fate. Fas/CD95 mediated signals kill bystander or autoreactive B cells, while the receptor for B cell activating factor of tumour necrosis factor family (BAFF-R) mediates survival signals for transitional/mature B cells. Toll-like receptor 9 (TLR9) induced signals stimulate B cells and may enhance the antigen-specific B cell response. The study aim was to reveal the collaboration between these signalling pathways in human B cells. Materials and methods B cells were isolated from peripheral blood of normal blood donors by negative selection, and stimulated by various combinations of ligands binding to BCR, BAFFR, TLR9 and Fas. Kinase activities were tested by protein profiler microarray and Western blot. Proliferation of 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) loaded cells was tested by flow cytometry, antibody production was measured by ELISA. Apoptosis was evaluated by determining the ratio of subdiploid cells by flow cytometry. Results The authors found that BAFF induced a small but significant reduction of Fas-mediated cell death; however, BAFF and BCR mediated rescue signals did not cooperate. TLR9 mediated signals significantly reduced apoptosis, and the rescuing effect of TLR9 was sensitive for inhibitors of protein kinase C and p38. TLR9 synergised with BCR not only in inhibiting apoptosis but also in inducing proliferation, indicating that TLR9 may support B cell growth in the presence and in absence of antigen. Antibody synthesis was also enhanced by the co-signals of TLR9 and BAFF-R. To reveal the mechanism behind the collaboration between various signals in B cells, phosphorylation of crucial signalling molecules was tested. Proteome profiler microarray has shown that activities of Erk2, JNK, p38 and Rsk1 were synergistically enhanced by the co-stimulation via BCR and TLR9. These results were confirmed by Western blots. Additionally, the authors found that the mitogen-activated protein kinase kinase kinase, transforming growth factor β (TGFβ) activated kinase 1 (Tak1) plays a crucial role in co-signalling. Inhibitor of Tak1 blocked p38 and Rsk1 phosphorylation, and diminished proliferation induced by BCR and TLR9. Conclusions These data suggest that BAFF may function independently of BCR, rescuing B cells from Fas-mediated death. It may enhance the risk of autoimmune diseases by promoting the survival of bystander B cells in the germinal centre. TLR9 mediates both antiapoptotic and proliferative signals, and it co-operates with BCR-induced signals at the level of Tak1-p38 pathway. Thus TLR9 may significantly contribute to the positive selection, activation and growth of B cells, and thus may help to break tolerance.