Rita A Moura

Alterations on peripheral blood B-cell subpopulations in very early arthritis patients

OBJECTIVE To characterize circulating B-cell subpopulations of arthritis patients with <6 weeks of disease duration. METHODS Peripheral blood samples were collected from very early untreated polyarthritis patients, with <6 weeks of disease duration, for flow cytometric evaluation of B-cell subpopulations. Samples from patients who were later diagnosed as RA [very early RA (VERA)] were also collected 4-6 weeks after starting a low dose of prednisone (5-10 mg) and 4 months after reaching the minimum effective dose of MTX. A matched healthy group was used as a control. RESULTS VERA patients have a lower percentage of total peripheral blood memory B cells (CD19(+)CD27(+)) and a significant decrease in the frequency of circulating pre-switch memory B cells (CD19(+)IgD(+)CD27(+)) as compared with controls. Therapy with corticosteroids or MTX was unable to restore the normal frequencies of these B-cell subpopulations. A significant decrease in peripheral pre-switch memory B cells is equally observed in other early arthritis patients. Furthermore, no significant differences are found in the frequencies of CD4(+) and CD8(+) T cells in all patient groups. CONCLUSIONS In very early polyarthritis patients, there is a reduction in circulating pre-switch memory B cells. The reasons that may account for this effect are still unknown. Short-term corticosteroids and MTX do not seem to have a direct effect on circulating B-cell subpopulations in VERA patients.

Cytokine pattern in very early rheumatoid arthritis favours B-cell activation and survival

OBJECTIVES B cells play an important role in the perpetuation of RA, particularly as autoantibody-producing cells. The ICs that further develop deposit in the joints and aggravate the inflammatory process. However, B-cell contribution in the very early stage of the disease remains unknown. The main goal of this work was to determine the concentration of cytokines potentially relevant for B-cell activation in serum from very early polyarthritis patients, with <6 weeks of disease duration, who latter on evolved into very early RA (VERA). METHODS A proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF) and IL-21 levels were measured by ELISA in the serum of VERA, other very early arthritis (VEA), established RA patients and controls. SF samples of established RA were also analysed. RESULTS VERA patients have higher levels of APRIL and BAFF as compared with VEA, established RA and controls. Furthermore, APRIL and BAFF levels are also significantly elevated in RA-SF when compared with serum. CONCLUSIONS The increased levels of APRIL and BAFF in VERA patients suggests that B-cell activation and the development of autoreactive B-cell responses might be crucial in early phases of RA. Therefore, APRIL and BAFF could be promising targets for therapy in the early phase of RA.

B-cell-activating factor receptor expression on naive and memory B cells: relationship with relapse in patients with rheumatoid arthritis following B-cell depletion therapy

Objectives To examine the expression of B-cell-activating factor receptor (BAFF-R) on naive CD27− and memory CD27+ B cells in normal individuals and patients with rheumatoid arthritis (RA) undergoing B-cell depletion therapy with rituximab. Patients and Methods BAFF-R expression on B-cell subsets was determined in normal controls (NC; n=11), active patients with RA pre-rituximab (pre-RX; n=15), relapsing patients either concordant for B-cell repopulation (C-R, n=13) or discordant, with relapse more than 3 months after repopulation (D-R, n=11) and patients in remission over 3 months postrepopulation (discordant non-relapsing (D-NR), n=5). Serum BAFF was measured by ELISA and analysed using Mann–Whitney. Results There was no significant difference between NC, pre-RX and D-NR patients in %BAFF-R-positive B cells or mean fluorescence intensity (MFI) in naive and memory B cells. Relapsing patients had significantly lower MFI and %BAFF-R-positive cells in both naive and memory compartments from NC and pre-RX (C-R and D-R; p<0.01). BAFF levels in pre-RX patients were within the normal range and did not correlate with BAFF-R expression in any patient group. D-NR patients had relatively lower proportions of pre and postswitch CD27+ B cells than pre-RX patients (D-NR vs pre-RX; p<0.05 for both) and also lower numbers of postswitch B cells than D-R patients (D-NR vs D-R, p<0.05). Conclusion BAFF-R expression was significantly reduced on both naive and memory B cells in patients at relapse, regardless of the relationship with B-cell repopulation or serum BAFF levels. Re-establishment of active disease was also associated with an increase in class-switch recombination. Factors responsible for lower levels of BAFF-R may relate to altered thresholds for autoreactive B-cell generation at relapse in patients with RA.

BAFF and TACI Gene Expression Are Increased in Patients with Untreated Very Early Rheumatoid Arthritis

Objective. B cells play important roles in rheumatoid arthritis (RA). Given the beneficial effect of B cell depletion therapy in RA as well as the observed alterations in B cell subpopulations in this disease, we evaluated whether changes in the expression of genes related to B cell survival and activation were already present in patients with untreated very early RA (VERA; < 6 weeks of disease duration). Methods. The expression of a group of B cell-related activation and survival genes was quantified in peripheral blood mononuclear cells from patients with VERA by real-time PCR and compared with untreated early RA (< 1 year), established treated RA, and other untreated early arthritis conditions. Serum B cell-activating factor belonging to the tumor necrosis factor family (BAFF) was quantified by ELISA. Results. BAFF gene expression and serum levels were highest in patients with VERA. The expression of BAFF receptor (BAFF-R) increased with disease progression, while transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) was elevated since the first weeks of RA onset. Paired box 5 gene expression was also increased at all RA stages. Chemokine (C-X-C motif) receptor 5 was elevated only in established RA. No differences were observed in B cell maturation antigen, activation-induced cytidine deaminase, B lymphocyte-induced maturation protein, and B cell lymphoma 2 expression. Conclusion. Disturbances in the expression of B cell-related activation and survival genes, particularly BAFF and TACI, occur from the onset of RA and precede changes in BAFF-R. These alterations can lead to the development of autoreactive B cells from the first weeks of RA onset.

Cytokine pattern in very early rheumatoid arthritis favours B cell activation and survival

B cells play an important role in the perpetuation of rheumatoid arthritis (RA), particularly as autoantibody producing cells. The immune complexes that further develop deposit in the joints and aggravate the inflammatory process. However, B cells contribution in the very early stage of the disease remains unknown.

Expression of the inherently autoreactive idiotope 9G4 on autoantibodies to citrullinated peptides and on rheumatoid factors in patients with early and established rheumatoid arthritis.

Background The pre-symptomatic stage of Rheumatoid arthritis (RA) is associated with pro-inflammatory cytokines and autoantibodies. High levels and epitope spread by Rheumatoid factors (RhF) and autoantibodies to citrullinated proteins signify progression towards disease expression. In established RA, the persistence of high autoantibody levels reflects production by both long-lived plasma cells and short-lived plasmablasts. Neither the relative contributions to pathogenesis by autoantibodies from either source, nor the factors responsible for deciding the fate of autoantigen specific ‘parent’ B-cells, is understood. Phenotypic markers identifying subsets of autoreactive B-cells are therefore of interest in understanding the origin and perpetuation of the autoimmune response in RA. One such phenotypic marker is the rat monoclonal antibody, 9G4, which recognises an idiotope on immunoglobuins derived from the inherently autoreactive VH-gene, VH4-34. We therefore investigated whether the 9G4 idiotope was expressed on autoantibodies in patients with RA. Methodology/Principal Findings Sera from 19 patients with established RA and those with <1year history of untreated polyarthritis either resolving into RA (n = 42) or non-RA diagnosis (n = 31) were included. Autoantibodies to cyclic citrullinated peptides (CCP), RhF and co-expression of the 9G4 idiotope were measured by ELISA. 9G4 recognised a population of anti-CCP antibodies in the majority of sera from patients with established disease and also in samples from patients with early disaese. 9G4+RhF levels were generally lower and not associated with positivity for, or levels of 9G4+CCP. Conclusions/Significance The persistence of 9G4+ immunoglobulins, of any isotype, in serum is rare. We describe here the novel finding of 9G4 expression on anti-CCP antibodies in patients from the earliest symptoms of RA through to established disease. Our results suggest that 9G4 expression on anti-CCP autoantibodies was not due to polyclonal expansion of VH4-34-encoded immunoglobulins. These studies may therefore provide a new focus for investigation into the evolution of the autoimmune response in RA patients.

B-cell phenotype and IgD-CD27- memory B cells are affected by TNF-inhibitors and tocilizumab treatment in rheumatoid arthritis.

Background The use of TNF-inhibitors and/or the IL-6 receptor antagonist, tocilizumab, in rheumatoid arthritis (RA) have pleiotropic effects that also involve circulating B-cells. The main goal of this study was to assess the effect of TNF-inhibitors and tocilizumab on B-cell phenotype and gene expression in RA. Methods Blood samples were collected from untreated early RA (ERA) patients, established RA patients under methotrexate treatment, established RA patients before and after treatment with TNF-inhibitors and tocilizumab, and healthy donors. B-cell subpopulations were characterized by flow cytometry and B-cell gene expression was analyzed by real-time PCR on isolated B-cells. Serum levels of BAFF, CXCL13 and sCD23 were determined by ELISA. Results The frequency of total CD19+ B cells in circulation was similar between controls and all RA groups, irrespective of treatment, but double negative (DN) IgD-CD27- memory B cells were significantly increased in ERA and established RA when compared to controls. Treatment with TNF-inhibitors and tocilizumab restored the frequency of IgD-CD27- B-cells to normal levels, but did not affect other B cell subpopulations. TACI, CD95, CD5, HLA-DR and TLR9 expression on B-cells significantly increased after treatment with either TNF-inhibitors and/ or tocilizumab, but no significant changes were observed in BAFF-R, BCMA, CD69, CD86, CXCR5, CD23, CD38 and IgM expression on B-cells when comparing baseline with post-treatment follow-ups. Alterations in B-cell gene expression of BAFF-R, TACI, TLR9, FcγRIIB, BCL-2, BLIMP-1 and β2M were found in ERA and established RA patients, but no significant differences were observed after TNF-inhibitors and tocilizumab treatment when comparing baseline and follow-ups. Serum levels of CXCL13, sCD23 and BAFF were not significantly affected by treatment with TNF-inhibitors and tocilizumab. Conclusions In RA patients, the use of TNF-inhibitors and/ or tocilizumab treatment affects B-cell phenotype and IgD-CD27- memory B cells in circulation, but not B-cell gene expression levels.

A1.74 Serum levels of CXCL13 are increased in untreated patients with rheumatoid arthritis from the first weeks of disease development

Background and Objectives CXCL13 (C-X-C motif ligand 13), also known as B-cell chemo-attractant, is an important B-cell chemokine that directly affects the migration and organization of B cells within follicles of lymphoid tissues. CXCL13 plays an important role as a major regulator of B-cell recruitment towards secondary lymphoid organs and inflammatory sites and it has recently been proposed as a serum biomarker of synovitis in patients with rheumatoid arthritis (RA). The aim of this study was to quantify CXCL13 serum levels in untreated very early polyarthritis patients to understand the dynamics of B-cell trafficking since early disease onset. Materials and Methods CXCL13 levels were measured by ELISA in serum samples from untreated very early RA (VERA) with <6 weeks of disease duration, and compared with arthritis due to other conditions of the same duration (non-RA) (VENRA), untreated early RA (ERA) (<1 year), other early arthritis patients (non-RA) (ENRA) (<1 year), established treated RA and healthy controls (HC). Statistical analysis was performed with Mann-Whitney, Kruskal-Wallis and Spearman tests (p < 0.05). Results We found that untreated polyarthritis patients with <6 weeks of disease duration (VERA and VENRA) had significantly higher CXCL13 serum levels in comparison with HC (p = 0.01 and p = 0.02, respectively). ERA and established RA patients also had significantly higher circulating levels of CXCL13 when compared to HC (p = 0.04 and p = 0.003, respectively), but no significant differences were observed in comparison with VERA. Moreover, no significant differences were found in ENRA patients when compared to HC. However, all patients diagnosed with RA (VERA, ERA and established RA) had significantly higher levels of CXCL13 when compared to ENRA (p < 0.05). VENRA also had significantly higher CXCL13 levels when compared to ENRA. Furthermore, no correlation was observed between CXCL13 serum levels with age, DAS28, ESR, VAS, tender and swollen joint counts. Conclusions CXCL13 serum levels were significantly increased in untreated polyarthritis patients who subsequently resolved into a clinical diagnosis of RA. This was found in both early RA cohorts (VERA and ERA). Although VERA and VENRA (<6 weeks of disease duration) had similar CXCL13 levels, only ERA and not ENRA had significantly higher levels suggesting that chronicity was strongly associated with CXCL13 in RA patients. Overall, our results might reflect an interaction between CXCL13-producing cells (activated macrophages, follicular dendritic cells, or Th17-cells) with B-cells in secondary lymphoid tissues such as the spleen, lymph nodes and possibly in inflammatory sites i.e., joints.

A5.13 Effect of Rituximab on B Cell Subpopulations Expressing the 9G4 Idiotype in Patients with Rheumatoid Arthritis

Background and Objectives Antibodies encoded by the VH4–34 gene are inherently autoreactive, binding to red blood cell determinants. An idiotope present on the majority of immunoglobulins derived from the VH4–34 gene can be detected using the rat monoclonal antibody 9G4. 9G4 also allows the detection of B cells expressing B cell receptors derived from VH4–34 gene. We therefore determined the effect of B cell depletion therapy with rituximab (RTX) on B cell subpopulations expressing the 9G4 idiotype in peripheral blood of patients with rheumatoid arthritis (RA) as a means of following the fate of a specific autoreactive B cell population. Materials and Methods B-cell subpopulations were characterised by flow cytometry using combinations of IgD, CD5, CD27 and CD38 in healthy controls (HC) (n = 7), patients with RA before (n = 10) and at clinical relapse after RTX (n = 17). The frequency of each 9G4+ B cell subpopulation was calculated after gating on 9G4+ cells. Results No significant differences were observed in the frequency of total 9G4+ B cells between both patient groups and HC, although the levels of 9G4+ B cells tended to be higher at relapse after RTX treatment. After RTX treatment, repopulating total B cells were predominantly transitional (IgD+CD38++) and naïve (IgD+CD38+), with lower frequencies of most memory B-cell subpopulations. At relapse, RA patients had significantly more 9G4+IgD−CD38− memory B cells compared to HC. This B cell subset was positively correlated with a 9G4+CD5+CD27+ subpopulation. The frequency of 9G4+IgD–CD38+ memory B cells also tended to be increased after RTX compared to HC and pre-RTX. Lower levels of 9G4+IgD+CD38+ naïve, 9G4+IgD+CD38– memory and 9G4+CD5–CD27+ memory B cells were found after RTX treatment compared with HC and pre-RTX. No significant differences were observed in 9G4+IgD+CD38++ transitional, or 9G4+IgD–CD38++ plasmablasts in RA patients before and after RTX treatment in comparison with HC. However, there was a positive correlation between the frequency of 9G4+IgD–CD38++ plasmablasts and time after RTX treatment. Conclusions Alterations in the frequency of memory B cell subpopulations expressing 9G4 idiotype occur in clinically relapsing RA patients after RTX treatment, particularly in IgD–CD38– memory B cell subset. It is possible that this 9G4+ B cell subpopulation escapes B cell depletion therapy, being rescued in niches such as secondary lymphoid organs, or in inflammatory sites. Our results also suggest that the normal tolerance mechanisms preventing 9G4+ B cells from becoming antibody producing cells is defective in patients with RA.

Expression of 9G4 idiotope on autoantibodies to citrullinated peptides in patients with early inflammatory arthritis and established rheumatoid arthritis

The expression of VH4 gene family during immunoglobulin heavy chain synthesis has been detected in autoimmune diseases. In particular, the rat monoclonal antibody 9G4 detects VH4-34-encoded immunoglobulins and B cells expressing these antibodies as surface receptor (autoreactive 9G4+ B cells).