Non-response to rituximab therapy in rheumatoid arthritis is associated with incomplete disruption of the B cell receptor repertoire.

OBJECTIVE: To gain more insight into the dynamics of lymphocyte depletion and develop new predictors of clinical response to rituximab in rheumatoid arthritis (RA). METHODS: RNA-based next-generation sequencing was used to analyse the B cell receptor (BCR) repertoire in peripheral blood and synovial tissue samples collected from 24 seropositive patients with RA treated with rituximab. Clonal expansion, mutation load and clonal overlap were assessed in samples collected before, at week 4 and at week 16 or 24 after treatment and correlated to the patients' clinical response. RESULTS: After 4 weeks of rituximab-induced B cell depletion, the peripheral blood BCR repertoire of treated patients consisted of fewer, more dominant and more mutated BCR clones. No significant changes in the synovial tissue BCR repertoire were detected until week 16 post-treatment, when a reduced clonal overlap with baseline and an increased mutation load were observed. In patients who were non-responders at month 3 (n=5) using the European League Against Rheumatism response criteria, peripheral blood samples taken at week 4 after rituximab treatment showed more dominant clones compared with moderate responders (n=9) (median (IQR): 36 (27-52) vs 18 (16-26); p<0.01) and more clonal overlap with the baseline (median (IQR): 5% (2%-20%) vs 0% (0%-0%); p≤0.01). CONCLUSION: Significant changes in BCR clonality are observed in peripheral blood of patients 4 weeks after rituximab treatment, while changes in synovial tissue were observed at later time points. Incomplete depletion of the dominant baseline peripheral blood BCR repertoire in the first month of treatment might predict clinical non-response at 3 months.

Effect of rituximab treatment on T and B cell subsets in lymph node biopsies of patients with rheumatoid arthritis.

OBJECTIVES: The exact underlying mechanism of rituximab treatment in patients with RA is poorly defined and knowledge about the effect of B cell depletion on immune cells in secondary lymphoid organs is lacking. We analysed lymphoid tissue responses to rituximab in RA patients. METHODS: Fourteen RA patients received 2 × 1000 mg rituximab intravenously, and lymph node (LN) biopsies were obtained before and 4 weeks after the first infusion. Tissues were examined by flow cytometry, immunohistochemistry and quantitative PCR. LN biopsies from five healthy individuals (HC) served as controls. RESULTS: LN biopsies of RA patients showed increased frequencies of CD21+CD23+IgDhighIgMvariable follicular B cells and CD3+CD25+CD69+ early activated, tissue resident T cells when compared with HCs. After treatment, there was incomplete depletion of LN B cells. There was a significant decrease in CD27-IgD+ naïve B cells, and CD27+IgD+ unswitched memory B cells including the CD27+IgD+IgM+ subset and follicular B cells. Strikingly, CD27+IgD- switched memory B cells persisted in LN biopsies after rituximab treatment. In the T cell compartment, a significant decrease was observed in the frequency of early activated, tissue resident T cells after rituximab treatment, but late activated T cells persisted. B cell proliferation inducing cytokine IL-21 was higher expressed in LN biopsies of RA patients compared with HC and expression was not affected by rituximab treatment. CONCLUSION: Rituximab does not cure RA, possibly due to persistence of switched memory B cells in lymphoid tissues suggesting that factors promoting B cell survival and differentiation need to be additionally targeted.

Brief report: a phase IIa, randomized, double-blind, placebo-controlled trial of apilimod mesylate, an interleukin-12/interleukin-23 inhibitor, in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the safety, tolerability, pharmacokinetics, and efficacy of apilimod mesylate, an oral interleukin-12 (IL-12)/IL-23 inhibitor, in patients with rheumatoid arthritis (RA). METHODS: We performed a phase IIa, randomized, double-blind, placebo-controlled proof-of-concept study of apilimod, in combination with methotrexate, in 29 patients with active RA (3:1 ratio of apilimod-treated to placebo-treated patients) in 3 stages. Patients received apilimod 100 mg/day or placebo for 4 weeks (stage 1) or 8 weeks (stage 2). In stage 3, patients received apilimod 100 mg twice a day or placebo for 8 weeks, with an optional extension of 4 weeks. Clinical response (Disease Activity Score in 28 joints [DAS28] and American College of Rheumatology [ACR] criteria) was assessed throughout; synovial tissue samples collected at baseline and on day 29 (stages 1 and 2) or day 57 (stage 3) were stained for cellular markers and cytokines for immunohistochemistry analysis. RESULTS: While only mild adverse events were observed in stages 1 and 2, in stage 3, all patients experienced headache and/or nausea. Among apilimod-treated patients (100 mg/day), there was a small, but significant, reduction in the DAS28 on day 29 and day 57 compared with baseline. ACR20 response was reached in only 6% of patients on day 29 and 25% of patients on day 57, similar to the percentage of responders in the placebo group. Increasing the dosage (100 mg twice a day) did not improve clinical efficacy. Consistent with clinical results, apilimod did not have an effect on expression of synovial biomarkers. Of importance, we also did not observe an effect of apilimod on synovial IL-12 and IL-23 expression. CONCLUSION: Our results do not support the notion that IL-12/IL-23 inhibition by apilimod is able to induce robust clinical improvement in RA.

Rituximab abrogates joint destruction in rheumatoid arthritis by inhibiting osteoclastogenesis.

OBJECTIVES: To examine how rituximab may result in the inhibition of joint destruction in rheumatoid arthritis (RA) patients. METHODS: Twenty-eight patients with active RA were treated with rituximab. Radiographs of hands and feet before and 1 year after therapy were assessed using the Sharp-van der Heijde score (SHS). Expression of bone destruction markers was evaluated by immunohistochemistry and immunofluorescence of synovial biopsies obtained before and 16 weeks after the initiation of treatment. Serum levels of osteoprotegerin, receptor activator of nuclear factor κB ligand (RANKL), osteocalcin and cross-linked N-telopeptides of type I collagen (NTx) were measured by ELISA before and 16 weeks post-treatment. RESULTS: After 1 year, the mean (SD) change in total SHS was 1.4 (10.0). Sixteen weeks after treatment there was a decrease of 99% in receptor activator of nuclear factor κB-positive osteoclast precursors (p=0.02) and a decrease of 37% (p=0.016) in RANKL expression in the synovium and a trend towards reduced synovial osteoprotegerin expression (25%, p=0.07). In serum, both osteoprotegerin (20%, p=0.001) and RANKL (40%, p<0.0001) levels were significantly reduced 16 weeks after treatment, but the osteoprotegerin/RANKL ratio increased (157%, p=0.006). A trend was found towards an increase of osteocalcin levels (p=0.053), while NTx concentrations did not change. CONCLUSIONS: Rituximab treatment is associated with a decrease in synovial osteoclast precursors and RANKL expression and an increase in the osteoprotegerin/RANKL ratio in serum. These observations may partly explain the protective effect of rituximab on the progression of joint destruction in RA.

Presence of lymphocyte aggregates in the synovium of patients with early arthritis in relationship to diagnosis and outcome: is it a constant feature over time?

OBJECTIVES: To evaluate the presence of lymphocyte aggregates in synovial tissue of patients with early arthritis in relationship to clinical outcome and to determine whether this is a stable feature over time. METHODS: Arthroscopic synovial biopsy samples were collected in a prospective cohort of disease-modifying antirheumatic drug-naïve patients with early arthritis (<1 year's disease duration) at baseline (n=93) and, if rheumatoid arthritis was suspected, after 6 months of follow-up (n=17). After 2 years of follow-up, definitive diagnosis and clinical outcome were assessed. Size of synovial lymphocyte aggregates was graded (score 1-3). Lymphoid neogenesis (LN) was defined by the presence of grade ≥2 aggregates and subclassified based on the presence of follicular dendritic cells (FDCs). RESULTS: LN was present in 36% of all patients and FDCs in 15% of patients with LN. Presence of lymphocyte aggregates differed over time. LN was associated with the degree of synovial inflammation. There was no relationship between the presence of lymphocyte aggregates at baseline and definitive diagnosis or clinical outcome after follow-up. CONCLUSIONS: Presence of lymphocyte aggregates is a dynamic phenomenon related to the degree of synovitis and can be detected in different forms of early arthritis. This feature does not appear to be related to clinical outcome.