Study Population. Patients with RA who agreed to enter a Phase IIIb study of tocilizumab entitled, “An open-label, randomized study to evaluate the safety, tolerability and efficacy of tocilizumab (TCZ) monotherapy or TCZ in combination with non-biologic disease modifying antirheumatic drugs (DMARDs) in patients with active rheumatoid arthritis who have an inadequate response to current non-biologic or biologic DMARDs (ACT-STAR)” were offered the opportunity to enter a separate laboratory-based research study to investigate the mechanism of action of tocilizumab in RA. The laboratory study was entitled “IL-6 and IL-21 in rheumatoid arthritis.” Patients who agreed to enter the laboratory study signed a separate consent form that was approved by the Institutional Review Board of The University of Vermont.
Peripheral blood was obtained from 8 patients with RA who participated in the ACTSTAR protocol. Patients enrolled in the study were adult men and women with moderate to severe disease activity of RA, who were currently experiencing an inadequate clinical response or experiencing safety or tolerability related issues to stable doses of non-biological or biological DMARD therapy. Patients had to be ≥18 years of age, with moderate-to-severe RA of ≥6 months' duration, with a swollen joint count (SJC) of ≥4 and a tender joint count (TJC) of ≥4, Eligible patients receiving permitted DMARDs (methotrexate, hydroxychloroquine, sulfasalazine, azathioprine, and leflunomide) must have received stable doses for ≥7 weeks prior to study entry. Oral glucocorticoids (≤10 mg/day prednisone or equivalent) and nonsteroidal antiinflammatory drugs (NSAIDs)/cyclooxygenase 2 inhibitors were permitted but dose changes were not allowed. Patients were excluded if treated with rituximab within 6 months before screening. In patients who were receiving biologic drugs, these were discontinued prior to study entry, but traditional DMARDs were continued. After the initial screening visit, the 8 patients enrolled in the laboratory study were randomized in a 1:1 ratio to receive monthly infusions of either 4 mg/kg or 8 mg/kg tocilizumab over 6 months. There were 7 women and 1 man, with ages ranging from 32-75 years, and disease duration from 3-19 years. Two patients had never received a biologic drug, while the other 6 patients either had an inadequate response or intolerance to one or more biologic drug. All 8 patients were receiving one or more oral DMARD (methotrexate, sulfasalazine or hydroxychloroquine). Five patients were randomized to tocilizumab 8 mg /kg. Two of the three patients randomized to 4 mg/kg had the dose of tocilizumab increased to 8 mg/kg at week 8 because they had less than 20 % improvement in joint counts at that time point.
Whole heparinized blood and serum samples were obtained at baseline prior to the first infusion (0 month), and 1 month (prior to the second infusion), 3 months (prior to the fourth infusion), and 6 months (prior to the last infusion). Tender and swollen joint counts were obtained at the same time points. Heparin containing blood samples were used for the isolation of peripheral blood mononuclear cells, and a gelatin-containing tube was used to obtain serum. The levels of rheumatoid factor (RF), anti-cyclic citrullinated peptide (CCP), and anti-nuclear antibody (ANA) were determined at the same time points by the clinical laboratory of Fletcher Allen Health Care.
Cell purification and culture. Mononuclear cells from peripheral blood (PBMC) were isolated by Ficoll density-gradient centrifugation (Histopaque). CD4 T cells were purified by positive selection using the CD4 MACS kit (Miltenyi Biotec) as recommended by the manufacturer. CD4 T cells were then sorted into CD4 CD45 RO+ and CD4 CD45RA+ cells after releasing anti-CD4 Ab from the cells using the CD45RA MACS kit (containing the “Release” reagent) as recommended by the manufacturer. Purity of the cells in both populations (>95%) was determined by cell surface staining and FACS as described below.
CD4 CD45RA and CD4 CD45RO cells were activated (106
cells/ml in RPMI with 5% fetal bovine serum) with immobilized anti-CD3 antibody (Ab) (3 μg/ml) and soluble anti-CD28 Ab (BD Bioscience) (1 μg/ml) as previously described 30
. Supernatants were collected after 24 h (CD4 CD45RO cells) or 48 h (CD4 CD45RA cells) and used for cytokine detection as described below.
B cells were obtained from peripheral blood of healthy volunteers by CD22 MACS microbead magnetic column selection (Miltenyi Biotec). Purity was > 97% and was determined by flow cytometry by staining for CD19 and CD20. We co-cultured B cells (2 - 2.5 × 105 cells/ml) on irradiated (50 Gray) CD40L-expressing L cell fibroblasts (5 × 104 cells/ml, > 96% surface CD154+) in 1 ml Iscove's Modified Eagle's Medium containing 8% fetal bovine serum and penicilin/streptomycin and with or without rhIL-4 (25 ng/ml, Peprotech) or rhIL-21 (10 ng/ml, Peprotech) in 24-well plates. After 6 days, we harvested supernatants and the levels of IgG4 in the supernatant were determined as described below.
Flow cytometry analysis. PBMC cells were stained with an anti-CD14 (monocytes), anti-CD19 (B cells) and anti-CD4, anti-CD45RA and anti-CD45RO Abs (Caltag). Stained samples were examined by flow cytometry using the LSRII flow cytometer (BD Bioscience).
Cytokine production and serum antibody analysis.
The levels of IL-2, IL-17, IL-21, IL-4 and IFNγ in the culture supernatants were determined using the Human Cytokine/Chemokine Milliplex MAP Kits following the recommendations of the manufacturer (Millipore Co.) and using the fluorescence plate reader and analysis software (BioRad). For analysis of IL-21 gene expression, total RNA was extracted from 4 x 105
freshly isolated CD4 CD45RO cells using the RNAeasy kit (Qiagen) as recommended by the manufacturer. First-strand cDNA synthesis was performed as previously described 30
. Quantitative RT-PCR was performed on cDNA using Assay on Demand probe/primer sets for IL-21 and HPRT (Applied Biosystems). Gene amplification was performed on an ABIPrism® 7700 instrument from Applied Biosystems. Expression of IL-21 was normalized to HPRT levels. Relative values were determined by the comparative Ct method. Patient 1 and 8 were not included in the analyses due to insufficient amount of RNA.
The levels of serum IgG1, IgG2, IgG3, IgG4, IgM and IgA were determined using the Human Immunoglobulin Isotype Milliplex MAP Kit following the recommendations by the manufacturer (Millipore). The levels of IgG4- and IgG1-specific anti-CCP Abs were determined using the QUANTA LiteTM CCP 3.1 ELISA kit (INOVA Diagnostic Inc.) containing a CCP-coated plate, but instead of using the horseperoxidase (HRP)-conjugated anti-human IgG/IgA Abs provided with the kit, we used a HRP-conjugated anti-human IgG4 (Invitrogen) or anti-human IgG1 (Invitrogen) Abs. Assay was performed as recommended by the manufacturer (INOVA Diagnostic Inc). Plates were read at 450 nm using an ELISA plate reader.
Statistical Analysis. Single-group repeated measures analysis of variance based on ranked data was used to determine differences over time of treatment in the various parameters examined. Repeated measures analysis of variance was also used to examine within-subject differences in fold reduction between IgG1 and IgG4, followed by tests of simple effects for examination of fold reduction over time of IgG1 and IgG4 separately. All analyses were performed using SAS 9.2.