Patients, materials and methods:
In a prospective open label trial patients with active RA qualifying for anti-cytokine therapy were treated either with tocilizumab or etanercept. The primary endpoint was set to 12 weeks with an extension period up to 1 year for tocilizumab treated patients. All patients met the American College of Rheumatology (ACR) criteria for RA[1].
Informed consent was obtained from all patients according to the protocol approved by the ethics committee of the University of Würzburg, Germany. Patients were required to have failed at least two conventional DMARD. Previous anti- TNF treatment was only allowed when stopped at least 6 month before entering the trial. Seven patients at the age of 30-69 years (median 51 years, 4 women, 3 men) were treated with infusions of tocilizumab at a dosage of 8 mg/kg every 4 weeks in combination with ongoing methotrexate (MTX). The median disease duration was 11 years (2-12 years). 6/7 patients had failed to respond to standard DMARDs, one patient also to a TNF-a antagonist (infliximab). 4/7 patients were RF positive and 3/7 patients were anti-CCP positive. All 7 patients were assessed at baseline and week 12. 3 patients recalled their consent for the additional blood samples (60ml) required for the study at later time points. DAS28 declined significantly from 4.6 at baseline to 1.7 at week 12 (p<0.001). In all of these patients DAS28 remission (<2.6) was observed at week 12. The clinical response was maintained up to 1 year. The inflammatory parameters ESR and CRP declined after the first infusion to normal values and stayed negative during the whole subsequent study period. There were no serious adverse events or serious infections during the study.
As a parallel group 4 RA patients treated with etanercept (50 mg s.c weekly) and ongoing MTX were analyzed in a comparable way (2 female, 2 men). The median age of these patients was 60 years (29-68). They had long disease duration of 12.5 (5-19) years. All patients were RF and anti-CCP antibody positive. DAS28 values declined from 3.9 to 1.4 at week 12. The characteristic of all patients are shown in table S1. The observed changes in pre- and post-switch memory B cells (% of lymphocytes) did not reach statistical significance.
Healthy controls
Peripheral blood was taken from 4 healthy volunteers (2 men, 2 women, mean age 41 years, 27-61 years) to sort pre- and post-switch memory B cells.
Single Cell sorting
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus separation and single B cell sorting was performed as described[2]. In detail, B cells were stained for CD19-APC, CD27-PE and IgD-FITC. B cells were gated by forward/ sidescatter and CD19 positivity. Pre-switch memory B cells were defined as CD19+/IgD+/CD27+ and post-switch memory by CD19+/IgD-/CD27+. Plasmablasts were included in the post-switch population. Their number was low and remained constant under treatment with values of around 0.3% of CD19 cells. Individual memory B cells were sorted in a 96 well plate containing lysis buffer by using FACS DiVA (Becton Dickenson, USA). Lysis buffer was comprised of Triton X-100, BSA, oligo (dT)15 primer, dithiothreitol, RNasin, and distilled H2O.
cDNA preparation and nested PCR
A mixture comprising RT-PCR buffer, reverse transcriptase and nucleotides from Titan One Tube RT-PCR System (Roche Diagnostics, Germany) was added into 96 well plate. cDNA synthesis was carried out at 50°C for 1 hour. VH3 and VH4 gene rearrangements were amplified by nested PCRs using family specific primers as previously described [3, 4]. The error rate due to the Taq polymerase used in the amplification process is estimated to be 1 × 10-4 mutations/bp[5]. VH family specific PCR products were separated via electrophoresis and further purified by using MinElute Gel Extraction kit (Qiagen, Hilden, Germany).
Sequencing and analysis
Gel extracted products of Ig-VH3/VH4 were further amplified by using BigDye Terminator Cycle Sequencing Ready Reaction kit followed by sequencing in genetic analyser ABI PRISM 310 (Applied Biosystems). Sequences were analyzed by matching their closest germline counterparts using the online programme JOINSOLVER [6].
Statistical analysis
Statistical analysis was performed by using GraphPad Prism 3.03 (GraphPad Software, San Diego, USA). Non-parametric Mann-Whitney U test and Chi square test were employed for statistical analysis. P values ≤0.05 were considered to be significant.
References
[1] Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988 Mar;31(3):315-24.
[2] Muhammad K, Roll P, Einsele H, Dorner T, Tony HP. Delayed acquisition of somatic hypermutations in repopulated IGD+CD27+ memory B cell receptors after rituximab treatment. Arthritis Rheum. 2009 Aug;60(8):2284-93.
[3] Ruzickova S, Pruss A, Odendahl M, Wolbart K, Burmester GR, Scholze J, et al. Chronic lymphocytic leukemia preceded by cold agglutinin disease: intraclonal immunoglobulin light-chain diversity in V(H)4-34 expressing single leukemic B cells. Blood. 2002 Nov 1;100(9):3419-22.
[4] Brezinschek HP, Brezinschek RI, Lipsky PE. Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction. J Immunol. 1995 Jul 1;155(1):190-202.
[5] Dorner T, Brezinschek HP, Brezinschek RI, Foster SJ, Domiati-Saad R, Lipsky PE. Analysis of the frequency and pattern of somatic mutations within nonproductively rearranged human variable heavy chain genes. J Immunol. 1997 Mar 15;158(6):2779-89.
[6] Souto-Carneiro MM, Longo NS, Russ DE, Sun HW, Lipsky PE. Characterization of the human Ig heavy chain antigen binding complementarity determining region 3 using a newly developed software algorithm, JOINSOLVER. J Immunol. 2004 Jun 1;172(11):6790-802.
Table:
Table S.1: Laboratory and clinical parameters of the patients treated with tocilizumab or etanercept (control group). Shown are median values ± SEM. B cell subsets were analysed by flow cytometry. Results are shown as % CD19+ lymphocytes.
DAS28 / CRP(mg/dl) / ESR mm/1h / CD27+IgD+ (%) / CD27+IgD-(%)tocilizumab
baseline n=7 / 4.6±0.4 / 0.5±0.6 / 12±8.7 / 15.6± 1.3 / 20.0± 2.2
week 12 n=7 / 1.7±0.4 / 0.07±0.009 / 2±0.6 / 14.2±1.3 / 17.4±1.6
week 24 n=4
one year n=3 / 2.2±0.2
1.5±0.4 / 0.06±0.2
0.4±0.30 / 4±7
5±3.3 / 12.2±1.6
10±0.5 / 16.0±2.8
16.0±1.0
etanercept
baseline n=4 / 3.9±0.8 / 0.5±1.5 / 13±3.9 / 14.3±1.4 / 22.5±1.8
week 12 n=4 / 1.4±0.2 / 0.2±0.008 / 5±1.4 / 12.2±2.4 / 18.0±2.1
One year n=4 / 1..9±0.4 / 0.2±0.07 / 9±2.3 / 15.5±1.19 / 20.5±1.3
Supplementary Figures:
A) B)
Figure S1: Mutational frequency of pre- (A) and post-switch (B) memory B cells in RA patients at baseline (BL) is comparable with normal donors (ND). A) Mean mutational frequencies of pre-switch memory B cells are 4.5±0.3, 4.1±0.2 and 4.3±0.3 in ND, BL of tocilizumab and BL of etanercept treated patients. B) Mean mutational frequencies of post-switch memory B cells are 6.1±0.3, 6.0±0.3 and 6.5±0.2 in ND, BL of tocilizumab and BL of etanercept treated patients. (n=number of individuals S=number of analyzed sequences).
Figure S2: Mutational frequencies of pre-switch memory B cells in individual patients under tocilizumab therapy.
Figure S3: Distribution of mutations, classified as unmutated (NMUT), low (LMUT) and high mutated (HMUT) sequences within pre-switch memory B cells. Analyses at different time points during tocilizumab treatment (baseline, week12, 24 and one year) indicate a reduction of highly mutated sequences and increase of unmutated and low mutated sequences.
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