Download Blood memory B cells are disturbed and predict the

ARTHRITIS & RHEUMATISM
Vol. 63, No. 12, December 2011, pp 3692–3701
DOI 10.1002/art.30599
© 2011, American College of Rheumatology
Blood Memory B Cells Are Disturbed and Predict the
Response to Rituximab in Patients With Rheumatoid Arthritis
Jérémie Sellam,1 Stéphanie Rouanet,2 Houria Hendel-Chavez,3 Karim Abbed,3
Jean Sibilia,4 Jacques Tebib,5 Xavier Le Loët,6 Bernard Combe,7 Maxime Dougados,8
Xavier Mariette,1 and Yassine Taoufik3
Objective. To examine blood B cell subsets in
patients with rheumatoid arthritis (RA) prior to B cell
depletion therapy and to assess their potential as predictors of clinical response to rituximab (RTX).
Methods. Blood B cell subsets were assessed by
flow cytometry in 208 RA patients included in an RTX
retreatment study (assessed prior to RTX treatment)
and in 47 age-matched controls. Expression of BAFF
receptor (BAFF-R) on B cells and serum B cell biomarkers was also measured. B cell subsets and BAFF-R
expression were compared between RA patient and
control populations. Univariate and multivariate analyses were performed to identify baseline factors associated with a European League Against Rheumatism
response 24 weeks after 1 cycle of RTX.
Results. Mean ⴞ SD counts of both CD27ⴚ naive
and CD27ⴙ memory B cells were decreased in RA
patients (188.6 ⴞ 121.4/mm3) compared with controls
(257.3 ⴞ 154.1/mm3) (P ⴝ 0.001) and were partially
restored in patients treated with methotrexate (MTX)
plus anti–tumor necrosis factor compared with patients
treated with MTX alone. Within the CD27ⴙ memory B
cells, the CD27ⴙIgDⴚ switched memory subtype was
selectively decreased, irrespective of treatment. The
frequency of CD27ⴙ memory B cells correlated inversely with levels of several B cell activation biomarkers in RA. Serum BAFF level and BAFF-R expression
was comparable in RA patients and controls. A low
baseline CD27ⴙ memory B cell frequency was associated with a greater clinical response to RTX (odds ratio
0.97 [95% confidence interval 0.95–0.99], P ⴝ 0.0015).
Conclusion. In B cell depletion therapy–naive RA
patients, a low frequency of CD27ⴙ memory B cells
correlated with levels of serum B cell activation biomarkers and may predict response to RTX. These
results suggest that low memory B cell frequency may be
indicative of a B cell–driven RA subtype that is more
sensitive to B cell depletion therapy.
ClinicalTrials.gov identifier: NCT01126541.
Supported by Roche France.
1
Jérémie Sellam, MD, PhD (current address: Hôpital SaintAntoine, AP-HP, and Université Pierre et Marie Curie Paris 6, Paris,
France), Xavier Mariette, MD, PhD: Hôpital Bicêtre, AP-HP,
INSERM U1012, and Université Paris-Sud 11, Le Kremlin Bicêtre,
France; 2Stéphanie Rouanet, PhD: Roche, Neuilly/Seine, France;
3
Houria Hendel-Chavez, PhD, Karim Abbed, MD, Yassine Taoufik,
PhD: Hôpital Bicêtre, AP-HP, and Université Paris-Sud 11, Le
Kremlin Bicêtre, France; 4Jean Sibilia, MD, PhD: EA 3432, Hôpitaux
Universitaires de Strasbourg and Université de Strasbourg, Strasbourg, France; 5Jacques Tebib, MD, PhD: Centre Hospitalier LyonSud, Pierre-Bénite, France; 6Xavier Le Loët, MD, PhD: Rouen
University Hospital and INSERM U905, Rouen, France; 7Bernard
Combe, MD, PhD: Lapeyronie University Hospital, Montpellier I
University, and UMR5535, Montpellier, France; 8Maxime Dougados,
MD: Université Paris Descartes, UPRES-EA 4058, and Hôpital
Cochin, AP-HP, Paris, France.
Drs. Mariette and Taoufik contributed equally to this work.
Drs. Sellam, Sibilia, and Mariette have received consulting
fees, speaking fees, and/or honoraria from Roche (less than $10,000
each). Drs. Sibilia, Tebib, Le Loët, Combe, Dougados, and Mariette
have received honoraria from Roche for serving on the scientific
committee of the SMART study (less than $10,000 each). Dr. Le Loët
has received consulting fees, speaking fees, and/or honoraria from
Abbott, Pfizer, Roche, Sanofi-Aventis, and Schering-Plough (less than
$10,000 each). Dr. Combe has received consulting fees, speaking fees,
and/or honoraria from Roche, Wyeth-Pfizer, Schering-Plough, and
UCB (less than $10,000 each). Dr. Dougados has received research
grants and honoraria from Roche to conduct studies and/or to
participate at symposia organized by Roche (less than $10,000 each).
Address correspondence to Jérémie Sellam, MD, PhD, Service de Rhumatologie, Hôpital Saint-Antoine, 184 Rue du Faubourg
Saint-Antoine, 75012 Paris, France (e-mail: jeremie.
[email protected]); or to Xavier Mariette, MD, PhD, Service de
Rhumatologie, Hôpital de Bicêtre, 78 Rue du Général Leclerc, 94275
Le Kremlin Bicêtre, France (e-mail: [email protected]).
Submitted for publication January 19, 2011; accepted in
revised form August 3, 2011.
In rheumatoid arthritis (RA), B cells can play a
number of roles critical for inducing or maintaining
autoimmune inflammation (1). Accumulating evidence
points to the disruption of B cell–regulated processes in
the pathogenesis of RA and other autoimmune disorders, and provides the rationale for B cell depletion
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MEMORY B CELLS IN RA AND CLINICAL RESPONSE TO RITUXIMAB
therapy in RA. Rituximab (RTX), a chimeric anti-CD20
monoclonal antibody effective for reducing the clinical
signs and symptoms of RA and inhibiting the progression of joint damage in patients with RA, markedly
depletes peripheral and, to a lesser extent, tissue B cells
(1,2).
As RTX specifically targets B cells, the monitoring of blood B cell subsets before and after B cell
depletion therapy may be valuable in the management of
RA before and during treatment. Recently, it has been
suggested that decreases in preplasma cells and memory
B cells might be associated with a better response to
RTX and with a late relapse in responders, respectively
(3–5). However, in contrast to studies of other systemic
autoimmune diseases such as Sjögren’s syndrome or
systemic lupus erythematosus (6–9) and despite several
studies focusing on the modification of peripheral blood
B cell homeostasis induced by RTX (3,4,10,11), few
studies have explored disturbances of peripheral B cell
subsets in RA patients compared with controls and the
potential influence of non–B cell depletion therapies
such as tumor necrosis factor (TNF) blockers or methotrexate (MTX) in RA patients (12–14).
BAFF, a cytokine secreted predominantly by
myeloid cells but also by synoviocytes, is involved in B
cell activation and survival in general (15–17) and in RA
(18,19). BAFF binds to B cells expressing B lymphocyte
stimulator receptor 3 (also called BAFF receptor
[BAFF-R]) and mediates a survival signal. An increase
in serum BAFF, together with a decrease in BAFF-R
expression, has already been observed in other autoimmune diseases, leading to the hypothesis that changes
in components of the BAFF/BAFF-R system could be
implicated in RA and be associated with the clinical
outcome after RTX treatment (20–23).
Thus, we aimed to investigate 1) the distribution
of peripheral blood B cell subsets in RA before B cell
depletion therapy and the influence of MTX or an
anti-TNF agent on absolute lymphocyte counts and
relative frequencies of B cell subsets, and 2) the predictive value of baseline blood B cell subset on the clinical
response after 1 course of RTX in patients with refractory RA.
PATIENTS AND METHODS
Patients. A total of 224 patients diagnosed as having
RA for at least 6 months and fulfilling the 1987 revised criteria
of the American College of Rheumatology (24) were included
in the SMART (eSsai MAbthera sur la dose de ReTraitement) study (NCT01126541). This is a 2-year national,
multicenter, randomized, open-label study evaluating the efficacy and tolerability of 2 doses of RTX for retreatment
following 1 initial course of RTX at the licensed dose (1,000
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mg on days 1 and 15). The biologic ancillary study presented
here includes 208 of the 224 patients and only concerns the
first 6 months after this first cycle and thus does not consider
the retreatment phase. A list of the SMART investigators is
provided in Appendix A.
All patients had active disease, as defined by a Disease
Activity Score in 28 joints (25) using C-reactive protein
(DAS28-CRP) of ⬎3.2 and ⱖ6 swelling joints (of 66) and ⱖ6
tender joints (of 68), or a CRP level of ⱖ10 mg/liter, or an
erythrocyte sedimentation rate (ESR) of ⱖ28 mm/hour. Each
patient was receiving a stable dose of MTX (ⱖ10 mg/week for
at least 4 weeks) and either had experienced an inadequate
response or intolerance to anti-TNF agents or had had contraindications to anti-TNF therapy. Patients previously receiving etanercept were required to have discontinued therapy for
at least 4 weeks; patients previously receiving infliximab or
adalimumab were required to have discontinued therapy for at
least 8 weeks. Steroids (ⱕ10 mg/day prednisone or equivalent)
and nonsteroidal antiinflammatory drugs were permitted if
their doses had remained stable for at least 4 weeks and 2
weeks, respectively, before enrollment.
Cross-sectional study comparing RA patients before B
cell depletion therapy and age-matched controls. Prior to
initiation of B cell depletion therapy, we compared peripheral
blood B cell subsets from the 208 RA patients receiving
anti-TNF and/or MTX (of the 224 included in the SMART
study) with those from 47 age-matched controls who were
hospitalized for degenerative rheumatic diseases and had not
been diagnosed as having neoplasia, active infections, or
inflammatory diseases. In this part of the study, RA patients
were stratified according to the length of time since their last
dose of anti-TNF. Patients taking MTX who received their last
administration of anti-TNF within 6 months of inclusion were
included in the anti-TNF plus MTX group. Patients taking
MTX who received their last anti-TNF injection more than 6
months prior to study initiation, or who had never received
anti-TNF because of a contraindication, were included in the
MTX group.
Longitudinal SMART study protocol. All RA patients
included in the SMART study received 1 course of RTX (a
1,000 mg infusion on days 1 and 15). Intravenous methylprednisolone (100 mg before each infusion) and 1,000 mg of
acetaminophen and 100 mg of diphendramine hydrochloride
(antihistamine) were also administered prior to each RTX
infusion. Treatment efficacy was evaluated 24 weeks after the
first RTX infusion according to European League Against
Rheumatism (EULAR) response criteria (26,27). This initial
response was used to classify patients as responders (good or
moderate) or nonresponders. Responders were subsequently
randomly assigned to receive retreatment with 1 of 2 RTX
doses (one or two 1,000 mg infusions), although we do not
report the data from this part of the clinical study, which
corresponds to the main part of the SMART study
(NCT01126541). In this biologic ancillary study, we investigated whether baseline repartition of peripheral blood B cell
subpopulations before the first cycle of RTX treatment predicts clinical response to this therapy at 24 weeks. This study
was approved by the local ethics committee (Groupe Hospitalier Pitié-Salpêtrière). All patients and controls provided informed consent for the entire trial.
Assessment of blood lymphocyte subsets. A peripheral
blood sample was obtained from all patients and controls at
3694
SELLAM ET AL
Figure 1. Representative flow cytometry studies of B cell subsets according to CD27/IgD classification, plasmablast identification, and expression
of BAFF receptor (BAFF-R). A, Frequency of CD19⫹ B cells in the lymphocyte gate. B, Frequency of memory CD27⫹ B cells in the CD19⫹ B
cell population. C, Frequency of IgD⫺CD38⫹⫹ plasmablasts in a large lymphocyte gate (upper left in the dot plot). D, Frequency of CD19⫹ B cell
subsets according to CD27/IgD classification. E, BAFF-R expression expressed as mean fluorescence intensity (MFI) in each B cell subset. The
numbers in each quadrant of dot plots or on the histograms represent the percentages of each subpopulation (A–D) or the value of BAFF-R MFI
(E).
enrollment for assessment of lymphocytes and B cell subsets
using flow cytometry. For this purpose, 4-color staining was
performed, and samples were analyzed using FACSCalibur
and FACSCanto II cytometers (Becton Dickinson). Lympho-
cyte subsets (CD3⫹ T cells, CD19⫹ B cells, and CD16⫹
and/or CD56⫹ natural killer [NK] cells) were enumerated
using the multitest reagent (Becton Dickinson). B cell subsets
were analyzed based on the presence of CD19, CD38, CD27,
MEMORY B CELLS IN RA AND CLINICAL RESPONSE TO RITUXIMAB
and IgD (Becton Dickinson). Likewise, we characterized B cell
subsets using the following IgD/CD27 classification (28–31):
memory CD19⫹CD27⫹ cells split into CD19⫹CD27⫹IgD⫺
switched memory B cells and CD19⫹CD27⫹IgD⫹ nonswitched memory B cells (also called “marginal zone–like” B
cells), naive CD27⫺IgD⫹ cells, and the recently characterized
CD27⫺IgD⫺ memory B cell subset. CD38 and IgD staining
was also used to evaluate CD38⫹⫹IgD⫺ plasmablasts, using
a wider gate for CD45⫹ cells exclusively in the RA patient
population for the longitudinal study (32,33). The expression
of BAFF-R in each CD27/IgD B cell subset was measured as
mean fluorescence intensity. A representative example is
shown in Figure 1.
Serum biomarker assessment. Serum samples obtained before the first RTX infusion were analyzed for several
biomarkers of B cell activation to identify potential factors
predictive of response to RTX (34). Specifically, we assessed
rheumatoid factor (RF) by nephelometry (BN Prospec; DadeBehring) and anti–cyclic citrullinated peptide (anti-CCP) IgG
antibodies by enzyme-linked immunosorbent assay (ELISA)
(DiaSorin). The cutoff values for positivity were 15 IU/liter for
RF and 25 IU/liter for anti-CCP antibodies. BAFF was measured by Quantikine ELISA (R&D Systems). Serum concentrations of IgG, IgA, and IgM (BN Prospec; Dade-Behring)
and free light chains (Freelite; The Binding Site) were assessed
by nephelometry. We also measured serum BAFF concentrations in samples from the control population to enable comparisons with the samples from RA patients.
Statistical analysis. Statistical analyses were performed using SAS software, version 9.1. All tests were 2-sided,
and the Type I error was set at 0.05. Continuous data are
described as the mean ⫾ SD or the median and range.
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To compare B cell subsets and BAFF-R expression in
samples from RA patients and controls at baseline, we used
Student’s t-test or analysis of variance for continuous data and
the chi-square test or Fisher’s exact test for qualitative variables. The correlation between B cell subsets and B cell
biomarkers or disease activity was assessed using Spearman’s
correlation coefficient.
For the longitudinal study investigating baseline factors predictive of response to RTX, the relationship between
EULAR response at 24 weeks and explanatory variables was
analyzed by logistic regression. The variables were selected
by univariate logistic regression within the frequencies of
peripheral blood B cell subsets and BAFF-R expression at
baseline. Variables significant after univariate regression
(P ⱕ 0.15) were then entered into a stepwise multivariate
model adjusted for the DAS28-CRP. Results are expressed as
the odds ratio (OR) and 95% confidence interval (95% CI).
P values less than 0.05 were considered significant. All analyses
were performed on the 208 patients who received at least
1 RTX infusion, had an available measure of EULAR response at week 24, and had available results of B cell subset
investigations.
RESULTS
Characteristics of the study population. Baseline
characteristics of the 208 RA patients (all patients,
anti-TNF plus MTX group, and MTX group) and 47
controls are reported in Table 1. Of the 66 patients in
Table 1. Baseline characteristics of the RA patients and healthy controls*
Age, mean ⫾ SD years
Women, no. (%)
Disease duration,
mean ⫾ SD years
DAS28-CRP, mean ⫾ SD
HAQ DI score, mean ⫾ SD
Prednisone ⬍10 mg/day,
no. (%)
MTX, mean ⫾ SD mg/week
Monoclonal antibody/etan.,
no. (%)
RF, no. (%), median (range)
IU/liter
Anti-CCP, no. (%), median
(range) IU/liter
CRP level, median (range)
mg/liter
Radiographic erosions, no. (%)
ESR, median (range) mm/hour
MTX duration, mean ⫾ SD
years
Time since last anti-TNF,
median (range) months
Controls
(n ⫽ 47)
RA patients
(n ⫽ 208)
Anti-TNF ⫹ MTX group
(n ⫽ 142)
MTX group
(n ⫽ 66)
58 ⫾ 15
29 (62)
NA
56 ⫾ 11
176 (85)
13 ⫾ 9
55 ⫾ 12
120 (85)
12 ⫾ 9
58 ⫾ 10
56 (85)
16 ⫾ 9
NA
NA
NA
5.8 ⫾ 0.9
1.8 ⫾ 0.6
165 (79)
5.8 ⫾ 0.9
1.7 ⫾ 0.6
110 (78)
5.8 ⫾ 0.9
1.9 ⫾ 0.6
55 (83)
NA
NA
14.1 ⫾ 3.7
NA
14.1 ⫾ 3.6
95 (67)/47 (33)
14.1 ⫾ 4.0
NA
NA
136 (65), 98.1 (17.0–2,430.0)
90 (63), 97.2 (17.5–1,460.0)
46 (70), 101.5 (17.0–2,430.0)
NA
159 (76), 740.0 (26.0–17,194.0)
103 (73), 695.0 (26.0–17,194.0)
56 (85), 839.0 (41.0–13,447.0)
2.0 (1.0–28.0)
11.0 (0.3–139.0)
13.3 (0.4–139.0)
9.4 (0.3–89.1)
NA
12.0 (2.0–82.0)
NA
184 (89)
28.0 (2.0–124.0)
5.1 ⫾ 4.7
122 (86)
27.5 (4.0–124.0)
4.8 ⫾ 4.3
62 (94)
28.0 (2.0–102.0)
5.6 ⫾ 5.3
NA
2.7 (0.6–73.6)
2.3 (0.6–5.6)
17.8 (6.2–73.6)
* RA ⫽ rheumatoid arthritis; anti-TNF ⫽ anti–tumor necrosis factor; MTX ⫽ methotrexate; NA ⫽ not applicable; DAS28-CRP ⫽ Disease Activity
Score in 28 joints using C-reactive protein; HAQ DI ⫽ Health Assessment Questionnaire disability index; etan. ⫽ etanercept; RF ⫽ rheumatoid
factor; anti-CCP ⫽ anti–cyclic citrullinated peptide; ESR ⫽ erythrocyte sedimentation rate.
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SELLAM ET AL
Table 2. Lymphocyte subsets in RA patients before rituximab therapy and in controls*
Lymphocytes/mm3
B cells/mm3
T cells/mm3
NK cells/mm3
B cells, %
CD27⫺ B cells, %
CD27⫹ B cells, %
CD27⫺IgD⫹ B cells,
CD27⫺IgD⫺ B cells,
CD27⫹IgD⫺ B cells,
CD27⫹IgD⫹ B cells,
CD38⫹⫹IgD⫺
plasmablasts, %
%
%
%
%
Controls
(n ⫽ 47)
RA patients
(n ⫽ 208)
Anti-TNF ⫹
MTX group
(n ⫽ 142)
MTX group
(n ⫽ 66)
P, RA patients
vs. controls
P, anti-TNF ⫹
MTX group vs.
MTX group
1,959.4 ⫾ 601.0
257.3 ⫾ 154.1
1,422.3 ⫾ 499.9
256.7 ⫾ 134.6
12.8 ⫾ 5.2
67.0 ⫾ 17.1
33.0 ⫾ 17.1
57.7 ⫾ 20.1
9.0 ⫾ 7.5
23.1 ⫾ 15.8
9.9 ⫾ 7.7
ND
1,787.8 ⫾ 853.7
188.6 ⫾ 121.4
1,379.1 ⫾ 702.3
196.8 ⫾ 131.1
10.6 ⫾ 5.3
70.7 ⫾ 17.0
29.3 ⫾ 17.0
61.8 ⫾ 19.8
7.5 ⫾ 6.4
17.9 ⫾ 11.8
10.6 ⫾ 9.6
3.3 ⫾ 3.8
1,872.7 ⫾ 880.4
211.3 ⫾ 128.7
1,437.5 ⫾ 717.0
201.7 ⫾ 138.9
11.5 ⫾ 5.3
71.4 ⫾ 15.6
28.6 ⫾ 15.6
63.4 ⫾ 18.4
6.7 ⫾ 4.4
17.7 ⫾ 10.3
10.6 ⫾ 9.4
2.9 ⫾ 2.9
1,602.5 ⫾ 767.8
137.2 ⫾ 83.5
1,251.6 ⫾ 657.5
185.8 ⫾ 112.2
8.6 ⫾ 3.9
69.3 ⫾ 19.9
30.8 ⫾ 19.9
58.2 ⫾ 22.3
7.8 ⫾ 9.1
18.4 ⫾ 14.7
10.5 ⫾ 10.0
4.3 ⫾ 5.2
0.20
0.001
0.69
0.007
0.009
0.19
0.19
0.21
0.16
0.01
0.66
NA
0.04
0.0004
0.09
0.48
0.0006
0.45
0.45
0.11
0.02
0.73
0.97
0.30
* Values are the mean ⫾ SD. NK ⫽ natural killer; ND ⫽ not done (see Table 1 for other definitions).
the MTX group (32% of all RA patients), 52 had
discontinued anti-TNF therapy more than 6 months
before the study and 14 had never received anti-TNF.
The 142 patients in the anti-TNF plus MTX group
(68% of all RA patients) did not respond to anti-TNF
received within the past 6 months in combination with
MTX. These 2 groups of RA patients had similar
baseline characteristics except for disease duration, which
was longer in the MTX group (P ⫽ 0.004). The control
group had a lower proportion of women compared with
the RA group (P ⬍ 0.001).
Subsets of blood lymphocytes in RA patients and
controls. Compared with controls, RA patients exhibited
global blood lymphopenia selectively involving B cells
and NK cells; no decrease in T cell counts was observed
(Table 2). Interestingly, mean ⫾ SD B cell and NK cell
counts were reduced to a lesser extent in the anti-TNF
plus MTX group (211 ⫾ 129/mm3 and 202 ⫾ 139/mm3,
respectively) than in the MTX group (137 ⫾ 84/mm3
and 186 ⫾ 112/mm3, respectively), although counts of
both cell subsets were reduced in each group compared
with controls (B cell count 257 ⫾ 154/mm3 and NK cell
count 257 ⫾ 135/mm3 in controls; P ⬍ 0.03 for both
versus MTX group and P ⬍ 0.02 for both versus
anti-TNF plus MTX group). In the anti-TNF plus MTX
group, no difference was observed between the soluble
Table 3. Correlation between the frequency of CD27⫹ memory B
cells and DAS28-CRP, serum B cell activation biomarkers, and
biomarkers of inflammation in RA patients*
Figure 2. Frequency of blood B cell subsets in patients with rheumatoid arthritis (RA) and healthy controls (HC). A, Frequency of CD27⫺
and CD27⫹ B cells. B, Frequency of switched CD27⫹IgD⫺ and
nonswitched CD27⫹IgD⫹ memory B cells. NS ⫽ not significant.
DAS28-CRP
Anti-CCP level
Serum IgG level
Serum IgA level
Serum IgM level
Serum ␬ FLC level
Serum ␭ FLC level
CRP level
ESR
Serum BAFF level
r
P
⫺0.07
⫺0.17
⫺0.31
⫺0.25
0.08
⫺0.33
⫺0.21
⫺0.18
⫺0.19
⫺0.23
0.37
0.05
⬍0.0001
0.0008
0.27
⬍0.0001
0.006
0.02
0.02
0.002
* FLC ⫽ free light chain (see Table 1 for other definitions).
MEMORY B CELLS IN RA AND CLINICAL RESPONSE TO RITUXIMAB
3697
Table 4. Leukocytes, lymphocytes, B cell subsets, and BAFF-R expression on B cell subsets according to the EULAR response at week 24 in
patients with rheumatoid arthritis*
Characteristic
Leukocyte count, no. (%)†
⬍10,000/mm3 (referent)
ⱖ10,000/mm3
Lymphocyte count, no. (%)‡
⬍1,500/mm3 (referent)
ⱖ1,500/mm3
CD3⫹ T cells, %
NK cells, %
CD19⫹ B cells, %
CD27⫺ naive B cells, %
CD27⫹ memory B cells, %
CD27⫺IgD⫺ B cells, %
CD27⫺IgD⫹ B cells, %
CD27⫹IgD⫺ B cells, %
CD27⫹IgD⫹ B cells, %
CD38⫹⫹IgD⫺ plasmablasts, %
BAFF-R expression on, MFI
CD27⫹IgD⫺ B cells
CD27⫹IgD⫹ B cells
CD27⫺IgD⫹ B cells
CD27⫺IgD⫺ B cells
EULAR
nonresponders
(n ⫽ 59)
EULAR
responders
(n ⫽ 149)
OR (95% CI)
35 (26)
21 (32)
102 (74)
44 (68)
–
0.72 (0.38–1.37)
23 (32)
25 (24)
76.2 ⫾ 8.3
12.8 ⫾ 8.7
9.4 ⫾ 4.4
64.2 ⫾ 19.0
35.9 ⫾ 19.0
6.9 ⫾ 4.4
54.5 ⫾ 23.1
21.4 ⫾ 14.1
13.1 ⫾ 12.1
4.1 ⫾ 4.8
49 (68)
78 (76)
76.2 ⫾ 7.9
11.6 ⫾ 6.3
11.0 ⫾ 5.3
73.2 ⫾ 15.5
26.8 ⫾ 15.5
7.7 ⫾ 7.0
64.5 ⫾ 17.7
16.6 ⫾ 10.5
9.6 ⫾ 8.3
3.0 ⫾ 3.3
–
1.47 (0.75–2.86)
1.00 (0.96–1.04)
0.98 (0.93–1.03)
1.07 (0.99–1.16)
1.03 (1.01–1.05)
0.97 (0.95–0.99)
1.02 (0.97–1.09)
1.03 (1.01–1.04)
0.97 (0.940–0.995)
0.97 (0.933–0.997)
0.93 (0.86–1.02)
474.6 ⫾ 210.8
514.4 ⫾ 228.5
658.2 ⫾ 270.3
471.2 ⫾ 224.3
473.0 ⫾ 225.1
548.2 ⫾ 240.7
636.5 ⫾ 296.0
512.5 ⫾ 265.1
1.000 (0.998–1.002)
1.001 (0.999–1.002)
1.000 (0.999–1.001)
1.001 (0.999–1.002)
P
–
0.32
–
0.26
0.99
0.34
0.07§
0.002§
0.002§
0.43
0.004§
0.02§
0.03§
0.11§
0.97
0.41
0.67
0.36
* Except where indicated otherwise, values are the mean ⫾ SD. EULAR ⫽ European League Against Rheumatism; OR ⫽ odds ratio; 95% CI ⫽
95% confidence interval; NK ⫽ natural killer; BAFF-R ⫽ BAFF receptor; MFI ⫽ mean fluorescence intensity.
† The denominator is the total number of patients with a count of ⬍10,000/mm3 or ⱖ10,000/mm3.
‡ The denominator is the total number of patients with a count of ⬍1,500/mm3 or ⱖ1,500/mm3.
§ Significant variables in univariate analysis (P ⱕ 0.15) included in the multivariate analysis.
receptor and monoclonal antibody therapies for B cell
count (P ⫽ 0.24). In addition, there was no difference in
B cell counts according to steroid use (always ⬍10
mg/day) in the anti-TNF plus MTX group (207 ⫾
132/mm3 with steroids versus 226 ⫾ 120/mm3 without;
P ⫽ 0.49) or in the MTX group (134 ⫾ 85/mm3 with
steroids versus 157 ⫾ 78/mm3 without; P ⫽ 0.58). In a
manner similar to the total B cell count, B cell frequency
was also decreased in RA patients, with a smaller
decrease noted in the anti-TNF plus MTX group than in
the MTX group (Table 2).
Cross-sectional study of B cell subsets in RA
patients before B cell depletion therapy and in controls.
The CD27 marker enabled a simple differentiation
between naive and memory B cells (except for the very
low fraction of CD27⫺IgD⫺ memory B cells). The
decrease in B cell count observed in the RA group
involved the CD27 naive and the CD27⫹ memory B
cells in equal proportions, leading to a conserved equilibrium between these 2 subpopulations in RA patients
(mean ⫾ SD 67 ⫾ 17% and 71 ⫾ 17% of CD27⫺ B cells
in controls and RA patients, respectively; P ⫽ 0.19)
(Figure 2A).
We further investigated the distribution of the
CD27⫹ memory B cell subsets into switched
CD27⫹IgD⫺ and nonswitched CD27⫹IgD⫹ subsets.
The CD27⫹IgD⫹ B cell frequency was comparable in
controls and RA patients, and within the RA patient
population between the 2 treatment groups. In contrast,
the frequency of switched CD27⫹IgD⫺ B cells was
significantly decreased in RA patients (18 ⫾ 12%)
compared with controls (23 ⫾ 16%) (P ⫽ 0.01), although there was no significant difference between the
RA treatment groups (P ⫽ 0.73) (Table 2 and Figure
2B). This difference was observed irrespective of
whether patients received steroids (19 ⫾ 13% with
prednisone versus 14 ⫾ 9% without; P ⫽ 0.54).
The frequency of the small fraction of memory
CD27⫺IgD⫺ B cells was comparable between RA patients and controls (P ⫽ 0.16). However, the frequency
of this subset was lower in the anti-TNF plus MTX
group than in the MTX group (P ⫽ 0.02) (Table 2).
BAFF and BAFF-R assessment in RA patients
and controls. Serum BAFF concentrations were comparable in RA patients (561 ⫾ 313 ng/ml) and controls
(mean ⫾ SD 480 ⫾ 118 ng/ml) (P ⫽ 0.21). In addition,
no difference in serum BAFF concentration was observed between the MTX group and the anti-TNF plus
MTX group. Moreover, the level of expression of
BAFF-R on each peripheral blood B cell subset accord-
3698
SELLAM ET AL
ing to the CD27/IgD classification was similar in the RA
patient and control populations (data not shown).
Correlation between decreased frequency of
memory B cells, B cell biomarkers, and biomarkers of
inflammation. We assessed whether several B cell biomarkers of disease activity and disease activity itself
correlated with the relative frequency of B cell subsets.
Accordingly, we used the DAS28-CRP in addition to the
several measurements undertaken in the SMART study
as previously described (34): anti-CCP, serum IgG, IgA,
and IgM, serum ␬ and ␭ free light chains, and serum
BAFF levels. Interestingly, we found that the decreased
frequency of CD27⫹ memory B cells was correlated
with several biomarkers of B cell activation (IgG, IgA, ␬
free light chains, ␭ free light chains, anti-CCP level, and
serum BAFF level) as well as with biologic markers of
inflammation (CRP level and ESR) (Table 3). However,
no correlation was found between CD27⫹ memory B
cells and disease activity.
Baseline predictive factors associated with response to RTX at week 24. A total of 149 patients (72%)
were classified as EULAR responders to RTX at week
24. These included 44 good responders (21%) and 105
partial responders (50%).
For the leukocyte subsets and B cell subsets, we
used the relative frequencies of the B cell subsets (%),
rather than absolute cell counts, to analyze correlation
with EULAR response. Results are presented in Table
4. By univariate analysis, higher frequencies of CD19⫹
B cells, CD27⫺ naive B cells, and CD27⫺IgD⫹ naive B
cells and lower frequencies of CD27⫹ memory B cells,
switched CD27⫹IgD⫺ B cells, nonswitched CD27⫹
IgD⫹ memory B cells, and CD38⫹⫹IgD⫺ plasmablasts
were associated with EULAR response and subsequently included in the multivariate analysis. The level
of BAFF-R expression was not associated with a particular profile of EULAR response. In the multivariate
analysis, a lower frequency of memory CD27⫹ B cells
was significantly associated with EULAR response at
week 24 (OR 0.97 [95% CI 0.95–0.99], P ⫽ 0.0015).
DISCUSSION
In this study, we have shown that B cell depletion
therapy–naive RA patients exhibit a global B cell lymphopenia attenuated by anti-TNF therapy; this quantitative decrease involves CD27⫺ naive and CD27⫹
memory B cells in equal proportions, leading to a
maintenance of the global equilibrium between these 2
subsets. Moreover, in the CD27⫹ memory B cell subset,
the decrease was selectively observed in CD27⫹IgD⫺
switched memory B cells, while the nonswitched mem-
ory CD27⫹IgD⫹ B cells remained unchanged in RA
patients compared with controls. In contrast to other
autoimmune diseases, the BAFF/BAFF-R system studied in the blood exhibited no significant abnormalities in
RA. The most interesting finding of our study is that the
decrease of global memory CD27⫹ B cells is associated
with a greater clinical response to RTX.
While the variations of B cell subsets after RTX
therapy have been extensively studied in RA and other
autoimmune diseases (3,4,10,11,35), few data are available concerning B cell homeostasis in RA patients
receiving other agents (12–14,22). Recently, de la Torre
et al have shown in a study with 11 healthy controls and
15 RA patients that blood B cell subset frequency
according to the IgD/CD27 classification was not disturbed, except for a slightly increased proportion of
CD27⫺IgD⫺ B cells in RA patients (22). In the present
study involving more than 200 RA patients, we could
not confirm disturbances of CD27⫺IgD⫺ B cells in RA
patients.
The first part of our study focused on B cell
subpopulations before B cell depletion therapy in RA
patients compared with controls, in addition to the
influence of anti-TNF and/or MTX therapy. The difference in the proportion of women between the RA
patient and control groups is unlikely to affect the
interpretation of data, since B cell subset frequency
might be affected by age but not by sex (36). Since the
range of time before receiving the last infusion or
injection of anti-TNF prior to the first infusion of RTX
was very broad, we chose to stratify these patients into 2
groups: the anti-TNF plus MTX group, in which patients
received their last anti-TNF injection during the previous 6 months; and the MTX group, including anti-TNF–
naive patients and patients who received their last
anti-TNF more than 6 months before study inclusion.
The cutoff of 6 months was determined based on the
half-life of each TNF blocker; this ensured that patients
in the MTX group had no significant residual amounts
of anti-TNF able to modify B cell subsets.
Surprisingly, while no abnormalities in T cell
counts were observed, absolute counts of peripheral
blood B cells were quantitatively lower in RA patients
than in controls, and this decrease in B cells involved
both naive and memory B cells in the same proportion,
thus explaining the conservation of equilibrium between
naive and memory B cells in RA. The B cell lymphopenia in RA was more marked in the MTX group, suggesting that anti-TNF therapy may, in part, counteract this
decrease. However, we cannot separate the impact of
the disease itself and that of the treatment on this B cell
lymphopenia, since we could not study untreated RA
MEMORY B CELLS IN RA AND CLINICAL RESPONSE TO RITUXIMAB
patients, who represent the “ideal” population to determine whether B cell disturbances observed here are
explained by the disease itself or by anti-TNF and/or
MTX therapy. There was no variation of this equilibrium
between the treatment groups or according to the type
of anti-TNF (monoclonal antibody or etanercept). The
B cell lymphopenia was not linked to the use of steroids.
The reported B cell lymphopenia was more
marked in RA patients who had received MTX than in
those who had previously received MTX plus anti-TNF.
Studying a small number of patients, Anolik et al have
shown that RA patients receiving etanercept exhibited a
decrease in the frequency of peripheral blood memory
CD27⫹ B cells through the blockade of the germinal
center reaction and the follicular dendritic cell network
(12). In another study with results similar to those in our
study, a decrease in the frequency of nonswitched
CD27⫹IgD⫹ memory B cells observed in a small sample of RA patients was partially restored by infliximab;
however, no data on the total B cell count were available
(14).
Despite the conservation of the equilibrium between CD27⫺ and CD27⫹ B cells, the decrease in the
frequency of memory CD27⫹ B cells selectively affected
switched CD27⫹IgD⫺ B cells, suggesting that these
cells have migrated to the target tissue of RA, the
synovium, or are sequestered at this site. Infiltration of
the synovial membrane by these cells has been shown
previously (14,37). Another explanation would be a
decrease in the generation of this subset induced either
by the disease itself or by the previous treatments before
B cell depletion therapy. This more profound decrease
in memory CD27⫹ B cells may reflect a more active
disease state. Thus, despite the lack of correlation
between the frequency of memory CD27⫹ B cells and
the DAS28 (possibly explained by the fact that all
included patients had active disease with a DAS28-CRP
of ⬎3.2), a correlation between decreased CD27⫹ B cell
frequency and serum biomarkers of B cell activation was
observed. The latter correlate with disease activity in
both established and early RA (38,39), and two of them
(presence of autoantibodies and serum IgG levels above
normal) are predictive of a response to RTX (34). Of
note, the present study also confirmed a positive correlation between serum free light chain levels and the
DAS28 in established RA (data not shown).
Thus, we can hypothesize that in active RA, B cell
activation is associated with both the production of
several B cell biomarkers and the migration or sequestering of the pathogenic memory B cells into the synovium. Indeed, the only serum B cell activation biomarker
that did not correlate with the frequency of memory
3699
CD27⫹ B cells was serum IgM level; this was likely to be
a reflection of the activation of naive B cells.
Since RTX selectively depletes B cells, monitoring this cell subset could be valuable for predicting the
response to RTX and any subsequent recurrence of
disease. Several studies have shown that the reemergence of memory B cells is associated with early relapse
in a small group of RA patients (3–5). The presence of
residual blood B cells 15 days after RTX infusion is also
associated with a poorer outcome (40). However, these
parameters are measured after RTX infusion and do not
represent real predictive tools. Only Vital et al have
recently shown that a lower absolute count of preplasma
cells at baseline was predictive of a better outcome in a
univariate analysis (5). Here, we have reported, from a
study of a larger population, that a low frequency of
global CD27⫹ memory B cells, switched CD27⫹IgD⫺
B cells, nonswitched CD27⫹IgD⫹ B cells, and, to a
lesser extent, CD38⫹⫹IgD⫺ plasmablasts was predictive of a greater clinical response to a single course of
RTX. According to multivariate analyses, a low frequency of memory CD27⫹ B cells was the best parameter predictive of efficacy of RTX. The OR of 0.97
(95% CI 0.95–0.99) does not seem impressive but it is
actually highly statistically significant (P ⫽ 0.0015), and
it means that for each absolute decrease in memory
CD27⫹ B cells of 1%, the likelihood of responding to
RTX increases by 3%, which is clinically meaningful.
Taking into account the cost and the complexity
of the assay, as recently noted in an updated consensus
statement on the use of RTX in patients with RA (41),
we cannot currently recommend the study of B cell
subpopulations in routine daily clinical practice. Moreover, no threshold of baseline CD27⫹ memory B cell
frequency could discriminate responders from nonresponders by receiver operating characteristic curve analysis (data not shown). As we have previously reported
(34), there are other markers, such as autoantibodies or
serum IgG level, that are simpler and less expensive to
use in clinical practice. The present results suggest some
explanations of the mechanism of action of RTX; since
the decrease in memory CD27⫹ B cells was associated
with surrogate biomarkers of disease activity (B cell
biomarkers and biomarkers of inflammation), we suggest that this decreased frequency of switched memory B
cells in the blood could be a surrogate marker of
activation of these memory B cells in the target tissue of
RA, the synovium, and may delineate a B cell–driven
subset of RA that is more sensitive to B cell depletion
therapy.
Increases in serum BAFF and decreases in
BAFF-R expression on B cells have been reported in
3700
SELLAM ET AL
several autoimmune diseases (20–23). Previously, we
showed that serum BAFF levels were not predictive of
response to RTX (34). In the present study, we confirmed the findings of some studies showing that BAFF
concentration did not differ significantly in RA patients
compared with controls (22,42). Moreover, BAFF-R
expression on B cell subsets was similar in RA patients
and healthy controls, as previously reported (22), and
here we have also shown that this expression was not
predictive of response to RTX.
In conclusion, RA patients not previously treated
with B cell depletion therapy exhibit a selective B cell
lymphopenia attenuated by anti-TNF treatment and
involving CD27⫺ and CD27⫹ B cell subsets in equal
proportions. The CD27⫹IgD⫺ switched memory B cell
frequency was especially decreased within CD27⫹ memory B cells. The low frequency of CD27⫹ memory B
cells may be predictive of response to RTX. These data
suggest that in RA, decrease in peripheral memory B
cell compartments may be associated with migration and
activation of these cells in the synovium. These features
may define a specific B cell–driven subtype of RA that is
more sensitive to B cell depletion therapy.
ACKNOWLEDGMENTS
We thank Dr. Rosemary Jourdan, Dr. Nadine Mackenzie (Roche France), and Dr. Jamila Filipecki (previously at
Roche France).
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Drs. Sellam, Mariette, and Taoufik
had full access to all of the data in the study and take responsibility for
the integrity of the data and the accuracy of the data analysis.
Study conception and design. Sellam, Hendel-Chavez, Abbed, Sibilia,
Tebib, Le Loët, Combe, Dougados, Mariette, Taoufik.
Acquisition of data. Sellam, Hendel-Chavez, Abbed, Sibilia, Tebib,
Le Loët, Dougados, Mariette, Taoufik.
Analysis and interpretation of data. Sellam, Rouanet, Hendel-Chavez,
Abbed, Sibilia, Tebib, Le Loët, Combe, Dougados, Mariette, Taoufik.
ROLE OF THE STUDY SPONSOR
Roche France designed the SMART study but did not
participate in the design, data collection, or interpretation of the
results of this ancillary study, which was proposed by an independent
scientific committee. Roche France supported the measurement of
serum biomarkers, the flow cytometry experiments, and the statistical
analysis. Roche France was not involved in the writing of the manuscript. Their agreement to submit the manuscript for publication was
not required, their approval of the content of the submitted manuscript
was not required, and publication of the manuscript was not contingent
upon their approval.
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APPENDIX A: THE SMART INVESTIGATORS
In addition to the authors, the SMART investigators (all in
France) are as follows: Dr. I. Azais (Poitiers), Dr. J. C. Balblanc
(Belfort), Dr. F. Berenbaum (Paris), Dr. P. Bertin (Limoges), Dr.
M.-C. Boissier (Bobigny), Dr. P. Bourgeois (Paris), Dr. A. Cantagrel
(Toulouse), Dr. P. Carli (Toulon), Dr. P.-Y. Chouc (Marseille), Dr. M.
Couret (Valence), Dr. L. Euller-Ziegler (Nice), Dr. P. Fardellone
(Amiens), Dr. P. Fauquert (Berck/Mer), Dr. R.-M. Flipo (Lille), Dr. P.
Gaudin (Echirolles), Dr. J.-L. Grauer (Aix-en-Provences), Dr. A.
Heraud (Libourne), Dr. P. Hilliquin (Corbeil), Dr. S. Hoang (Vannes),
Dr. E. Houvenagel (Lomme), Dr. D. Keita (Paris), Dr. K. Lassoued
(Cahors), Dr. L. Le Dantec (Lievin), Dr. J.-M. Le Parc (Boulogne),
Dr. L. Lequen (Pau), Dr. F. Lioté (Paris), Dr. C. Marcelli (Caen),
Dr. O. Meyer (Paris), Dr. J.-L. Pellegrin (Pessac), Dr. A. Perdriger
(Rennes), Dr. G. Rajzbaum (Paris), Dr. S. Redeker (Abbeville), Dr.
J.-M. Ristori (Clermont-Ferrand), Dr. A. Saraux (Brest), Dr. G.
Tanguy (La Roche sur Yon), Dr. T. Thomas (Saint-Priest-en-Jarez),
Dr. L. Zabraniecki (Toulouse), and Dr. C. Zarnitski (Montivilliers).