ARTHRITIS & RHEUMATISM
Vol. 65, No. 4, April 2013, pp 1097–1106
© 2013, American College of Rheumatology
Rituximab Therapy for Primary Sjo ¨gren’s Syndrome
An Open-Label Clinical Trial and Mechanistic Analysis
E. William St.Clair,1Marc C. Levesque,2Eline T. Luning Prak,3
Frederick B. Vivino,3Chacko J. Alappatt,3Meagan E. Spychala,4
Josiah Wedgwood,†James McNamara,5Kathy L. Moser Sivils,6
Lytia Fisher,7and Philip Cohen,7for the Autoimmunity Centers of Excellence
Objective. To study the safety and clinical efficacy
of rituximab therapy for primary Sjo ¨gren’s syndrome,
as well as to investigate its mechanisms.
Methods. Patients with primary Sjo ¨gren’s syn-
drome were enrolled in an open-label trial, were given
rituximab (1 gm) infusions on days 1 and 15, and were
monitored through week 52. The primary end point
was safety, with secondary end points evaluating clinical
and biologic efficacy. Blood was obtained for enumera-
tion of lymphocyte subsets, measurement of serum
autoantibody and BAFF levels, and analysis of gene
Results. Twelve female patients with primary
Sjo ¨gren’s syndrome were administered rituximab. They
had a median age of 51 years (range 34–69 years) and a
median disease duration of 8.0 years (range 2–18 years).
We observed no unexpected toxicities from the ritux-
imab therapy. Modest improvements were observed at
week 26 in patient-reported symptoms of fatigue and
oral dryness, with no significant improvement in the
objective measures of lacrimal and salivary gland func-
tion. The recovery of blood B cells following the nadir
from rituximab therapy was characterized by a predom-
inance of transitional B cells and a lack of memory
B cells. While blood B cell depletion was associated
with an increase in serum BAFF levels, no significant
changes were observed in the levels of serum anti-Ro/
SSA, anti-La/SSB, and anti–type 3 muscarinic acetyl-
choline receptor autoantibodies or in the blood inter-
Conclusion. In patients with primary Sjo ¨gren’s
syndrome, a single treatment course of rituximab was
not associated with any unexpected toxicities and led to
only modest clinical benefits despite effective depletion
of blood B cells.
Primary Sjo ¨gren’s syndrome is among the most
common of the connective tissue diseases. For women,
its prevalence in the UK has been estimated to be
0.1–0.6% (1). The disease is characterized by the pres-
ence of keratoconjunctivitis sicca (dry eyes), xero-
stomia (dry mouth), serum antinuclear antibodies, and
chronic salivary gland inflammation, as well as the
occurrence of systemic features, such as profound fatigue,
ClinicalTrials.gov identifier: NCT0012101829.
Supported by the Autoimmunity Centers of Excellence, a
consortium funded by the National Institute of Allergy and Infectious
Diseases, NIH (grant U19-AI-056363). Additional support was pro-
vided by Genentech, Inc., and the Pennsylvania Department of Health.
1E. William St.Clair, MD: Duke University Medical Center,
Durham, North Carolina;2Marc C. Levesque, MD, PhD: University
of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;3Eline T.
Luning Prak, MD, PhD, Frederick B. Vivino, MD, Chacko J. Alappatt,
MD: University of Pennsylvania Perelman School of Medicine, Phila-
delphia;4Meagan E. Spychala, DrPH: Rho, Inc., Chapel Hill, North
and Infectious Diseases, NIH, Bethesda, Maryland;6Kathy L. Moser
Sivils, PhD: Oklahoma Medical Research Foundation, Oklahoma City;
7Lytia Fisher, BS, Philip Cohen, MD: Temple University School of
Medicine, Philadelphia, Pennsylvania.
†Dr. Wedgwood is deceased.
Dr. Levesque has received consulting fees from Genentech
(less than $10,000) and grant support from Genentech. Dr. Luning
Prak has received consulting fees, speaking fees, and/or honoraria
from Biogen Idec (less than $10,000). Dr. Vivino has received con-
sulting fees, speaking fees, and/or honoraria from Amplimmune
and Daiichi-Sankyo (less than $10,000 each) and has provided ex-
pert testimony on behalf of Daiichi-Sankyo. Dr. Alappatt has re-
ceived consulting fees, speaking fees, and/or honoraria from Takeda,
Novartis, Amgen, and URL Pharma (less than $10,000 each).
Address correspondence to E. William St.Clair, MD, Depart-
ment of Medicine, Duke University Medical Center, Box 3874 DUMC,
Durham, NC 27710. E-mail: email@example.com.
Submitted for publication August 26, 2012; accepted in
revised form December 27, 2012.
5James McNamara, MD: National Institute of Allergy
pheral neuropathy, and leukocytoclastic vasculitis (2,3).
Patients with primary Sjo ¨gren’s syndrome are also at
increased risk of developing B cell lymphoma (4).
The treatment of primary Sjo ¨gren’s syndrome is
largely based on alleviation of symptoms and includes
the use of topical cyclosporine (for the management of
dry eyes), sialogogues (oral muscarinic agonists), hy-
droxychloroquine, and low doses of prednisone (5).
Patients with more serious systemic manifestations may
require more intensive therapy with glucocorticoids and
other immunosuppressive agents. However, no drugs
have been shown in well-designed clinical trials of pa-
tients with primary Sjo ¨gren’s syndrome to reduce dis-
ease activity or prevent damage.
The potential clinical utility of rituximab therapy
has recently been investigated in primary Sjo ¨gren’s
syndrome (6–10), owing to its proven efficacy in other
chronic inflammatory diseases, such as rheumatoid ar-
thritis (11,12) and systemic vasculitis (13), and its effects
on potential disease-inciting B cells. The importance of
abnormal B cell responses in the mechanisms of primary
Sjo ¨gren’s syndrome is strongly suggested by the presence
of serum autoantibodies, most notably, anti-Ro/SSA
and anti-La/SSB antibodies (3). The benign and malig-
nant B cell monoclonal proliferations in the blood and
salivary gland tissues of patients with primary Sjo ¨gren’s
syndrome (14), as well as the abnormalities in B cell
memory (15), provide further evidence that B cells play
an important role in the pathophysiology of this condi-
tion. We therefore conducted an open-label study of
rituximab, a potent B cell depleter, to evaluate the safety
and possible clinical efficacy of this approach in primary
Sjo ¨gren’s syndrome, as well as to determine its effects on
blood B cell subsets, autoantibodies, cytokines, and gene
PATIENTS AND METHODS
Study design and treatment. The study was a prospec-
tive, open-label, single-arm, phase I study of rituximab therapy
for patients with primary Sjo ¨gren’s syndrome (ClinicalTrials.
gov identifier NCT0012101829). Twelve patients received two
1,000-mg infusions of rituximab 2 weeks apart, using a standard
protocol, with escalation of the infusion rate to a maximum of
400 mg/hour. All patients were pretreated with 50 mg of oral
diphenhydramine, 650 mg of oral acetaminophen, and 100 mg
of intravenous methylprednisolone ?30 minutes before each
of the infusions. The patients returned for followup visits at
weeks 4, 8, 14, 26, 30, 36, and 52. Diphtheria and tetanus toxoid
as well as a pneumococcal polyvalent-23 vaccine were admin-
istered at week 26 to 8 of the patients for assessment of
The study was approved by the institutional review
boards (IRBs) at Duke University Medical Center and the
University of Pennsylvania. All patients provided informed
Patient population. Eligible patients were adults be-
tween the ages of 18 and 75 years who met the classification
criteria for primary Sjo ¨gren’s syndrome as defined by the
American–European Consensus Group (16). In addition, pa-
tients had one or more of the following systemic manifesta-
tions: fatigue (?50 mm on a 100-mm visual analog scale
[VAS]), joint pain (?50 mm on a 100-mm VAS), severe parotid
gland swelling, peripheral neuropathy, interstitial lung dis-
ease, leukocytoclastic vasculitis, interstitial nephritis, or other
extraglandular disease causing organ-system dysfunction. Pa-
tients of reproductive potential agreed to use an acceptable
method of birth control during treatment and for 12 months
Concomitant therapy with nonsteroidal antiinflamma-
tory drugs, cevimeline, pilocarpine, ophthalmic cyclosporine,
and hydroxychloroquine was allowed, provided the dosages
were maintained at the baseline levels. Concurrent therapy
with prednisone (?10 mg/day) was permitted if the dosage had
been stable for at least 2 weeks prior to study entry and was
kept constant during the study.
Patients were excluded from the study if they had
previously been treated with rituximab or if they had been
recently treated with the following medications: cyclo-
phosphamide within 24 weeks, methotrexate, azathioprine,
cyclosporine, or mycophenolate mofetil within 4 weeks, etan-
ercept within 4 weeks, adalimumab within 8 weeks, or inflix-
imab within 12 weeks. Those taking potent anticholinergic
agents, such as tricyclic antidepressants, antihistamines, pheno-
thiazine, and antiparkinsonian drugs were not allowed to
participate in the study. Patients were also excluded if they
had active infection, chronic or persistent infection that might
be worsened by immunosuppressive therapy (e.g., human
immunodeficiency virus, hepatitis B or C, tuberculosis), known
coronary artery disease or a history of significant cardiac
arrhythmias or severe congestive heart failure, pregnancy,
ongoing oral anticoagulant therapy, a history of alcohol or
substance abuse, prior head and neck radiation therapy, his-
tory of sarcoidosis, history of a positive result on purified
protein derivative test without documentation of treatment for
active or latent tuberculosis, history of severe pulmonary
disease (forced vital capacity ?50% predicted, diffusing capac-
ity for carbon monoxide ?50% predicted, resting oxygen
saturation ?95%), history of malignancy, except for resected
basal cell or squamous cell carcinoma of the skin, cervical
dysplasia, or in situ cervical cancer grade 1 within the last 5
years, abnormal laboratory results (absolute neutrophil count
?1,000/mm3, platelets ?100,000/mm3, hemoglobin ?9 gm/dl,
serum creatinine ?2.0 mg/dl, aspartate aminotransferase or
alanine aminotransferase ?2 times the upper limit of normal),
or administration of a live vaccine within the previous 3 months.
Study end points. The primary safety end point was the
proportion of patients experiencing a grade 3, grade 4, or grade
5 adverse event (AE) according to the Common Terminology
Criteria for Adverse Events of the National Cancer Institute
that was judged by the investigator to be possibly, probably,
or definitely related to rituximab therapy. The other goal of
the study was to obtain preliminary evidence of clinical and
1098 ST.CLAIR ET AL
biologic activity using measures of exocrine gland function and
other disease features, as well as immune system function. For
the clinical and biologic end points, we focused our analysis on
the changes between weeks 0 and 26.
Clinical assessments. Safety was evaluated at each visit
by monitoring for AEs, including changes in the values of
routine laboratory parameters. Assessments of clinical efficacy
were performed at weeks 8, 14, 26, 36, and 52 and included a
Sjo ¨gren’s Syndrome Symptom Survey (17), physician’s and
patient’s global assessments of disease activity (100-mm VAS),
unanesthetized Schirmer’s test, slit-lamp examination with
lissamine green staining, and measurement of unstimulated
and stimulated whole salivary flow rate. For the assessment of
salivary flow, patients were instructed to withhold the morning
dose of secretagogue and take nothing by mouth for at least
60 minutes prior to measurement of salivary flow. Unstimu-
lated salivary flow was determined by having the patient
expectorate into a preweighed 50-cm3centrifuge tube for 15
minutes. Saliva samples were weighed on an analytical balance
to quantify the volume over the 15-minute collection period
(1 gm ? 1 ml). Subsequently, patients were treated with 5 mg
of oral pilocarpine to stimulate salivary flow, and 60 minutes
later, another 15-minute saliva sample was collected. In addi-
tion, quality of life was examined at weeks 0 and 26 using the
Short Form 36 (SF-36) health survey (18).
Flow cytometry. Venous blood that was less than 24
hours old was processed for flow cytometry as described
previously (19,20). After washing and Fc blockade, cells were
stained with the following fluorochrome-conjugated antibodies
in multiple 4-color combinations (all antibodies were pur-
chased from BD Biosciences): fluorescein isothiocyanate–
conjugated CD27 (M-7271) and CD3 (SK7); phycoerythrin-
conjugated CD38 (HIT2), CD8 (HIT8a), CD16 (3G8), and
CD56 (B159); PerCP–Cy5.5–conjugated CD20 (L27), CD4
(SK3), and CD8 (SK1); and allophycocyanin-conjugated CD19
(HIB19), CD45 (H130), and CD14 (M5E2). After staining
and lysis of red blood cells, the white blood cell pellets were
washed and fixed in paraformaldehyde. Data from stained
cells were acquired with a FACSCalibur instrument and ana-
lyzed using CellQuest Pro software version 5.2 (both from BD
Biosciences). Approximately 10,000 CD19? lymphocytes were
analyzed per tube or, in samples with low numbers of B cells,
the maximum number of events was acquired by draining the
For comparison with the patients in the clinical trial,
blood was obtained, with IRB approval, from 15 subjects (11
women and 4 men) at the University of Pennsylvania. These
subjects served as controls. Their median age was 39.5 years
(interquartile range 27–49 years). Control subjects were not
part of the current trial and were younger than the patients
enrolled in the study herein.
B cell subset counts. Fresh whole blood (less than
24 hours old) was analyzed by flow cytometry as described
above. To calculate the absolute counts of B cell subsets, the
absolute lymphocyte count (in cells/?l of whole blood) was
obtained by Coulter analysis or through the complete blood
cell count at each of the local sites. The B cell fraction was
obtained by multiplying the absolute lymphocyte count by the
CD19? fraction. B cell subset counts were computed by
multiplying the B cell absolute count by the fraction of B cells
in each of the subsets. CD38 and CD27 staining was used to
define B cell subsets, which were then classified as follows:
transitional (CD38??CD27?), mature naive (CD38?
CD27?), mature activated memory (CD38?CD27?), resting
memory (CD38?CD27?), plasmablast (CD38??CD27??),
and double negative (CD38?CD27?), as described previously
Measurement of autoantibodies. Serum IgM rheuma-
toid factor was determined by nephelometry. Serum antibodies
to Ro/SS-A and La/SS-B were measured by enzyme-linked
immunosorbent assay (ELISA) in the Clinical Immunology
Laboratory at the Hospital of the University of Pennsylvania.
For assay of anti–type 3 muscarinic acetylcholine receptor
(anti-M3R) antibodies, patient sera were incubated with
Flp-In Chinese hamster ovary cells that had been transfected
with a human M3R construct, as previously described (21).
Transfected or nontransfected control cells were incubated
with 5 ?l of serum for 2 hours on ice, washed once, and stained
with fluorescein isothiocyanate–conjugated goat anti-rabbit
antibodies to human IgG or IgA. After washing, cells were
analyzed with a FACScan flow cytometer (Becton Dickinson).
The results are expressed as the mean fluorescence intensity
Determination of serum BAFF levels. ELISA was
performed using anti-human BAFF monoclonal antibody
clone B4H7.2 for coating and biotin-labeled anti-human BAFF
clone A9C9.1 for detection. Standard curves were constructed
with recombinant BAFF (all from Biogen Idec). To control for
interassay variations in each freshly prepared standard curve, a
2-point interassay control standard was applied. Serum was
diluted 1:10 (or higher if necessary for high BAFF levels) and
tested in triplicate.
Gene expression profiling. Peripheral blood was col-
lected into 2 PAXgene RNA tubes (PreAnalytiX), and total
RNA was extracted using PAXgene 96 Blood RNA kits
(Qiagen). Excess globin transcripts were removed using Am-
bion GlobinClear (Life Technologies), following the manufac-
turer’s protocols. RNA concentrations and quality were as-
sessed using an Agilent 2100 Bioanalyzer (quality threshold
28S:18S ribosomal RNA ratios ?1.0; RNA concentration
?70 ng/?l and no more than 14 ?l for optimum cleaning).
Biotinylated, amplified RNA was produced from 300 ng of
RNA using a modification of the Eberwine protocol (22) as
described in the Illumina TotalPrep RNA Amplification kit
(Ambion). The complementary RNA was hybridized overnight
at 58°C to Human WG-6 Expression BeadChip microarrays
(Illumina), washed under high-stringency conditions, labeled
with streptavidin–Cy3, and scanned.
Raw intensity values were background subtracted us-
ing Illumina BeadStudio software. Probe level analysis was
performed using the BeadConductor Lumi package for R, a
package specifically written to process Illumina microarray
data (www.bioconductor.org). The raw gene expression data
were subjected to variance-stabilizing transformation; robust
splines were then applied for normalization. The 8 samples
with data for each time point (baseline and 8 weeks, 26 weeks,
and 52 weeks following rituximab) were included in the
analysis. The expression data were not normalized for the
numbers of B cells in the blood. Paired t-tests and P values
were calculated at each 8-week, 26-week, and 52-week time
point compared to baseline. IPA 9.0 software (Ingenuity
Systems) was used to map transcripts to canonical pathways.
RITUXIMAB THERAPY FOR PRIMARY SJO ¨GREN’S SYNDROME1099
Statistical analysis. The sample size of 12 patients was
chosen to obtain sufficient information about safety in this
disease population, with a secondary objective of obtaining
preliminary information about its possible clinical and biologic
activity. A sample size of 12 patients is the minimum size
required to generate a one-sided 90% confidence interval that
excludes a prevalence of 30% for an event if no more than
one treatment-related AE of unacceptable severity is observed.
The frequencies of different B cell subsets were compared
between patients with primary Sjo ¨gren’s syndrome and healthy
controls, using the Mann-Whitney 2-tailed exact test. P values
for all other tests were based on Wilcoxon’s signed rank test
and examined the null hypothesis that the median difference
score between the baseline visit and the week 26 visit is equal
to 0. P values less than or equal to 0.05 were considered
statistically significant. No adjustments were made for multiple
comparisons. Calculations were performed using SAS version
9.1 software, or higher (SAS Institute).
Recruitment took place between April 2005 and
July 2006, with 6 patients enrolled from Duke University
Medical Center and 6 from the University of Pennsyl-
vania. All 12 patients received the full dose of rituximab,
completed the study followup through week 52, and
were included in the safety and efficacy analyses. Their
baseline characteristics are shown in Table 1. Most of
the enrolled participants had relatively low baseline
rates of unstimulated and stimulated whole salivary flow.
Eight patients were taking hydroxychloroquine, while
only 3 patients were receiving oral prednisone ?10 mg/
day. Four of the patients were receiving an oral secre-
tagogue (2 taking pilocarpine and 2 taking cevimeline).
Safety. The rituximab infusions were generally
well-tolerated in this study (Table 2). Two patients
experienced a serious adverse event (SAE), including 1
patient who had a grade 2 reaction to a pneumococcal
vaccine, consisting of local (pain, swelling, and numb-
ness) and systemic (fever, myalgia) symptoms that re-
sulted in an emergency department evaluation and
treatment with parenteral and oral antibiotics. Of note,
another patient in the study had a grade 2 vaccination
reaction, and an additional patient had a grade 2 vacci-
nation reaction associated with fever and chills; neither
of these AEs was considered an SAE. None of the other
5 patients who received both a pneumococcal and a
diphtheria and tetanus toxoid vaccine had AEs from
those immunizations. In all 3 cases, these AEs resolved
without apparent sequelae. No subsequent vaccinations
were administered following these AEs, owing to the
unanticipated severity of these reactions. The other
SAE was a squamous cell carcinoma of the skin that
occurred 301 days after administration of rituximab that
was considered to be possibly related to the study drug.
Characteristics of the 12 patients with primary Sjo ¨gren’s
Age, median (range) years
Race, no. (%)
Disease duration, median (range) years
Ocular symptoms, no. (%)
Oral symptoms, no. (%)
Ocular signs, no. (%)
Schirmer I test score, median (range) mm
Lissamine green staining score, median
(range) (0–18 scale)
Whole salivary flow rate, median (range)
Minor LSG focus score ?1 per 4 mm2,
Fatigue (VAS score ?50 mm), no. (%)
Joint pain (VAS score ?50 mm), no. (%)
Severe parotid gland swelling, no. (%)
Other extraglandular disease, no. (%)
Peripheral neuropathy, no. (%)
Interstitial lung disease, no. (%)
Antibodies, no. (%)
IgM rheumatoid factor
Short Form 36 health survey, median (range)
* LSG ? labial salivary gland; VAS ? visual analog scale (0–100 mm).
Adverse events in the 12 patients with primary Sjo ¨gren’s
No. of AEs reported
No. (%) of patients with AEs
No. of SAEs
No. of SAEs related to rituximab
No. (%) of patients with SAEs
No. (%) of AEs, by severity
Mild (grade 1)
Moderate (grade 2)
Severe (grade 3)
Life-threatening (grade 4)
Fatal (grade 5)
No. (%) of patients with AEs, by severity
Mild (grade 1)
Moderate (grade 2)
Severe (grade 3)
* AEs ? adverse events; SAEs ? serious adverse events.
† Patient was diagnosed as having squamous cell carcinoma of the skin
301 days after rituximab therapy (considered possibly related to the
1100ST.CLAIR ET AL
We did not observe any serum sickness–like reactions in
Clinical efficacy. The results showed significant
but modest levels of improvement between week 0 and
week 26 in the both the physician’s (median decrease
26 mm; P ? 0.012) and patient’s (median decrease
8.5 mm; P ? 0.009) global rating of disease activity.
Although positive trends toward subjective improve-
ment in dryness were observed in many items of the
survey, only the changes in the ratings of tongue dryness
(median decrease 11.1 mm; P ? 0.007), level of thirst
(median decrease 35.5 mm; P ? 0.005), level of oral
discomfort (median decrease 23.5 mm; P ? 0.02), and
level of overall fatigue (median decrease 18.4 mm; P ?
0.042) reached statistical significance. There was no
significant improvement in joint pain (median decrease
4.0 mm; P ? 0.077). We also did not find any statistically
significant improvement between week 0 and week 26
in the unstimulated (median change 0.01; P ? 0.287) or
stimulated (median change 0.005; P ? 0.718) whole
salivary flow. There were also no significant changes in
tear production, as measured by the unanesthetized
Schirmer’s test, or ocular surface dryness, as determined
by a modified von Bijsterveld scoring system (0–18
scale). Although no significant improvement was ob-
served between week 0 and week 26 in the summary
measures on the SF-36 for physical and mental func-
tioning, a statistically significant increase was found
during this period in the scores on the vitality scale
(P ? 0.006).
Peripheral blood B cell depletion and reconstitu-
tion. The absolute peripheral blood CD19? lymphocyte
(B cell) counts at baseline ranged from 45 cells/?l to
341 cells/?l (median 178). Following rituximab treat-
ment, all 12 patients showed ?95% depletion of blood
B cells by week 8 or week 14 (Figure 1). The blood B cell
counts had a nadir at weeks 8 and 14 and began to rise
by week 26; they returned to ?62% of baseline values by
week 52 in 8 of the 12 patients.
The subsets of circulating B cells were analyzed
according to the surface expression of CD38 and CD27,
a previously validated approach (20,23). Using these
markers, we defined 6 different subsets: transitional B
cells (CD38??CD27?), mature naive B cells (CD38?
CD27?), mature activated memory B cells (CD38?
CD27?), resting memory B cells (CD38?CD27?), plas-
mablasts (CD38??CD27??), and so-called double-
negative B cells (CD38–CD27?). Compared to our
healthy controls, the patients with primary Sjo ¨gren’s
syndrome at baseline had a greater number of transi-
tional B cells and a lower number of memory B cells
(data available upon request from the corresponding
author). Therefore, the distribution of circulating B cell
subsets in the 12 patients from our study was similar to
that in patients with primary Sjo ¨gren’s syndrome de-
scribed previously (10,15).
After rituximab therapy, the initial wave of re-
populating blood B cells comprised mainly transitional
B cells (Figure 2 and Table 3). At week 26, the median
number of transitional B cells (CD38??CD27?) in the
circulation was 10.3 cells/?l (65% of total CD19? B
cells). By comparison, the naive mature B cells at week
26 comprised, on average, only 8.3% (median 0.57
cells/?l) of the total CD19? B cells. Mature activated
and resting memory B cells (CD38?CD27? and
CD38?/CD27?) were relatively rare during this initial
phase of reconstitution, comprising ?4% (median) of
the total circulating CD19? B cells. By week 52, the
median number of circulating transitional B cells had
increased to 21.3 cells/?l (19.3% of total CD19? B
cells). While at the same time, the median number of
mature naive B cells (CD38?CD27?) had increased to
Figure 1. Total numbers of blood CD19? B cells before and after
treatment with rituximab in patients with primary Sjo ¨gren’s syndrome.
Absolute B cell counts were obtained by multiplying the absolute
lymphocyte count obtained from the complete blood cell count by the
CD19? fraction obtained by flow cytometry. Plotted are the whole
blood CD19? lymphocyte counts as a function of time. Time zero
is the baseline assessment and corresponds to the day of the first
rituximab infusion. Each symbol represents a different patient; lines
connect the individual data points.
RITUXIMAB THERAPY FOR PRIMARY SJO ¨GREN’S SYNDROME1101
75.9 cells/?l (73.1% of total CD19? B cells). The median
number of circulating mature activated (CD38?
CD27?) and resting memory (CD38–CD27?) B cells
remained diminished, both in absolute and in relative
terms, at week 52, representing only 3.2% and 0.4%,
respectively, of the total CD19? B cells. Thus, 52 weeks
following rituximab therapy, the more mature B cell
subsets had not yet fully repopulated the circulating
Blood T cell, natural killer (NK) cell, and mono-
cyte levels. Rituximab therapy was not associated with
any substantial changes in the numbers or percentages
of blood CD3?, CD4?, and CD8? T cells, CD16?
CD56? NK cells, or CD14? and CD11b monocytes
(data not shown).
Autoantibody levels. Rituximab therapy had little
effect on the serum levels of anti-Ro/SSA and anti-La/
SSB antibodies (data not shown). However, there was a
trend toward a decrease in the levels of serum rheuma-
toid factor, but this difference failed to reach statistical
significance (median 150.0 units/liter [25th, 75th percen-
tiles 20, 1930] at week 0 versus median 87 units/liter
[25th, 75th percentiles 20, 1560]; P ? 0.109) at week 26.
Serum antibodies to M3R have been reported to
occur in primary Sjo ¨gren’s syndrome and may be asso-
ciated with impaired cholinergic transmission (24,25).
Baseline data for serum anti-M3R antibodies were avail-
able for 11 of the 12 patients in the study. In general, we
observed no significant changes in the serum levels of
anti-MR3 antibodies between baseline and week 26,
except for a lone patient whose values decreased from
an MFI of 35 to an MFI of 17. Of note, this particular
patient did not show an increase in either the unstimu-
lated (0.12 ml/minute at baseline and 0.06 ml/minute at
week 26) or stimulated (0.33 ml/minute at baseline and
0.36 ml/minute at week 26) whole salivary flow rate over
this time period.
Serum BAFF levels. BAFF is a key survival factor
for B cells and is important for the maintenance of
Analysis of peripheral blood B cell subsets before and after
B cell subset, time point
No. of cells/?l,
Mature naive, CD38?CD27?
Mature activated memory, CD38?CD27?
Resting memory, CD38?CD27?
Double negative, CD38?CD27?
* Time points are the number of weeks following rituximab adminis-
tration. B cell subsets were determined as described in Patients and
Methods. Data on B cell subsets were not available for patient 08-096
at baseline or for patient 04-098 at week 26.
Figure 2. Analysis of blood B cell subsets following rituximab therapy
in patients with primary Sjo ¨gren’s syndrome. The average percentages
of B cells in each of the different B cell subsets (CD19? lymphocytes)
in all 12 patients are plotted as a function of time following rituximab
treatment. Time zero is the baseline assessment and corresponds to the
day of the first rituximab infusion. B cell subsets were defined
according to the expression of CD27 and CD38, as described in
Patients and Methods.
1102ST.CLAIR ET AL
peripheral B cell homeostasis (26). Serum BAFF levels
are elevated in primary Sjo ¨gren’s syndrome compared
with healthy controls (27) and have been shown to
increase after B cell depletion therapy and then gradu-
ally return to baseline following reconstitution of the
circulating pool (28,29). We found in our study that
median serum BAFF levels followed similar kinetics,
rising substantially while circulating CD19? B cells were
maximally depleted and then returning toward base-
line with reconstitution of the circulating B cell pool
(Figures 3A and B).
Effect of rituximab therapy on the interferon
(IFN) signature and expression of other gene tran-
scripts. In 8 patients, we analyzed the effects of ritux-
imab therapy on IFN signature transcripts, which are
up-regulated in primary Sjo ¨gren’s syndrome (30), as well
as the expression of other gene transcripts by comparing
the transcript levels at baseline with those at weeks 8, 26,
and 52 (31). Overall, we found a significant change in the
expression of 94, 77, and 342 genes at these time points,
respectively, compared to baseline (P ? 0.001). Among
63 IFN-related transcripts (IFNs, interferon regulatory
factors [IRFs], and interferon inducible [IFI] genes),
only IRF-4, IRF-8, interferon-induced transmembrane
protein 1 (IFITM-1), IFI-30, and IFITM-4P showed
statistically significant changes (P ? 0.01) between base-
line and any of these 3 subsequent time points.
We observed a significant decrease after ritux-
imab therapy in the expression of several B cell–related
genes, including CD79A, LOC652493 (Ig ?-chain V-I
region HK102-like), IGKV3D-20 (Ig? variable 3D-20),
FCRLA, LOC647450 (similar to Ig ?-chain V–I region
HK101 precursor), LOC652775 (similar to Ig ?-chain
V–V region L7 precursor), VPREB3, and BLK (data
available upon request from the corresponding author).
Since the expression data were not normalized for the
numbers of blood B cells, these changes probably reflect
the depleting effects of rituximab. These changes were
most pronounced at week 8 and returned toward base-
line values by week 52.
We also performed a pathway analysis, with the
top 3 canonical pathways mapping to primary immuno-
deficiency signaling (P ? 0.01 for baseline versus
week 8), altered T cell and B cell signaling (P ? 0.01
for baseline versus week 8 and versus week 26), phos-
phatidylinositol 3-kinase signaling in B lymphocytes
(P ? 0.01 for baseline versus week 26), and B cell
Figure 3. Relationship between blood B cell depletion and serum BAFF levels. A, Median serum BAFF levels (relative [Rel.] units) are significantly
elevated above baseline at weeks 8 and 14, the time of maximal blood B cell depletion, and slowly decline to near baseline levels by week 52,
consistent with reconstitution of the blood B cell pool. B, The relationship between the numbers of blood B cells and serum BAFF levels is plotted
for individual patients at weeks 0, 8, 14, 26, 36, and 52.
RITUXIMAB THERAPY FOR PRIMARY SJO ¨GREN’S SYNDROME 1103
development (P ? 0.01 for baseline versus week 8 and
versus week 26).
Our results show that two 1-gm infusions of
rituximab given 2 weeks apart produce effective deple-
tion of circulating B cells in patients with primary
Sjo ¨gren’s syndrome, with kinetics and pattern of B cell
subset reconstitution similar to those observed in an-
other study of patients with the same disease (10). The
corresponding increases in serum BAFF levels following
the depletion of blood B cells confirm earlier observa-
tions (31). The exploratory analysis of gene transcripts
and pathways provides further evidence that rituximab
therapy substantially alters B cell responses in this
Importantly, we did not detect any unexpected
safety signals, except for the possibility of exaggerated
vaccine reactions. Three of the patients in our trial had
an unusually severe reaction to the pneumococcal vac-
cine given at week 26. However, these vaccines were
administered to 68 patients with rheumatoid arthritis
who were treated with rituximab in a randomized open-
label trial without apparent untoward effects beyond
the usual occurrence of itching, rash, and soreness at the
injection site, and malaise (32). Serum sickness has
occurred in some patients with primary Sjo ¨gren’s syn-
drome following a rituximab infusion (6,7,9), but such an
event was not observed in the current trial. We at-
tempted to minimize this risk by premedicating patients
with 100 mg of methylprednisolone in addition to di-
phenhydramine and acetaminophen.
Despite the effective depletion of blood B cells,
rituximab therapy was not associated with striking clin-
ical benefits in the current trial. Any improvements
observed in an open-label study must be interpreted
with caution because of the inherent subjectivity of many
of the disease measures and the possibility of observer
bias. Previous studies investigated the potential clinical
efficacy and safety of rituximab therapy for primary
Sjo ¨gren’s syndrome (6–9), but they too, were limited by
their small sample size, open-label design in some cases,
and the lack of standardized treatment outcomes.
The results of the present study are consistent
with those of a 122-patient, randomized, placebo-
controlled study of rituximab therapy in primary Sjo ¨-
gren’s syndrome, which was reported in the form of
an abstract (33). In that study, rituximab therapy was
not significantly more effective than placebo in improv-
ing by ?30 mm the scores on at least 2 of the 4 100-mm
VAS scales evaluating dryness, pain, fatigue, and global
disease activity. Since relatively few patients in our study
had extraglandular manifestations beyond constitutional
symptoms and joint pain, we were unable to explore the
effects of rituximab therapy on severe systemic features.
Rituximab therapy has been shown in some cases, how-
ever, to benefit such systemic features as refractory
pulmonary disease, synovitis, and mixed cryoglobuline-
mia (34). To date, rituximab treatment has not been
consistently associated with an increase in lacrimal and
salivary gland function. It has been suggested that
patients whose lacrimal and salivary glands have mini-
mal secretory capacity due to long-standing disease may
be particularly refractory to disease-modifying treat-
ment (6). Indeed, 7 of the 12 patients in our trial had
stimulated salivary flow rates ?0.1 ml/minute at entry,
and it may be argued that their glandular function had
limited potential for improvement.
Rituximab therapy did not appear to affect the
serum levels of anti-SSA/Ro and anti-SSB/La anti-
bodies, although serum rheumatoid factors trended
lower, as has been seen in earlier studies (8,9). Ritux-
imab treatment also had no impact on the serum levels
of anti-M3R antibodies. This result is of interest because
of the possible role of these autoantibodies in the
mechanisms of impaired salivary flow. We also hypoth-
esized that rituximab treatment would alter the blood
IFN signature in primary Sjo ¨gren’s syndrome, based on
a previous study showing that it can induce type I IFN
activity in patients with rheumatoid arthritis (35). How-
ever, we were unable to demonstrate such an effect in
our study, which is not unexpected, based on the estab-
lished relationship between type I IFN and BAFF.
Elevated serum levels of BAFF, which result from B cell
depletion, are not known to induce type I IFN activity.
Rather, type I IFN has been shown to induce BAFF
In summary, rituximab treatment for primary
Sjo ¨gren’s syndrome in this small open-label trial was
associated with no unexpected toxicities, except possibly
for exaggerated vaccine reactions. It led to only modest
improvements in symptoms and no beneficial changes
in lacrimal or salivary gland function. In addition to
detailed studies of blood B cell subsets, exploratory
analyses of gene transcripts and pathways in the peri-
pheral blood suggest rituximab therapy substantially
alters B cells, while having little impact on the IFN
signature. Larger randomized placebo-controlled clini-
cal trials such as those reported by Devauchelle-Pensec
and colleagues (33) are needed to further evaluate the
1104 ST.CLAIR ET AL
clinical efficacy and safety of rituximab therapy for
primary Sjo ¨gren’s syndrome.
The authors wish to acknowledge Dr. Thomas A.
McGraw (Duke University Medical Center) for assistance with
the conduct of the study, Dr. Yang-Zhu Du (University of
Pennsylvania) for technical assistance with the flow cytometry
experiments, Dr. Dennis Wallace (formerly of Rho, Inc.) for
assistance with the study design, and Beverly Welch (National
Institute of Allergy and Infectious Diseases) for project man-
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. Dr. St.Clair had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. St.Clair, Levesque, Vivino, Wedgwood,
Acquisition of data. St.Clair, Levesque, Prak, Vivino, Alappatt,
Wedgwood, Sivils, Fisher, Cohen.
Analysis and interpretation of data. St.Clair, Levesque, Prak,
Spychala, McNamara, Sivils, Cohen.
ROLE OF THE STUDY SPONSOR
The study was designed by the investigators and coordinated
by the National Institutes of Allergy and Infectious Diseases (NIAID;
the study sponsor) and Rho, Inc., which managed the collection and
quality control of the data and performed the statistical analyses.
Rituximab was provided at no cost by Genentech, which had no other
role in the design, conduct, or analysis of the study. Publication of this
article was not contingent upon approval by the NIAID, Rho, Inc., or
1. Bowman SJ, Ibrahim GH, Holmes G, Hamburger J, Ainsworth JR.
Estimating the prevalence among Caucasian women of primary
Sjo ¨gren’s syndrome in two general practices in Birmingham, UK.
Scand J Rheumatol 2004;33:39–43.
2. Fox RI. Sjo ¨gren’s syndrome. Lancet 2005;366:321–31.
3. Ramos-Casals M, Solans R, Rosas J, Camps MT, Gil A, del
Pino-Montes J, et al, GEMESS Study Group. Primary Sjo ¨gren’s
syndrome in Spain: clinical and immunologic expression in 1010
patients. Medicine (Baltimore) 2008;87:210–9.
4. Theander E, Henriksson G, Ljungberg O, Mandl T, Manthorpe R,
Jacobsson LT. Lymphoma and other malignancies in primary
Sjo ¨gren’s syndrome: a cohort study on cancer incidence and
lymphoma predictors. Ann Rheum Dis 2006;65:796–803.
5. Ramos-Casals M, Tzioufas AG, Stone JH, Siso A, Bosch X.
Treatment of primary Sjo ¨gren’s syndrome: a systematic review.
6. Pijpe J, van Imhoff GW, Spijkervet FK, Roodenburg JL, Wolbink
GJ, Mansour K, et al. Rituximab treatment in patients with
primary Sjo ¨gren’s syndrome: an open-label phase II study. Arthri-
tis Rheum 2005;52:2740–50.
7. Devauchelle-Pensec V, Pennec Y, Morvan J, Pers JO, Daridon C,
Jousse-Joulin S, et al. Improvement of Sjo ¨gren’s syndrome after
two infusions of rituximab (anti-CD20). Arthritis Rheum 2007;57:
8. Dass S, Bowman SJ, Vital EM, Ikeda K, Pease CT, Hamburger J,
et al. Reduction of fatigue in Sjo ¨gren’s syndrome with rituximab:
results of a randomized, double-blind, placebo-controlled pilot
study. Ann Rheum Dis 2008;67:1541–4.
9. Meijer JM, Meiners PM, Vissink A, Spijkervet FK, Abdulahad W,
Kamminga N, et al. Effectiveness of rituximab treatment in
primary Sjo ¨gren’s syndrome: a randomized, double-blind, placebo-
controlled trial. Arthritis Rheum 2010;62:960–8.
10. Abdulahad WH, Meijer JM, Kroese FG, Meiners PM, Vissink A,
Spijkervet FK, et al. B cell reconstitution and T helper cell balance
after rituximab treatment of active primary Sjo ¨gren’s syndrome: a
double-blind, placebo-controlled study. Arthritis Rheum 2011;63:
11. Edwards JC, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A,
Emery P, Close DR, et al. Efficacy of B-cell-targeted therapy with
rituximab in patients with rheumatoid arthritis. N Engl J Med
12. Cohen SB, Emery P, Greenwald MW, Dougados M, Furie RA,
Genovese MC, et al, for the REFLEX Trial Group. Rituximab for
rheumatoid arthritis refractory to anti–tumor necrosis factor ther-
apy: results of a multicenter, randomized, double-blind, placebo-
controlled, phase III trial evaluating primary efficacy and safety at
twenty-four weeks. Arthritis Rheum 2006;54:2793–806.
13. Stone JH, Merkel PA, Spiera R, Seo P, Langford CA,
Hoffman GS, et al. Rituximab versus cyclophosphamide for
ANCA-associated vasculitis. N Engl J Med 2010;363:221–32.
14. Youinou P, Devauchelle-Pensec V, Pers JO. Significance of B cells
and B cell clonality in Sjo ¨gren’s syndrome [review]. Arthritis
15. Hansen A, Gosemann M, Pruss A, Reiter K, Ruzickova S,
Lipsky PE, et al. Abnormalities in peripheral B cell memory of
patients with primary Sjo ¨gren’s syndrome. Arthritis Rheum 2004;
16. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexan-
der EL, Carsons SE, et al, and the European Study Group on
Classification Criteria for Sjo ¨gren’s Syndrome. Classification cri-
teria for Sjo ¨gren’s syndrome: a revised version of the European
criteria proposed by the American-European Consensus Group.
Ann Rheum Dis 2002;61:554–8.
17. Vivino FB, Al-Hashimi I, Khan Z, LeVeque FG, Salisbury PL,
Tran-Johnson TK, et al. Pilocarpine tablets for the treatment of
dry mouth and dry eye symptoms in patients with Sjo ¨gren’s
syndrome: a randomized, placebo-controlled, fixed-dose, multi-
center trial. Arch Intern Med 1999;159:174–81.
18. Talamo J, Frater A, Gallivan S, Young A. Use of the short form
(SF36) for health status measurement in rheumatoid arthritis. Br J
19. Abdallah KO, Luning Prak ET. B cell monitoring of transplant
patients treated with anti-CD20. Clin Transpl 2006:427–37.
20. Sutter JA, Kwan-Morley J, Dunham J, Du YZ, Kamoun M,
Albert D, et al. A longitudinal analysis of SLE patients treated
with rituximab (anti-CD20): factors associated with B lymphocyte
recovery. Clin Immunol 2008;126:282–90.
21. Gao J, Cha S, Jonsson R, Opalko J, Peck AB. Detection of
anti–type 3 muscarinic acetylcholine receptor antibodies in the
sera of Sjo ¨gren’s syndrome patients by use of a transfected cell line
assay. Arthritis Rheum 2004;50:2615–21.
22. Van Gelder RN, von Zastrow ME, Yool A, Dement WC, Barchas
JD, Eberwine JH. Amplified RNA synthesized from limited
quantities of heterogeneous cDNA. Proc Natl Acad Sci U S A
23. Sekiguichi DR, Smith SB, Sutter JA, Goodman NG, Propert K,
Louzoun Y, et al. Circulating lymphocyte subsets in normal adults
are variable and can be clustered into subgroups. Cytometry B Clin
RITUXIMAB THERAPY FOR PRIMARY SJO ¨GREN’S SYNDROME1105
24. Waterman SA, Gordon TP, Rischmueller M. Inhibitory effects
of muscarinic receptor autoantibodies on parasympathetic neuro-
transmission in Sjo ¨gren’s syndrome. Arthritis Rheum 2000;43:
25. Park K, Haberberger RV, Gordon TP, Jackson MW. Antibodies
interfering with the type 3 muscarinic receptor pathway inhibit
gastrointestinal motility and cholinergic neurotransmission in
Sjo ¨gren’s syndrome. Arthritis Rheum 2011;63:1426–34.
26. Mackay F, Figgett WA, Saulep D, Lepage M, Hibbs ML. B-cell
stage and context-dependent requirements for survival signals
from BAFF and the B cell receptor. Immunol Rev 2010;237:
27. Groom J, Kalled SL, Cutleer AH, Olson C, Woodcock SA,
Schneider P, et al. Association of BAFF/BLyS overexpression
and altered B cell differentiation with Sjo ¨gren’s syndrome. J Clin
28. Pers JO, Devauchelle V, Daridon C, Bendaoud B, Le Berre R,
Bordron A, et al. BAFF-modulated repopulation of B lymphocytes
in the blood and salivary glands of rituximab-treated patients with
Sjo ¨gren’s syndrome. Arthritis Rheum 2007;56:1464–77.
29. Cambridge G, Stohl W, Leandro MJ, Migone TS, Hilbert DM,
Edwards JC. Circulating levels of B lymphocyte stimulator in
patients with rheumatoid arthritis following rituximab treatment:
relationships with B cell depletion, circulating antibodies, and
clinical relapse. Arthritis Rheum 2006;54:723–32.
30. Emamian ES, Leon JM, Lessard CJ, Grandits M, Baechler EC,
Gaffney PM, et al. Peripheral blood gene expression profiling in
Sjo ¨gren’s syndrome. Genes Immun 2009;4:285–96.
31. Lavie F, Miceli-Richard C, Ittah M, Sellam J, Gottenberg J,
Mariette X. Increase of B cell-activating factor of the TNF
family (BAFF) after rituximab treatment: insights into a new
regulating system of BAFF production. Ann Rheum Dis 2007;66:
32. Bingham CO III, Looney RJ, Deodhar A, Halsey N, Green-
wald M, Codding C, et al. Immunization responses in rheumatoid
arthritis patients treated with rituximab: results from a controlled
clinical trial. Arthritis Rheum 2010;62:64–74.
33. Devauchelle-Pensec V, Mariette X, Jousse-Joulin S, Berthelot JM,
Perdriger A, Hachulla E, et al. Tolerance and efficacy of rituximab
in primary Sjo ¨gren syndrome: final results of a randomized con-
trolled trial [abstract]. Arthritis Rheum 2012;64 Suppl:S1079.
34. Seror R, Sordet C, Guillevin L, Hachulla E, Masson C, Ittah M,
et al. Tolerance and efficacy of rituximab and changes in serum
B cell biomarkers in patients with systemic complications of
primary Sjo ¨gren’s syndrome. Ann Rheum Dis 2007;66:351–7.
35. Vosslamber S, Raterman HG, van der Pouw Krann TC, Schreurs
MW, von Blomberg BM, Nurmohamed MT, et al. Pharmacologi-
cal induction of interferon type I activity following treatment with
rituximab determines clinical response in rheumatoid arthritis.
Ann Rheum Dis 2011;70:1153–9.
36. Brkic Z, Maria NI, van Helden-Meeuwsen CG, van de Merwe JP,
van Daele PL, Dalm VA, et al. Prevalence of interferon type I
signature in CD14 monocytes of patients with Sjo ¨gren’s syndrome
and association with disease activity and BAFF gene expression.
Ann Rheum Dis 2012. E-pub ahead of print.
In the article by Shao et al in the March 2013 issue of Arthritis & Rheumatism (pages 780–791), the name
of one of the authors was incorrectly shown as Ward Wakeland. The author’s correct name is Edward K.
We regret the error.
1106 ST.CLAIR ET AL