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Serologic changes following B lymphocyte depletion therapy for rheumatoid arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 8, August 2003, pp 2146–2154
DOI 10.1002/art.11181
© 2003, American College of Rheumatology
Serologic Changes Following B Lymphocyte Depletion Therapy
for Rheumatoid Arthritis
Geraldine Cambridge,1 Maria J. Leandro,1 Jonathan C. W. Edwards,1 Michael R. Ehrenstein,1
Martin Salden,2 Mark Bodman-Smith,3 and Anthony D. B. Webster4
unpredictable (range 0–17 months). Relapse was, however, closely correlated with rises in the level of at least
one autoantibody. Increased autoantibody levels were
rarely observed in the absence of clinical change.
Conclusion. Following B lymphocyte depletion in
patients with RA, a positive clinical response occurred
in correlation with a significant drop in the levels of
CRP and autoantibodies. Antibacterial antibody levels
were relatively well maintained. B lymphocyte return
preceded relapse in all patients. There was also a
temporal relationship between clinical relapse and rises
in autoantibody levels. Although these observations are
consistent with a role for B lymphocytes in the pathogenesis of RA, the precise mechanisms involved remain
unclear.
Objective. To explore the changes in serologic
variables and clinical disease activity following B lymphocyte depletion in 22 patients with rheumatoid arthritis (RA).
Methods. B lymphocyte depletion was attained
using combination therapy based on the monoclonal
anti-CD20 antibody rituximab. Levels of a serologic
indicator of inflammation, C-reactive protein (CRP), of
antimicrobial antibodies, of autoantibodies including
IgA-, IgM-, and IgG-class rheumatoid factors (RF), and
of antibodies to cyclic citrullinated peptide (anti-CCP)
were assayed.
Results. The majority of patients showed a
marked clinical improvement after treatment with rituximab, with benefit lasting up to 33 months. Levels of
total serum immunoglobulins fell, although the mean
values each remained within the normal range. Whereas
the IgM-RF response paralleled the changes in total
serum IgM levels, the levels of IgA-RF, IgG-RF, and IgG
and anti-CCP antibodies decreased significantly more
than did those of their corresponding total serum
immunoglobulin classes. The kinetics for the reduction
in CRP levels also paralleled the decreases in autoantibody levels. In contrast, levels of antimicrobial antibodies did not change significantly. B lymphocyte return
occurred up to 21 months posttreatment. The time to
relapse after B lymphocyte return was often long and
The relative contribution of the different elements of the immune system to the pathogenesis of
rheumatoid arthritis (RA) remains controversial (1,2).
The genetic link with HLA–DR4 (3) implies a T cell–
dependent process. It is now evident that T cell–B cell
interaction is a complex 2-way process, in which the
function of each cell type is dependent on the other (4).
Moreover, this interaction may, in special cases, be
atypical, as for example, with rheumatoid factor (RF) B
cells, which can potentially present any T cell with its
cognate antigen and in return receive a positive survival
signal (5).
A specific subset of autoreactive B lymphocyte
clones, capable of self-perpetuation, has recently been
proposed to be involved in disease persistence in RA (2).
In this model, a dual role for B lymphocytes, and in
particular, those committed to producing RF, has been
suggested. First, they may differentiate into plasma cells
that produce autoantibodies capable of forming small
immune complexes. Interaction of such small immune
complexes with the immunoglobulin receptor Fc␥ receptor type IIIa (Fc␥RIIIa) on macrophages, in joints,
Supported by the Arthritis Research Campaign, UK.
1
Geraldine Cambridge, PhD, Maria J. Leandro, MD,
Jonathan C. W. Edwards, MD, Michael R. Ehrenstein, MD, PhD:
University College London, London, UK; 2Martin Salden, PhD:
Euro-Diagnostica, Arnhem, The Netherlands; 3Mark Bodman-Smith,
PhD: Guy’s Hospital, London, UK; 4Anthony D. B. Webster, MD:
Royal Free Hospital, London, UK.
Address correspondence and reprint requests to Jonathan
C. W. Edwards, MD, Centre for Rheumatology, Arthur Stanley
House, 40-50 Tottenham Street, London W1T 4NJ, UK. E-mail:
jo.edwards@ucl.ac.uk.
Submitted for publication June 7, 2002; accepted in revised
form April 10, 2003.
2146
B LYMPHOCYTE DEPLETION IN PATIENTS WITH RA
and in other tissues may be responsible for the production of proinflammatory cytokines (6,7). The Fc␥RIIIdependent arthritis of the K/BxN transgenic mouse may
be a useful model for this mechanism (8). Second,
daughter plasma cells may perpetuate the survival of
parent RF B lymphocytes by providing a constant supply
of self-complexed IgG (2).
Therapeutic B lymphocyte depletion provides a
new opportunity to assess the roles of B lymphocytes in
the pathogenesis of RA and other autoimmune diseases.
B lymphocyte depletion has recently been introduced
as a therapy for a range of autoantibody-associated
disorders, including RA, IgM-associated neuropathies,
immune thrombocytopenic purpura, autoimmune hemolytic anemia, systemic lupus erythematosus, and dermatomyositis (9–15). Encouraging results have been
reported, and a randomized controlled trial of this
therapy in RA is currently in progress.
Selective B lymphocyte depletion has been made
possible by the availability of the chimeric anti-CD20
monoclonal antibody rituximab (16–21). CD20 is a B
lymphocyte–restricted antigen that is expressed on B
lymphocyte precursors and mature B lymphocytes. It is
lost during differentiation into plasma cells. Rituximab
has been proven to be very effective in depleting normal
and malignant B lymphocytes in vivo. In humans, peripheral B cell depletion occurs within days, and studies in
primates have shown that up to 70% of B cells in
lymphoid organs are also rapidly cleared (16,17,21). In
autoimmune diseases, rituximab has been used in many
cases as monotherapy (10–12). However, B lymphocyte
depletion with rituximab alone is probably incomplete,
and in patients with lymphoma, long-term benefit from
rituximab is increased by combination with other drugs
(20). For these reasons, in the treatment of RA, it has
initially been used in combination with cyclophosphamide and corticosteroid (9).
We have treated 23 patients with RA with rituximab, alone or in combination with other agents. The
detailed clinical responses in these patients have been
reported previously (9,15). The majority of patients
showed substantial clinical improvement, which was
sustained for up to 33 months and up to 17 months after
the return of circulating B lymphocytes. However, in all
but 2 patients, the disease subsequently relapsed. The
key objective of the present study is to understand the
mechanism of this relapse. Critically, this requires an
understanding of whether disease persistence depends
on the existence of specific T cell clones, specific B cell
clones, or the continued production of antibodies by
long-lived plasma cells.
2147
Circulating antibody levels are, at present, the
only practical indices for monitoring the autoimmune
response in RA. The most prevalent autoantibody reactivity in RA is RF, with IgG-RF being particularly
implicated in the formation of small immune complexes
(2,22), but a significant proportion of patients also have
raised levels of antibodies to cyclic citrullinated peptides
(anti-CCP) (23) and to proteins such as the immunoglobulin heavy-chain binding protein (anti-BiP) (24). In
the absence of T cell responses to IgG, there is particular
interest in the possible roles of T cell responses to these
other antigens and their interrelationships with RF B
cells. For 22 of our 23 patients with RA (described in
refs. 9 and 15), we have serial immunologic data covering the period of B lymphocyte depletion, during which
no other therapy was introduced. We report herein
serial observations of the total serum immunoglobulin
and specific autoantibody levels together with the levels
of protective antibodies to pathogens in these patients,
and we report the relationships of those changes to
regression and relapse of inflammation following B
lymphocyte depletion.
PATIENTS AND METHODS
Patients. Twenty-two patients with active RA in whom
total circulating B lymphocyte depletion was achieved for ⱖ3
months were studied. All patients satisfied the American
College of Rheumatology (ACR; formerly, the American
Rheumatism Association) diagnostic criteria for RA (25). Two
patients were male and 20 were female. The mean age was 58
years (range 33–81 years) and the mean disease duration was
18 years (range 5–40 years). The study was approved by the
local hospital ethics committee. All patients gave informed
consent. Serum samples were kept at ⫺80°C until tested for
autoantibodies and antimicrobial antibodies. Relapse was determined clinically and was defined as an increase in pain of at
least 20% on visual analog scale and an increase in the swollen
joint count among ⬎2 joints on 2 consecutive visits.
Treatment protocol. Patients were treated in 5 cohorts
with a range of doses of rituximab, with or without intravenous
cyclophosphamide and/or oral prednisolone, over a 2–4-week
period (9,15). Disease-modifying antirheumatic drugs (excluding steroids) were discontinued from day 0. All patients
received nonsteroidal antiinflammatory agents or analgesics as
needed.
Assessment. Patients were assessed prior to treatment,
monthly for the first 6 months after treatment, and then every
2 months until 1 year, and subsequently every 2–3 months.
Circulating B lymphocytes (CD19-positive cells, determined by
flow cytometry; normal range 0.03–0.40 ⫻ 109/liter) and total
lymphocytes were measured before treatment, monthly after
treatment in the first 6 months, and then every 2 months until
they returned to normal levels. Serial laboratory measurements in sera included the C-reactive protein (CRP) concentration, total immunoglobulin levels, IgM-, IgG-, and IgA-RF
2148
CAMBRIDGE ET AL
Table 1. Relationship between B lymphocyte return, levels of circulating autoantibodies, and relapse in responder and nonresponder patients with
rheumatoid arthritis*
Patient†
Autoantibodies present
at baseline
ACR response at
6 months
posttreatment, %
Time to B cell
return, months
Time to clinical
relapse, months
Time to autoantibody
rise, months
(responding antibody)
1
2
3
4
5
7
9
10
11
12
13
14
16
17
18
19
8
6
20
21
22
23
M, A, CCP
M, A, CCP
M, G
M, G, A, CCP
M, G, A, CCP
M, A
M, G, A
M, G, A, CCP
M, G, A
M, CCP
M, A, CCP
M, G, A, CCP
M, G, A
M, G, A
M, G, A, CCP
M, G, A, CCP
CCP
M, A, CCP
BiP
M, G, A
M, G, A
M, A, CCP
70
70
70
50
50
20
70
70
70
50
70
50
20
50
70
⬍20
⬍20
⬍20
⬍20
⬍20
⬍20
⬍20
20.8
6.9
8.8
7.4
11.1
8.3
6.7
8.5
10.4
6.5
12.0
10.2
9.5
6.0
5.8
8.5
3.5
4.6
6.9
9.2
5.1
7.0
33.5
6.9
25.6
7.8
21.2
7.2
7.4
9.0
⬎24.0
11.5
⬎19.0
12.2
17.3
13.2
7.4
NA
NA
NA
NA
NA
NA
NA
30 (M, A, CCP)
6 (M, A, CCP)
25.6 (M)
6 (A, CCP, M)
21.2 (M, G, A)
6.5 (M)
5.8 (M, A)
8.3 (A, M, G)
NYK
9.9 (M, CCP)
NYK
12.2 (M)
–
14.3 (M, A)
6 (M, G, A, CCP)
NA
NA
NA
NA
NA
NA
NA
* American College of Rheumatology (ACR) responders were those who achieved ⱖ20% improvement at 6 months posttreatment. M ⫽
IgM–rheumatoid factor (RF); A ⫽ IgA-RF; CCP ⫽ cyclic citrullinated peptide; G ⫽ IgG-RF; NYK ⫽ not yet known; NA ⫽ not applicable; BiP ⫽
immunoglobulin heavy-chain binding protein.
† Patient numbers are described in ref. 15. Serum from patient 15 was not available for study.
levels, and levels of anti-CCP antibodies. The CRP concentration was measured by nephelometry (normal range 0–5 mg/
liter). Total serum immunoglobulin levels were measured by
immunoturbidometry (normal range for IgA 0.7–4.0 gm/liter,
IgG 7.0–16.0 gm/liter, IgM 0.4–2.3 gm/liter).
Measurement of autoantibodies. Serial samples from
each patient were analyzed together on the same day. IgM-RF,
IgG-RF, and IgA-RF were measured by enzyme-linked immunosorbent assay (ELISA) using the RF-isotype detection kit
developed by Dr. M. Teodorescu (26) and supplied by TheraTest Laboratories (catalog no. EL-RF/3; Chicago, IL). This
test is based on the binding of RFs to rabbit IgG. The
TheraTest ELISA utilizes horseradish peroxidase (HRP)–
conjugated rabbit anti-human IgG (Fab⬘)2 to detect IgG-RF,
and prior to testing for IgG-RF, samples are digested with
pepsin to avoid interference from IgM-RF and IgA-RF (26).
Completeness of pepsin digestion was checked by confirming
the absence of binding of anti–IgM-HRP to pepsin-digested
samples and controls. Results are expressed in IU/ml. The
cutoff values for normal control sera were ⬍25 IU/ml, ⬍20
IU/ml, and ⬍35 IU/ml for IgM-RF, IgG-RF, and IgA-RF,
respectively. IgG anti-CCP antibodies were measured by
ELISA (Immunoscan RA; Euro-Diagnostica, Arnhem, The
Netherlands), and results are expressed as units equivalent to
the standard serum, as determined by reading on a calibration
curve.
Measurement of antimicrobial antibodies. Specific
IgG antibodies to tetanus toxoid (anti-TT) and to pneumococ-
cal capsular polysaccharides (anti-PCP; combination of 23
common serotypes) were measured by ELISA (Binding Site
Limited, Birmingham, UK). Levels ⬎0.1 IU/ml for anti-TT
antibodies were considered to be optimally protective; 90% of
the population have anti-PCP levels ⬎35 mg/liter.
Statistical analysis. For comparison of absolute values
before and after treatment, the Wilcoxon signed rank test for
paired data was used (1-tailed). Wilcoxon rank sum analysis
was applied when comparing median levels of parameters for
responders and nonresponders. When normalized values were
compared, Student’s paired t-test was applied.
RESULTS
Pretreatment parameters and analysis of results.
Twenty of the 22 patients with RA were seropositive for
RF; all of these patients were found by ELISA to have
IgM-RF, 18 (82%) had IgA-RF, and 13 (59%) had
IgG-RF (Table 1). Thirteen patients had anti-CCP
antibodies, including 1 of the RF-seronegative patients.
The other seronegative patient was positive for anti-BiP
antibodies. Since all patients treated with the different
protocols achieved B lymphocyte depletion in the peripheral blood, results comparing the changes in CRP and
total serum immunoglobulin levels with autoantibody
B LYMPHOCYTE DEPLETION IN PATIENTS WITH RA
2149
Figure 2. Minimum (nadir) levels reached for IgA-RF, IgM-RF, and
IgG-RF, anti-CCP antibodies, and total IgA, IgM, and IgG serum
immunoglobulins, expressed as the mean and SD percentage of
pretreatment values. P values were calculated using Student’s paired
t-test. See Figure 1 for definitions.
Figure 1. Time course of changes in IgA–, IgM–, and IgG–
rheumatoid factor (RF) as well as anti–cyclic citrullinated peptide
(anti-CCP) antibodies and C-reactive protein (CRP). Bars show the
mean ⫾ SD percentage of pretreatment (before rituximab combination therapy) values for 6 months (26 weeks) following B lymphocyte
depletion in responder (A) and nonresponder (B) patient groups.
and antimicrobial antibody levels were combined and
analyzed together (Figures 1–3 and Figure 5). For
comparison of the serologic response to treatment,
patients were classified as responders (those achieving
and maintaining an ACR level of improvement [27] of
ⱖ20% at 6 months) (15 of 22 patients) or nonresponders
(those attaining an ACR level of improvement ⬍20% at
6 months) (Tables 1 and 2 and Figures 1 and 4).
Serologic parameters in responder versus nonresponder patient groups. When the pretreatment median
values of the serologic parameters in responders and
nonresponders were compared (Table 2), there was no
significant difference between the groups (P ⬎ 0.01, by
Wilcoxon rank sum). Similarly, there was no difference
between the median nadir levels in responders and
nonresponders, except, as may be expected, when CRP
levels were compared (P ⬍ 0.01). There was, however, a
highly significant difference between the drop in autoantibody levels and the drop in CRP levels in the
responding patients, but not in the nonresponding patients. In each patient in the responder group, serum
Figure 3. Time taken (weeks) following treatment for levels of each
parameter to decrease by 50% (T50) and 80% (T80) of initial
pretreatment values. Bars show the median and SEM. See Figure 1 for
definitions.
2150
CAMBRIDGE ET AL
Table 2. Serologic parameters in patients before treatment and at
nadir*
Parameter, response to
therapy (number of
patients per group)
IgM-RF
Responders (15)
Nonresponders (5)
IgG-RF
Responders (10)
Nonresponders (3)
IgA-RF
Responders (13)
Nonresponders (5)
Anti-CCP
Responders (8)
Nonresponders (4)
CRP
Responders (15)
Nonresponders (7)
Pretreatment
value
Lowest level
attained after
treatment
P†
126 ⫾ 78.2
122 ⫾ 16.8
50 ⫾ 38.2
90 ⫾ 15.4
77 ⫾ 25.9
50 ⫾ 24.1
15.5 ⫾ 12.6
24.5 ⫾ 13.5
0.002
NS
147 ⫾ 194.3
158 ⫾ 28.2
78 ⫾ 86.1
118 ⫾ 18.5
0.0002
NS
950 ⫾ 340.9
2,282 ⫾ 1,145
236 ⫾ 122.1
1,350 ⫾ 894
0.008
NS
37.9 ⫾ 8.4
39.7 ⫾ 18
2 ⫾ 1.3‡
13.7 ⫾ 5.4
0.00006
NS
Comparison of the effect of B lymphocyte depletion on the levels of total serum immunoglobulins and
autoantibodies. Following treatment, B lymphocytes
were undetectable (⬍0.005 ⫻ 109/liter) in the peripheral
blood of patients for a mean of 8.4 months (range
3.5–20.8 months) (Table 1). Total serum immunoglobulins decreased moderately, with IgM dropping from a
mean of 1.5 ⫾ 1.03 gm/liter to 0.9 ⫾ 0.9 gm/liter, IgA
from a mean of 2.7 ⫾ 1.5 gm/liter to 2.0 ⫾ 1.1 gm/liter,
and IgG from a mean of 11.9 ⫾ 3.3 gm/liter to 8.8 ⫾ 2.3
gm/liter. In only 3 patients did the IgG levels drop below
the lower limit of normal, whereas in 8 patients the IgM
0.00006
NS
* Responders were defined as having American College of Rheumatology ⱖ20% improvement at 6 months. Values are the median ⫾
SEM IU/ml, except values for C-reactive protein (CRP), which are the
median ⫾ SEM mg/liter. NS ⫽ not significant (see Table 1 for other
definitions).
† Pretreatment values versus values at nadir for each group of patients
tested, using Wilcoxon signed rank test with significance level of 0.1%.
‡ P ⫽ 0.009 versus nonresponders, by Wilcoxon rank sum test.
levels of all classes of RF and of anti-CCP antibodies
decreased relative to their pretreatment values (Table
2), with all achieving statistical significance at the 0.1%
level (by Wilcoxon signed rank paired test).
For comparison of the kinetics of the fall in
serologic parameters following B lymphocyte depletion,
posttreatment values at each time point were expressed
as a percentage of pretreatment values. The results are
shown in responding (Figure 1A) and nonresponding
(Figure 1B) patients. In the responder group, a gradual
decline in the CRP and autoantibody levels was observed, with an apparent plateau reached at ⬃12 weeks
posttreatment (Figure 1A). In the nonresponder group
(Figure 1B), the course of CRP and autoantibody decline was more erratic, although the levels of IgA-RF
and IgM-RF did reach an apparent plateau, also at ⬃12
weeks posttreatment. Comparison of these percentages
of pretreatment values for any of the autoantibodies and
CRP at the specific posttreatment time points revealed
no significant difference between the 2 groups of patients, except for the changes in IgA-RF at 4 weeks (P ⫽
0.03) and in CRP at 26 weeks (P ⫽ 0.04) posttreatment.
Similarly, as shown in Table 2, the only significant
difference in the absolute values of all measured parameters between the responder group and the nonresponder group was in the CRP levels (at nadir).
Figure 4. Levels of IgG-RF (A), IgM-RF (B), IgA-RF (C), and
anti-CCP antibodies (D) in responding patients at pretreatment, when
each patient had reached the maximum attained level of improvement
in the American College of Rheumatology response (ACRmax), and
at or shortly after relapse. See Figure 1 for other definitions.
B LYMPHOCYTE DEPLETION IN PATIENTS WITH RA
levels dropped below the normal range (data not
shown).
In order to compare the effect of B lymphocyte
depletion on the levels of individual autoantibodies and
their respective classes of serum immunoglobulins, the
data were normalized by expressing the lowest value
attained for each parameter as a percentage of the
pretreatment value. Differences between the percentage
drop in the 3 classes of RF measured became apparent
(Figure 2). The percentage drop in IgG-RF levels was
greatest and was also significantly greater than that in
IgA-RF levels (P ⫽ 0.007, by Student’s t-test), but not
when compared with that in IgM-RF levels (P ⫽ 0.09).
There was no significant difference between the decrease in IgA-RF and the decrease in IgM-RF, and no
difference was found between the decreases in any of the
RF classes and the decrease in anti-CCP antibodies.
When decreases in the different autoantibodies and in
their respective serum immunoglobulin classes were
compared (Figure 2), the mean percentage decrease in
IgA-RF (P ⫽ 0.00002) and IgG-RF (P ⫽ 0.000009), but
not in IgM-RF (P ⬎ 0.05), was significantly greater than
that found in the corresponding serum immunoglobulinclass levels. IgG anti-CCP also decreased significantly
more than the drop in levels of circulating IgG (P ⫽
0.001).
Temporal relationship of serologic parameters
following B lymphocyte depletion. Figure 3 compares the
T50 (median time taken for serum levels to fall by 50%
of the pretreatment levels) and T80 (the equivalent for
80% of pretreatment levels) for each autoantibody and
for CRP. The T50 for IgG-RF, anti-CCP, and CRP was
within 5 weeks of treatment. The T80 for the fall in all
autoantibody levels was longer than that for the CRP
levels, although the T80 for IgG-RF and anti-CCP was
achieved sooner than that for IgA-RF and IgM-RF.
Effect of B lymphocyte depletion on protective
antibody levels. Anti-TT and anti-PCP antibodies were
measured at 3 time points: baseline, 3 months after
treatment, and at or shortly after the time of B lymphocyte return. Before treatment, 7 patients had subprotective levels of anti-TT antibodies (⬍0.1 IU/ml) and 11
patients had subprotective levels of anti-PCP antibodies
(⬍50 mg/liter). At 3 months, anti-TT antibody levels had
decreased a mean of 22.7% with no further decrease
evident at the time of B lymphocyte return (Figure 5).
Comparison with the percentage decrease in serum IgG
at the same time points showed no difference. In those
subjects whose baseline antibody levels were within the
protective range, the levels remained protective at the
second and third time points. The levels of anti-PCP
2151
Figure 5. Percentage of pretreatment levels of anti–tetanus toxoid
and anti–pneumococcal capsular polysaccharide antibodies at 3
months after B lymphocyte depletion and at or near B lymphocyte
return. Bars show the mean and SD. For comparison, levels of serum
total IgG at the same time points are also shown.
antibodies did not change significantly with treatment
(Figure 5). However, in 3 patients with baseline levels
within the lower protective range, the anti-PCP levels
had decreased to subprotective values at or shortly after
B lymphocyte repopulation (data not shown). In 1
patient, anti-PCP levels increased 4-fold at 3 months, at
the time of a respiratory infection.
Factors associated with relapse. At the time of
submission, all except 2 patients (patients 11 and 13 in
Table 1) had undergone clinical relapse. All patients
who have relapsed have done so at or up to 17 months
after B lymphocytes were again detectable in the circulation (⬎0.005 ⫻ 109/liter). In all patients except 1
(patient 16 in Table 1), clinical relapse was preceded by
a detectable rise in autoantibody levels. Rises in autoantibody levels, usually IgM-RF, in the absence of
relapse were rarely seen (on 4 occasions only) and were
transient in nature (data not shown). Figures 4A–D
show the absolute values of IgA-, IgM-, and IgG-RF and
anti-CCP antibodies in sera from individual responding
patients at the pretreatment time point, at the time when
patients had reached their maximum ACR levels of
improvement, and at relapse. Levels of all autoantibodies were found to decrease following treatment and, in
most cases, to show a relative increase in titer at or close
to the time of relapse. There was no clear preference for
rises in any particular class of RF or anti-CCP antibodies
in association with relapse. In addition, no new autoan-
2152
CAMBRIDGE ET AL
responder group data shown in Figure 1A, the reduction
in inflammatory scores, as measured by a fall in CRP
levels, had the general appearance of an exponential
decay curve. The pattern of changes in autoantibody
levels generally paralleled the CRP profiles in each
patient. Although the slopes of the curves differed from
patient to patient, there tended to be an early drop in
autoantibody levels and CRP levels to a plateau, which
was sustained until relapse.
DISCUSSION
Figure 6. Serial studies of autoantibody (IgA-RF, IgM-RF, IgG-RF,
anti-CCP), antimicrobial antibody (anti–tetanus toxoid [anti-TT] and
anti–pneumococcal capsular polysaccharide [anti-PCP]), and CRP
levels in 2 representative patients (patients 1 [A] and 4 [B] in Table 1).
Results for each parameter are expressed as a percentage of the
pretreatment values. See Figure 1 for other definitions.
tibody classes or specificities were found to emerge
(Table 1).
Serial studies of 2 responding patients. Figures
6A and B show 2 examples of the change in serologic
variables during the time following B lymphocyte depletion. The patient shown in Figure 6A (patient 1 in Table
1) remained well for more than 12 months after B
lymphocyte repopulation. The levels of IgA-RF and
IgM-RF were seen to rise before relapse, with a rise in
anti-CCP antibodies also occurring coincident with relapse. In the patient shown in Figure 6B (patient 4 in
Table 1), detectable circulating B lymphocytes together
with a rise in autoantibodies coincided with clinical
relapse. As exemplified by the time courses of serologic
responses in these 2 representative patients and the
We found that B lymphocyte depletion had a
selective effect on different circulating antibody populations in patients with RA. Sera from patients with a
positive clinical response to treatment, in which an ACR
response of ⱖ20% was achieved at 6 months, showed a
significant fall in autoantibody and CRP levels, and this
was not observed in nonresponders. Although total
serum immunoglobulin levels also fell, the percentage
decrease in IgA-RF, IgG-RF, and IgG anti-CCP antibodies was significantly greater in all patients. This was
not the case for antimicrobial antibodies. Our findings
extend the preliminary observations in patients with
other autoimmune conditions, in whom a positive clinical response was associated with a significant fall in
autoantibody levels following B lymphocyte depletion
(9–11,28).
In patients with lymphoma who were treated with
rituximab, either alone or in combination with other
drugs, the mean total immunoglobulin levels had been
found to remain within the normal range (18,19,29,30).
This was taken as an indication that B lymphocyte
depletion therapy would not produce significant benefit
to autoimmunity if its effect were to be through reduction of autoantibody levels. Rituximab treatment in
other autoimmune diseases has also not been found to
result in a major drop in circulating immunoglobulin
levels, with the possible exception of hemolytic anemia
in children (31). In our group of patients, we did see a
significant decrease in serum immunoglobulin levels,
particularly in IgM, following B lymphocyte depletion,
even though the mean values for all classes remained
within the normal range. It is possible that in the patients
studied herein, the greater drop in serum total immunoglobulins might reflect a large contribution of autoantibodies to circulating immunoglobulin levels.
Treon and Anderson (32) have suggested that
only autoimmune conditions associated with pathogenic
antibodies of the IgM class, e.g., polyneuropathies,
might respond to rituximab treatment. In such condi-
B LYMPHOCYTE DEPLETION IN PATIENTS WITH RA
tions and in lymphoma, they suggested that IgM-class
antibodies may be derived from CD20-positive plasmablasts. In our studies of patients with RA, we observed
that the levels of all autoantibodies measured decreased,
and for IgA-RF, IgG-RF, and IgG anti-CCP, the decrease was proportionately greater than the decrease in
their respective total immunoglobulin classes. In addition, despite the half-life of IgG being longer than that
of other immunoglobulin classes, IgG-RF and IgG antiCCP antibodies decreased more rapidly than did
IgA-RF or IgM-RF. In contrast to what was observed
with autoantibodies, the levels of anti-PCP antibodies
did not change significantly following treatment, and the
anti-TT response closely corresponded to the changes in
total serum IgG levels.
Such a selective effect on autoantibody production suggests that their production may be more dependent on the constant generation of new plasma cells
from CD20-positive B lymphocytes. It is now known that
some plasma cells have short lifespans, but other plasma
cells may be able to live for extended periods of time
(33). It is also possible that the clones responsible for
antimicrobial antibodies are resident in the spleen,
where they may be slowly turning over into plasma cells.
Autoreactive clones are possibly in a more dynamic
situation because of their constant generation and, consequently, many more may be entering the circulation.
Their location in tissues other than secondary lymphoid
organs may also render autoantibody-committed clones
more susceptible to B lymphocyte depletion.
After rituximab administration, B lymphocyte
depletion in patients occurs rapidly (within days) in the
peripheral blood (17). Animal studies have shown that
depletion of B cells in lymphoid tissue is rapid and
unlikely to continue beyond 2 weeks (16). In this study,
although patients showed differences in the timing and
degree of their clinical response as measured by the
ACR (15), the drop in CRP, which is an indicator of
cytokine production, followed similar patterns in responding patients. This consisted of a gradual decrease
in the levels of CRP over weeks to months that then
reached a plateau until relapse, which was usually preceded by a rise in autoantibody levels. The reduction in
autoantibody levels also occurred gradually and was
reflected in the median times taken for them to decrease
to 50% and 80% of pretreatment levels. These results
are consistent with the effect of B lymphocyte depletion
on reducing the progenitors of daughter plasma cells
and thereby reducing autoantibody production. This
contrasts with the rapid clinical and serologic (CRP)
response of patients with RA to anti–tumor necrosis
2153
factor ␣ treatment, which reflects the swift removal of a
key effector in the inflammatory process (34). In our
study, when responders were compared with nonresponders, only the former showed significant drops in
autoantibody levels (as well as in CRP) in response to B
lymphocyte depletion. Whether this simply reflected
differences in the relative degree of B lymphocyte
depletion in lymphoid organs in nonresponder patients
when compared with responders could not be determined, since no tissue samples were obtained.
As shown in the representative serial studies and
by the cumulative data from responding patients, the
kinetics of the serologic (CRP) response to treatment
paralleled the decline in circulating autoantibody levels.
However, B lymphocyte return always preceded relapse.
Relapse was often preceded by or coincided with a rise
in 1 or more autoantibodies. On only 4 occasions were
rises in autoantibodies (usually, small, transient rises in
IgM-RF) detected without associated clinical manifestations of more active disease. The often long gap (up to
17 months) between B lymphocyte repopulation and
relapse also suggested that relapse was not solely related
to the presence of B cells.
The long period between B lymphocyte return
and relapse seen in some patients treated with B lymphocyte depletion suggests that generation of new B cell
clones capable of engaging in a vicious cycle of expansion may be a rate-limiting step in the recrudescence of
disease. This may involve the generation of sufficient
pathogenic B cell clones able to either restimulate
autoreactive T cells or be precursors of autoantibodyproducing plasma cells. It remains uncertain whether the
reappearance of autoantibodies following B lymphocyte
depletion represents the re-expansion of preexisting B
lymphocyte clones or whether new clones are generated.
The exact degree of B lymphocyte depletion achieved in
solid lymphoid tissues in our patients remains unknown.
There is also no information on what might happen to
surviving B lymphocytes and to plasma cells during a
period of B lymphocyte depletion. Therapeutic B lymphocyte depletion provides an excellent opportunity to
learn more about these and other aspects of B lymphocyte and plasma cell biology and their roles in disease
pathogenesis.
ACKNOWLEDGMENTS
The authors want to thank Dr. Chris Bunn and the
Immunopathology Laboratory, Royal Free Hospital for doing
the antimicrobial antibody tests, and the Arthritis Research
Campaign, which generously supported this work.
2154
CAMBRIDGE ET AL
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