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Brief report
Rituximab treatment results in impaired secondary humoral immune
responsiveness
Lizet E. van der Kolk, Joke W. Baars, Martin H. Prins, and Marinus H. J. van Oers
In lymphoma patients, treatment with chimeric CD20 monoclonal antibodies (rituximab) results in a depletion of normal and
malignant B cells, persisting for 6 to 9
months. This B-cell depletion leads neither to a decrease in immunoglobulin
levels nor an increase in the number of
infectious complications. However, the
effect of rituximab treatment on the immune responsiveness is unknown. In 11
patients with relapsed, low-grade lymphoma, we investigated the effect of rituximab treatment on the humoral immune
response to 2 primary antigens and 2
recall antigens. After rituximab treatment,
the humoral immune response to the recall antigens was significantly decreased
when compared with the response before
treatment. Already before rituximab treatment, none of these patients was able to
mount a response to the primary antigens. These findings are relevant regarding the feasibility of rituximab in maintenance treatment and may also offer a
rationale for the treatment of antibodymediated autoimmune diseases with rituximab. (Blood. 2002;100:2257-2259)
© 2002 by The American Society of Hematology
Introduction
Rituximab, the chimeric CD20 monoclonal antibody (mAb), has
become an important treatment modality in B-cell malignancies.1,2
It targets the CD20 antigen, expressed on more than 95% of normal
and malignant B cells. In patients with relapsed, low-grade
non-Hodgkin lymphoma (NHL), peripheral blood B-cell depletion
occurred within 24 to 48 hours after the first infusion of rituximab.
Recovery of B-cell counts started 6 to 9 months after the
completion of therapy, and normal levels were obtained after 9 to
12 months.1
The prolonged period of rituximab-induced B-cell depletion
might compromise the immune system. However, no studies
directly addressing the immune responsiveness of patients treated
with rituximab have been published. We investigated the humoral
immune responses of patients with low-grade lymphoma to 2
primary antigens (keyhole limpet hemocyanin [KLH] and
hepatitis A vaccine [HAV]) and 2 recall antigens (tetanus toxoid
[TT] and poliomyelitis vaccine [PV]), both before and after
treatment with rituximab.
Antigen
One thousand micrograms KLH (Calbiochem, San Diego, CA; dissolved in
NaCl as described previously4) was administered subcutaneously. HAV
(1440 IE of inactivated hepatitis A virus strain HM-175; Havrix) was
obtained from SmithKline Beecham (Rijswijk, The Netherlands) and
administered intramuscularly. TT (0.5 mL tetanus vaccine containing
80 IE/mL tetanus toxoid) and inactivated PV (type 1, at least 30 DE [PV1];
type 2, at least 6 DE [PV2]; type 3, at least 24 DE [PV3]) were obtained
from the RIVM (Utrecht, The Netherlands) and were administered intramuscularly and subcutaneously, respectively.
Measurement of antibody titers
Anti-KLH–specific immunoglobulin levels (IgG) and anti-TT antibodies
were measured with an enzyme-linked immunosorbent assay (ELISA).4
Anti–hepatitis A virus–specific IgG levels were measured by ELISA
(HAVAB 2.0 Quantitative; Abbott Laboratories, Diagnostics, Abbott Park,
IL). Titers of neutralizing antibodies against PV1, PV2, and PV3 were
measured by the poliovirus-neutralizing antibody test.5 For KLH, a positive
response was defined as a ratio of IgG antibodies (after immunization
versus before immunization) greater than 1.25.4 Seroconversion to HAV
was defined as an increase in HAV titers to more than 20 mIU/mL.6
Immunization schedule
Study design
Patients
The present immunization study was part of a phase I/II study evaluating
the safety and efficacy of the combination of rituximab (375 mg/m2 weekly
for 4 weeks) and granulocyte–colony-stimulating factor (G-CSF; 5 ␮g/kg
per day, administered on 3 consecutive days starting 2 days before each
infusion).3 Inclusion criteria for the immunization study were those of the
phase II study. Both studies were approved by the ethics committee, and all
patients gave written informed consent according to the rules of the institute.
From the Department of Hematology and the Department of Clinical
Epidemiology and Biostatistics, Academic Medical Center, Amsterdam, and the
Department of Medical Oncology, Antoni van Leeuwenhoek Hospital/
Netherlands Cancer Institute, Amsterdam, The Netherlands.
Because it was expected that this group of patients with pretreated
low-grade lymphoma would have impaired immune responsiveness when
compared with healthy volunteers,7,8 it was considered more appropriate to
use the patients as their own controls. To avoid boosting effects (ie, turning
a primary antigen into a recall antigen and inappropriately amplifying recall
responses), in each patient different vaccines were needed for the immunizations performed before and after rituximab treatment. Therefore, 2
primary antigens (KLH and HAV) and 2 recall antigens (TT and PV) were
used. Each patient simultaneously received one primary and one recall
antigen 2 weeks before treatment and crossed over to the other 2 vaccines
Medicine, F4-222, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands;
e-mail: [email protected].
Submitted June 18, 2001; accepted May 8, 2002.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: M. H. J. van Oers, Academic Medical Center, Department of Internal
© 2002 by The American Society of Hematology
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
2257
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2258
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
VAN DER KOLK et al
Table 1. Immunizations received by each patient
Before rituximab treatment
After rituximab treatment
Patient
Primary
antigen
Recall
antigen
Primary
antigen
A
HAV
PV
*
TT
B
†
TT
KLH
PV
C
*
TT
HAV
PV
D
HAV储
TT
KLH
PV
E
*
TT
‡
‡
F
KLH
PV
HAV储
TT
G
HAV储
TT
KLH
PV
Results and discussion
Recall
antigen
H
HAV储
PV
§
§
I
KLH
PV
HAV储
TT
J
KLH
TT
HAV
PV
K
HAV
PV
KLH
TT
Patients
All patients had progressive disease at the time of first immunization. Median age was 53 years (range, 27-70 years), and the median
number of prior chemotherapy regimens was 1 (range, 1-3). Not
every patient received all 4 immunizations because of various
reasons (Table 1).
Effect of rituximab treatment on peripheral blood B-cell counts
and immunoglobulin levels
*No KLH immunization was performed because of logistics problems at the
initiation of the immunization study.
†Patient was known to be HAV-seropositive; therefore, HAV immunization was
not performed.
‡No immunizations were performed after rituximab treatment because of
non–study-related disease.
§No immunizations were performed after rituximab treatment because of
logistics problems.
储These patients were found later to be HAV-seropositive.
4 weeks after treatment. To correct for a possible influence of one of the
vaccines on the response to the other vaccines, 4 different immunization
schedules had to be used (Table 1). Patients were sequentially assigned to 1
of the 4 groups. Before immunization and 2 weeks after immunization,
serum samples were collected and stored at ⫺20°C until use. Pretreatment
immunizations were performed during the necessary pretreatment work-up.
Therefore, the start of rituximab treatment was not delayed in any of
the patients.
Statistics
Antibody responses are expressed as the ratio between the antibody titers 14
days after immunization and those before immunization. To compare the
responses to the 2 different primary antigens and recall antigens within one
patient, for each vaccine the ratios of antibody responses were ranked. Each
of the patients received a rank obtained for his or her response to, for
example, TT (independent of the moment of immunization, before or after
rituximab treatment), and a rank was obtained for his or her response to PV.
The rank a patient obtained before rituximab (eg, for TT) and the rank that
this same patient obtained after rituximab (for PV1, PV2, and PV3, or vice
versa) formed pairs. The pairs of ranks achieved by all patients were
analyzed using the Wilcoxon signed rank test. P ⬍.05 was considered
significant.
In all patients, complete depletion of B cells from the peripheral
blood was observed 72 hours after the first infusion. One month
after the last rituximab treatment (ie, at time of second immunization), B cells were still absent in all but one patient (patient H).
Immunoglobulin levels (IgG, IgA, and IgM) remained stable
during treatment and follow-up (data not shown).
Response to recall antigens
Nine patients were immunized with TT and PV. Antibody responses, expressed as the ratio of titers before and after immunization, are shown in Table 2. To allow for comparison of the
responses to TT and PV within one patient, ratios to TT and ratios
to PV were ranked (Table 2). Responses to the poliovirus subtypes
PV1, PV2, and PV3 are separately documented and analyzed in
combination with TT. For example, the 3 pairs of ranks obtained by
patient A were 9-2, 9-2, and 7-2 for PV1-TT, PV2-TT, and PV3-TT,
respectively.
Pairs of ranks obtained by each patient were analyzed using the
Wilcoxon signed rank test (n ⫽ 9 paired samples). The response to
recall antigens after rituximab treatment was significantly lower
than the response before treatment (P ⫽ .078, P ⫽ .013, and
P ⫽ .012 for TT in combination with PV1, PV2, and PV3,
respectively).
Primary immune responses
In 11 patients the primary immune response could be evaluated—in
5 patients before and in 6 patients after rituximab treatment (Table
1). None of these patients developed a humoral immune response
to either primary antigen before or after rituximab treatment (data
not shown). Five patients were found to have high anti-HAV
Table 2. Immune responses to recall antigens
Before rituximab treatment
Patient
A
F
After rituximab treatment
PV1
PV2
PV3
TT
PV1
PV2
PV3
32* (9)
128 (9)
64 (7)
—
—
—
—
0.9 (2)
8 (6.5)
256 (8.5)
—
—
—
—
1.1 (4)
2 (4.5)
4 (3)
—
—
—
—
0.7 (1)
256 (8.5)
—
—
—
—
4.1 (8.5)
1 (1.5)
—
2 (4.5)
I
0.5 (1)
K
16 (8)
32 (8)
TT
B
—
—
—
2.8 (6)
2 (4.5)
1 (2.5)
C
—
—
—
4.1 (8.5)
4 (7)
8 (6.5)
D
—
—
—
1 (3)
2 (4.5)
G
—
—
—
3.8 (7)
2 (4.5)
1 (2.5)
16 (5)
—
J
—
—
—
1.9 (5)
1 (2)
2 (4.5)
8 (4)
—
0.5 (1)
32 (6)
1 (1.5)
—
—
Ratios to PV1, PV2, and PV3 are listed separately.
*Antibody responses to TT, PV1, PV2, and PV3 in the 9 patients immunized before and after rituximab treatment are expressed as ratios of post- and preimmunization
titers. Ratios to TT, PV1, PV2, and PV3 were ranked. Ranks are depicted in parentheses. Paired ranks, consisting of the rank obtained by each patient before and after
rituximab treatment, were analyzed by Wilcoxon signed rank test.
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BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
antibody titers, indicating that these patients either had experienced
hepatitis A infection or had been immunized against hepatitis A.
In the present study, we have demonstrated that the humoral
immune response to recall antigens decreases significantly with
rituximab treatment. Surprisingly, although all patients responded
to recall antigens, none of the patients responded to the primary
antigens before or after rituximab treatment. At time of vaccination, all patients had progressive disease requiring treatment.
Disease status at the time of vaccination might explain this strongly
impaired immune response to primary antigens observed in
our patients.9
The observed significantly decreased response to recall antigens
after rituximab treatment might be attributed to a decrease in the
amount of memory B cells after rituximab treatment. Previous
studies showed that although rituximab treatment induces B-cell
depletion for 6 to 9 months, this does not lead to a decrease in
immunoglobulin levels, possibly because of the presence of
long-lived plasma cells.10 However, our observation predicts that
prolonged treatment with rituximab may eventually lead to a
decrease in circulating plasma cells because of a decreased
replenishment from the pool of memory B cells. This might have
consequences for the immunoglobulin levels and, consequently, the
INFLUENCE OF RITUXIMAB ON HUMORAL IMMUNE RESPONSE
2259
incidence of infectious complications during rituximab maintenance therapy.
Recently, rituximab treatment was found to be effective in
several antibody-mediated autoimmune diseases,11,12 with some
responses ongoing for more than 9 to 14 months.12 We suggest that
rituximab, by depleting (memory) B cells, interrupts the ongoing
humoral autoimmune response in autoimmune diseases. Although
plasma cells surviving rituximab treatment may continue to
produce (auto-)antibodies for a certain period of time, deletion of
the autoreactive B-cell clone eventually leads to a decrease in
antibody production.
Our data might have implications for studies investigating the
feasibility of rituximab as maintenance therapy. Furthermore, these
findings provide a rationale for the treatment of antibody-mediated
autoimmune diseases with rituximab.
Acknowledgments
We thank Dr T. Out and Prof Dr R.A.W. van Lier for critical
reading of the manuscript and for helpful discussions and F. de
Wilde for measurements of KLH- and tetanus-specific antibodies
by ELISA.
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the treatment of chronic idiopathic thrombocytopenic purpura (ITP) [abstract]. Blood. 1999;94:49.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2002 100: 2257-2259
Rituximab treatment results in impaired secondary humoral immune
responsiveness
Lizet E. van der Kolk, Joke W. Baars, Martin H. Prins and Marinus H. J. van Oers
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