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Perspectives
Am“B”valent: anti-CD20 antibodies unravel the dual role of B cells in
immunopathogenesis
Olivier Thaunat,1-4 Emmanuel Morelon,1-4 and Thierry Defrance1,3,4
1Université
de Lyon, Lyon 1, Villeurbanne; 2Hospices Civils de Lyon, Hôpital Edouard Herriot, Service de Transplantation Rénale et d’Immunologie Clinique,
Lyon; 3Inserm U851, Lyon; and 4Institut Fédératif de Recherche 1, 28 Biosciences, Lyon, France
Accumulating evidence has designated
B cells as central players in the pathogenesis of immune diseases. In the late 1990s,
anti-CD20 monoclonal antibodies were developed for the treatment of B-cell nonHodgkin lymphomas, offering the opportunity to efficiently deplete the B-cell
compartment for therapeutic immunointerventions. Several studies have since estab-
lished the beneficial effect of this drug on
the course of a wide range of immune diseases. However, paradoxically, it has also
been reported that rituximab sometimes
worsens the symptoms of the very same
conditions. The explanation that reconciles
such apparently conflicting results has recently emerged from basic studies, which
demonstrate that (1) B cells are also en-
dowed with immune-regulatory properties
and (2) the opposing contributions of B cells
may overlap during the course of the disease. Caution should therefore be exercised
when considering B-cell depletion because
the therapeutic effect will depend on the
relative contributions of the opposing B-cell
activities at the time of the drug administration. (Blood. 2010;116(4):515-521)
“Bad boy” B cells
Historically, the role of B lymphocytes in the pathogenesis of
immune diseases has been associated mainly with their capacity to
produce harmful antibodies after differentiation into plasma cells.
This conception was based on seminal experiments that demonstrated that the mere transfer of antibodies was sufficient to
recapitulate the symptoms of myasthenia gravis, Graves disease,
Goodpasture disease, among others.1 In contrast to these diseases,
other autoimmune conditions, in which the role of antibodies has
not been recognized, have traditionally been termed “T cell–
mediated” diseases. Among the latter, type 1 diabetes, in which
T lymphocytes are crucial for the destruction of the ␤ islets, has
long been considered as archetypal. However, recent investigations
in nonobese diabetic mice have shown that more than 50%2 of the
lymphocytes infiltrating islets of Langerhans are B cells3 and that
these B cells are critically necessary for the development of
diabetes.4 Another clue that B cells exert pathogenic roles through
“antibody-independent” mechanistic pathways came from genetically modified lupus-prone mice. Although B-cell depletion leads
to abrogation of the disease in this model, transgenic mice, whose
B cells cannot secrete immunoglobulin, still developed nephritis.5
Thus, in many immune diseases, even including those not driven by
antibodies, B cells have been demonstrated to play an essential
pathogenic role.
Among the antibody-independent pathogenic roles of B cells,
accumulating evidence points to their capacity to present antigen.6
Upon recognition of specific antigen, the B-cell membrane is
reorganized resulting in the aggregation of B-cell receptor in an
immunologic synapse that functions as a platform for internalization of the complex.7 Internalized antigen is degraded and subsequently exposed on the B-cell surface in association with major
histocompatibility complex molecules for presentation to T cells.
This surface presentation of antigen, in the presence of various
costimulatory molecules, elicits the T-cell assistance required for
B-cell maturation, which in turn allows B cells to drive optimal
T-cell activation and differentiation into memory subsets8 (Figure 1
left panel). Of note, B cells are endowed with unique properties as
antigen-presenting cells because they have an antigen-specific
receptor, allowing extraction and presentation of antigen, even if it
is membrane tethered9 or present in limiting quantities.10 Furthermore, B cells also have the capacity to clonally expand, thereby
becoming the numerically dominant antigen-presenting cells.
In addition to antigen presentation, activated B cells also
produce a wide range of cytokines and chemokines that modulate
the maturation, migration, and function of other immune effectors.11,12 In particular, B cells have been shown to play a critical
role in lymphoid neogenesis,13 that is, the process by which ectopic
functional lymphoid structures (“tertiary lymphoid tissues”) appear
de novo during chronic inflammation.14,15 Several studies16-18 have
demonstrated that tertiary lymphoid tissues are permissive microenvironments for the induction of immune responses and have led to
the hypothesis that lymphoid neogenesis may contribute to the
exacerbation of a wide range of chronic inflammatory diseases.19
Submitted January 26, 2010; accepted March 16, 2010. Prepublished online as
Blood First Edition paper, March 25, 2010; DOI 10.1182/blood-2010-01-266668.
© 2010 by The American Society of Hematology
BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
Anti-CD20 monoclonal antibody for
therapeutic B-cell depletion
Given the multiple pathogenic roles attributed to B lymphocytes,
therapeutic strategies that aim at depleting this cell population were
expected to be beneficial in a wide range of immune diseases.
Pioneer attempts to deplete B cells in the 1980s relied on
xenogenic polyclonal antibodies directed against the surface immunoglobulin receptor.20 Finally, it was only in the late 1990s that
progress achieved in the treatment of lymphoproliferative disorders
offered clinicians the opportunity to efficiently target the B-cell
compartment for therapeutic immunointerventions.
CD20 is a transmembrane protein that functions as a Capermeable cation channel, whose expression is restricted to B cells
515
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516
THAUNAT et al
BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
Figure 1. Janus-faced B cells. Janus, the Roman god of beginnings and endings, is most often depicted as having 2 faces facing opposite directions. Like him, B cells can
either elicit (right panel) or terminate (left panel) an immune response, after their activation by combination of ligation of the B-cell receptor, CD40, and Toll-like receptors
(TLRs). (Left panel) B cells present the antigen along with costimulatory signals, leading to the activation and the proliferation of T effectors. In turn, activated T cells provide
CD40 ligand (CD40L) for the differentiation of B cells into antibody-producing plasma cells. The cytokines produced by B cells may participate into the polarization of
T effectors. Finally, B cells play a critical role for the development of T-cell memory. (Right panel) B cells regulate immune response by provision of IL10 that suppresses the
activation and the expansion of T effectors directly, and indirectly through the differentiation of T regulatory cells and the suppression of dendritic cell function.
from the pre-B-cell to the immunoblast stage.21 Rituximab, a
chimeric monoclonal antibody composed of human immunoglobulin G1 kappa antibody with variable regions isolated from a murine
anti-CD20 clone (IDEC-2B8),22 was initially introduced for the
treatment of B-cell non-Hodgkin lymphomas.23 A single course of
rituximab successfully depleted peripheral human B lymphocytes
for periods ranging from 3 months to more than 1 year through
mechanisms involving Fc- and complement-dependent killing as
well as other signals inducing apoptosis24 (Figure 2 top panel).
Several studies have highlighted the influence of circulatory
dynamics and microenvironment on the efficiency of rituximab
depletion.25,26 As a result, the reduction of B-cell numbers after
rituximab appears to be less complete in secondary lymphoid
tissues than in peripheral blood, and variations in the extent and
kinetics of the depletion have been reported among B-cell subsets.1
Successes of therapeutic B-cell depletion
The most frequent hematologic malignancy is non-Hodgkin lymphoma, 85% of which are of B-cell origin. Rituximab has been
approved for the treatment of B-cell non-Hodgkin lymphoma in
1997. Since then, several randomized, phase 3 trials have reported
significant survival benefits associated with rituximab, in combination with chemotherapy, in patients with diffuse large B-cell
lymphoma and follicular lymphoma. Furthermore, these benefits
have been demonstrated for rituximab in combination with a wide
variety of chemotherapy regimens, across many patient subtypes,
and in different treatment and disease settings (for a recent review
please see Hagemeister27), leading to the conclusion that anti-CD20
monoclonal antibodies represent one of the most important advance in the treatment of B-cell lymphoma in the past 30 years.
In chronic lymphocytic leukemia, the leukemic counterpart of
small lymphocytic lymphoma, the addition of rituximab to fludara-
bine-based chemotherapy has significantly increased complete
response and progression-free survival rates for both untreated and
relapsed or refractory chronic lymphocytic leukemia.27
The association between autoimmune diseases and hematologic
malignancies has long been recognized. Although autoimmune
conditions associated with hematologic malignancies can affect
any organ, they seem to predominantly target blood constituents.28
The pathophysiology of these autoimmune manifestations is complex and remains incompletely understood. Autoimmune anemia,
for example, either can result from monoclonal antibodies produced by the lymphoma clone (ie, cold hemagglutinin disease) or
can be the consequence of polyclonal autoantibodies produced by
the residual nonmalignant B cells in chronic lymphocytic leukemia. Several reports suggest that treatments, in particular chlorambucil and fludarabine, might trigger the onset of autoimmune
manifestations.29,30 In contrast, rituximab has been shown to be an
effective treatment for chronic lymphocytic leukemia–associated
autoimmune hemolytic anemia,28,31 and for mixed cryoglobulinemia and cold agglutinins secondary to non-Hodgkin lymphoma.32
These observations along with the favorable safety profile of the
rituximab in patients treated for hematologic malignancies33 have
catalyzed the application of the drug in primary immune diseases.
Several clinical studies have since established the beneficial effect
of B-cell depletion on the course of a wide range of immune
diseases.
Rheumatoid arthritis, a multisystem disorder that predominantly causes inflammation in synovial joints, is the autoimmune
condition in which the effect of rituximab has been the most
studied.34 In the pivotal phase 2a study, approximately 80% of
patients with active rheumatoid arthritis (despite methotrexate
treatment) showed clinical benefit after rituximab administration—a percentage similar to the one obtained with anti–tumor
necrosis factor agents.35 Furthermore, a recent phase 3 trial has
reported that even patients with an inadequate response to anti–
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BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
DUAL ROLE OF B CELL IN IMMUNOPATHOGENESIS
517
Figure 2. Mechanisms of action of anti-CD20 antibodies. (Top panel) Apoptosis of malignant B-cell clones occurs upon cross-linking of rituximab-CD20 complexes in the
lipid rafts. This activates signaling pathways involving the Src kinases and their regulatory molecules. Complement-mediated cytolysis involves the ability of anti-CD20
immunoglobulin G1 bound to their antigen to bind C1 and trigger the classical complement pathway. Antibody-dependent cell cytotoxicity requires interaction between the Fc
portion of rituximab and appropriate receptors on effector cells. (Bottom panel) Another possible mechanism by which rituximab could promote the destruction of malignant
B-cell clones is the restoration of the protective antitumoral response. The destruction of the nonmalignant B cells endowed with immune-regulatory properties could facilitate
the development of antitumoral T-cell clones.
tumor necrosis factor agents showed significant improvement after
rituximab therapy,36 a result that prompted the Food and Drug
Administration to approve the drug for the treatment of refractory
rheumatoid arthritis.
In chronic idiopathic thrombocytopenic purpura, an acquired
hemorrhagic condition associated with accelerated platelet consumption due to antiplatelet autoantibodies, a phase 2 study has reported
a 40% good response rate to rituximab.37
Based on previous encouraging case reports, a phase 2 controlled clinical trial has recently been conducted in multiple
sclerosis. In patients with relapsing-remitting multiple sclerosis, a
single course of rituximab reduced inflammatory brain lesions and
clinical relapses for 48 weeks.38
Numerous small, open-label studies and isolated cases have also
reported favorable outcomes after rituximab administration in a
wide range of autoimmune diseases, including vasculitis,39
autoantibody-associated neuropathies,40 Graves disease,41 myasthenia gravis,42 myositis,43 blistering skin disorders,44,45 mixed cryoglobulinemia,46 thrombotic thrombocytopenic purpura,47 Sjögren
syndrome,48 and anti–factor VIII syndrome.49 There are ongoing
phase 2 and 3 trials of rituximab for each of these autoimmune
disorders.50
Finally, recent advances in solid organ transplantation have
unraveled new mechanisms of allograft damage. Unexpected
clusters of CD20⫹ B cells have been discovered in rejected
grafts,18,51 and C4d deposition, indicating classic complement
pathway activation, is now routinely seen in refractory rejection.52
Rituximab has therefore emerged as a rational choice for therapy in
transplantation to abrogate B cell–mediated events.53,54
Limits of therapeutic B-cell depletion
Given the central role of B cells, autoantibodies, and immune
complexes in the pathophysiology of systemic lupus erythematosus
(SLE), it was widely anticipated that anti-CD20 would be efficient
in treating SLE. However, despite the successes reported in some
open-label preliminary studies,55,56 rituximab failed to meet its
primary and secondary end points in 2 large controlled trials of
nonrenal SLE57 and renal lupus nephritis.58 These disappointing
results are sometimes attributed to the fact that the highly
heterogeneous clinical presentation of SLE makes it difficult to
devise quantitative measures of response to therapy. However, the
latter justification is an unlikely explanation for either the paradoxical exacerbation of the clinical condition59-61 or the onset of new
immune diseases (including psoriasis,62,63 vasculitis,64-66 interstitial
pneumonitis,67-69 and autoimmune cytopenia70,71) that are sometimes observed after rituximab administration. One could argue
that these small nonrandomized studies focus on a very rare
adverse effect of rituximab, because this problem was not identified
in the huge cohort of patients treated for hematologic malignancies.33 We rather favor the alternative explanation that rituximabinduced autoimmune complications are underreported. It is indeed
a well-established fact that the quality and quantity of drug safety
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518
THAUNAT et al
reporting are inadequate,72 a matter that seems even more common
for drugs with excellent efficacy outcomes.72 One can indeed
conceive the reluctance of clinicians to report side effects of a drug
that has changed the standard of care for patients suffering from
life-threatening diseases such as non-Hodgkin lymphoma or chronic
lymphocytic leukemia. Furthermore, the widely recognized association between autoimmune diseases and hematologic malignancies
already discussed makes very difficult the formal incrimination of
the drug for these patients. The same concern also exists for
patients receiving rituximab for an autoimmune disease, the
occurrence of distinct autoimmune conditions in the same patients
being a very common feature.73 Thus, the current lack of a
consistent demonstration of the increased incidence of autoimmune
phenomena after rituximab administration does not exclude the
possibility that a carefully conducted meta-analysis might still be
able to detect this adverse event.
In this context, the results of the study recently published by
Clatworthy et al74 are particularly interesting because they clearly
establish the “paradoxical” immune stimulatory effect of rituximab.
Indeed, this randomized controlled trial, which compared rituximab with an anti-CD25 monoclonal antibody as induction therapy
in patients undergoing renal transplantation, had to be suspended
because the authors observed a 6-fold increased incidence of acute
cellular rejection in the rituximab group (83% vs 14%).
Based on the worsening of autoimmunity observed in chronic
lymphocytic leukemia patients receiving fludarabine,29,30 a drug
that induces a profound lymphopenia, it is tempting to speculate
that this paradoxical immune stimulation is rather a general feature
after B-cell depletion than a specific adverse effect of rituximab.
Finally, the ambivalence of B-cell depletion on the course of
immune diseases has been recently demonstrated in experimental
autoimmune encephalomyelitis.75 Experimental autoimmune encephalomyelitis (EAE) is an autoimmune central nervous system
disease that can be induced in certain susceptible murine strains
after immunization with myelin to model human multiple sclerosis.
Whereas CD20 antibody–mediated B-cell depletion during EAE
disease progression dramatically reduced the symptoms, the same
treatment given before EAE induction substantially exacerbated
the disease (Figure 3). Interestingly, administration of the drug at
other time points had no significant effect (Figure 3).
Altogether, these data demonstrate that it is more difficult than
anticipated to forecast the effect of B-cell depletion on the course of
an immune disease: the same therapy can lead to opposite
outcomes depending on the timing of its administration.65
BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
Figure 3. B-cell depletion has ambivalent effects on the course of immune
diseases. The figure is a schematic representation of the findings of Matsushita et
al.75 During the course of EAE, B cells play opposite overlapping roles. Depending on
the timing of anti-CD20 antibody administration, the net clinical effect can be
deleterious, neutral, or beneficial. Dashed lines indicate the severity of EAE in
untreated control animals.
experienced a greater variation in disease onset and severity, and
failed to recover completely. After this seminal contribution,
independent groups made consistent observations in other experimental models of autoimmune diseases, including collageninduced arthritis78 and a model of spontaneous colitis, pathologically reminiscent of human ulcerative colitis.79 Interestingly,
whereas the pathogenic T-cell response involves the same T helper
1 (Th1) cells and Th17 proinflammatory T-cell populations in EAE
and collagen-induced arthritis, the colitis model differs in that the
inflammation appears to be driven by Th2 cells. Thus the B-cell
compartment has the capacity to control organ-specific inflammation that may be driven by Th1, Th2, or Th17 effectors.
Dissection of the underlying mechanisms revealed that B cells
limit immune disease progression by providing interleukin-10
(IL-10)75,78,80-82 that, in turn, directly suppresses the differentiation
of pathogenic T cells, promotes the development of regulatory
T cells,83 and constrains dendritic cell functions84 (Figure 1 right
panel).
The recent demonstration that IL-10–producing B cells exist in
humans, and that B cells from patients with multiple sclerosis85 and
lupus86 produce decreased amounts of IL10, suggests a general role
of B cells in clinical immune homeostasis.
Evidence for an immune-regulatory role of
B cells
Which B cells control the immune responses?
The explanation that reconciles such apparently conflicting results
has recently emerged from basic studies that demonstrate an
immune-regulatory role of B cells.
The first clue that B cells can regulate immune responses was
provided by seminal in vivo experimental studies by Shimamura et
al, demonstrating that adoptive transfer of antigen-activated B cells
could induce tolerance in naive mice through the induction of
suppressor T cells.76 Despite these data, the role of B cells in the
regulation of immune diseases remained overlooked for another
decade, until Wolf et al reported that mice lacking B cells suffered
an unusually severe and chronic form of EAE.77 Although spontaneous recovery is the norm in wild-type mice, the authors observed
that genetically B cell–deficient mice of the same background
B cells can be stimulated to produce IL-10 by a combination of
ligation of the B-cell receptor by the antigen and of CD40 by CD40
ligand. Some reports also point to a critical nonredundant role of
certain Toll-like receptors (TLR2 and TLR4) in driving the
regulatory activity in B cells.87
The involvement of the B-cell receptor, CD40, and TLRs in the
regulatory function of B cells raises a conceptual difficulty. Indeed,
these signals are the very same as the ones involved in the
activation of B cells in most immune responses. One hypothesis
would therefore be the existence of a peculiar “B-reg” subset,
endowed with the unique function to regulate immune processes. In
the mouse, B cells are classically divided into B-1 cells that reside
in pleural and peritoneal cavities and B-2 cells that populate
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BLOOD, 29 JULY 2010 䡠 VOLUME 116, NUMBER 4
DUAL ROLE OF B CELL IN IMMUNOPATHOGENESIS
secondary lymphoid organs. Peritoneal B-1 cells, known to produce particularly large amounts of IL10 after stimulation, have
been ascribed with regulatory function in some studies.88,89 However, because this subset was excluded from the transfer experiments demonstrating the regulatory role of B cells,78,80,90 it is likely
that B-2 cells also contain a subset regulating immune diseases.
Interestingly, a rare population of splenic B cells characterized
by a unique phenotype that associates features from B-1 (expression of CD5) and B-2 (high expression of CD1d, like the marginal
zone B cells) cells has recently been reported to play a critical role
in the regulation of murine EAE.82 However, the B-cell subsets
involved in suppression of other experimental immune diseases do
not have exactly the same phenotype.81,91-94
Ambiguity therefore remains regarding the B-cell subpopulation(s) involved in the regulation of immune responses. An
alternative hypothesis is that the immune-suppressive activity is
not a unique property of a single B-cell subset but is perhaps
exerted by different B-cell subsets, depending on the integration of
available signals in the microenvironment.
Clinical implications
Collectively, these studies demonstrate that Janus-faced B cells
play both pathogenic and regulatory activities in immunopathogenesis (Figure 1) and that these opposing contributions may overlap
during the course of the disease. Consequently, the therapeutic
effect of B-cell depletion depends on the relative contributions of
the opposing B-cell activities at the time of drug administration
(Figure 3). It would be of utmost importance to define simple
criteria or reliable markers that would help to predict whether
administration of rituximab would be beneficial for the patient.
Although there are clues about directions that should be taken in
the future, there is no easy answer to this question yet. The recent
identification of a human B-cell subset with regulatory properties
in the peripheral blood of lupus patients86 represents an important
step but will likely be insufficient because this subset appears to be
qualitatively rather than quantitatively deficient in these patients.86
A functional assay, which would measure the response of T cells to
a normalized stimulation in presence or absence of the B cells,
could be theoretically useful but its development would require an
important amount of work, the limitations of functional assay for
519
routine immunomonitoring being well known (minor changes of
test conditions potentially having a major impact on the test results,
necessity to work with freshly isolated cells, etc).
Of note, immune stimulation resulting from the removal of
B-cell regulation could also be used in a therapeutic perspective.
This strategy could be particularly useful in cancer, where B cells
have been shown to inhibit the induction of T cell–dependent
protective antitumor immunity.95 Accordingly, the administration
of anti-CD20 antibodies slowed the growth of nonhematopoietic
solid tumors (not expressing CD20) and enhanced the efficiency of
a tumor vaccine in a murine model.96 It is thus conceivable that the
mode of action of rituximab in hematologic malignancies also
relies in part on such a mechanism (Figure 2 bottom panel).
Conclusions
The optimization of B-cell depletion strategies for the treatment of
immune diseases therefore not only should focus on the identification of new targets and the development of more depleting drugs,
but also requires a better understanding of the complex temporal
interplay between pathogenic and regulatory B cells. Instead of a
mere depletion of the B-cell compartment, future therapy should
instead attempt to reset the regulatory balance—to which B cells
can clearly contribute.
Acknowledgments
This work was supported by grants from the CENTAURE Transplantation Research Network and the Hospices Civils de Lyon.
Authorship
Contribution: O.T., E.M., and T.D. wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Olivier Thaunat, Service de Transplantation
Rénale et d’Immunologie Clinique, Hôpital Edouard Herriot,
5 place d’Arsonval, 69437 Lyon Cedex 03, France; e-mail:
[email protected].
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2010 116: 515-521
doi:10.1182/blood-2010-01-266668 originally published
online March 25, 2010
Am''B''valent: anti-CD20 antibodies unravel the dual role of B cells in
immunopathogenesis
Olivier Thaunat, Emmanuel Morelon and Thierry Defrance
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