Importance of Cellular Microenvironment
and Circulatory Dynamics in B Cell
Immunotherapy
This information is current as
of June 15, 2017.
Qian Gong, Qinglin Ou, Shiming Ye, Wyne P. Lee, Jennine
Cornelius, Lauri Diehl, Wei Yu Lin, Zhilan Hu, Yanmei Lu,
Yongmei Chen, Yan Wu, Y. Gloria Meng, Peter Gribling,
Zhonghua Lin, Kathy Nguyen, Thanhvien Tran, Yifan
Zhang, Hugh Rosen, Flavius Martin and Andrew C. Chan
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2005; 174:817-826; ;
doi: 10.4049/jimmunol.174.2.817
http://www.jimmunol.org/content/174/2/817
The Journal of Immunology
Importance of Cellular Microenvironment and Circulatory
Dynamics in B Cell Immunotherapy1
Qian Gong,* Qinglin Ou,* Shiming Ye,* Wyne P. Lee,* Jennine Cornelius,§ Lauri Diehl,*§
Wei Yu Lin,* Zhilan Hu,* Yanmei Lu,‡ Yongmei Chen,† Yan Wu,*† Y. Gloria Meng,‡
Peter Gribling,* Zhonghua Lin,* Kathy Nguyen,* Thanhvien Tran,* Yifan Zhang,*
Hugh Rosen,¶ Flavius Martin,* and Andrew C. Chan2*
M
onoclonal Abs represent a cornerstone in the therapeutic armamentarium for cancers and autoimmune disorders. These therapeutic mAbs neutralize pathogenic
cytokines (e.g., anti-TNF mAbs for rheumatoid arthritis), inhibit
cellular proliferation (e.g., herceptin for Her-2⫹ breast cancers),
induce agonistic or inhibitory receptor activities to regulate cellular functions (e.g., anti-CD3 mAb for transplantation rejection),
and eliminate malignant and pathogenic cells by targeting cell surface Ags (1–3). Rituximab, an anti-CD20 mAb is a prototype of
the latter category and is efficacious in the treatment of B cell
malignancies and autoimmune disorders by depleting malignant
and, presumably, pathogenic B cells, respectively (4, 5).
CD20, a four transmembrane glycoprotein expressed on both
normal and malignant B cells, is regulated during B cell differentiation and extinguished when B cells undergo terminal differentiation into plasma cells (6 – 8). Although B cell depletion by rituximab reportedly involves immune effector cells, complement
proteins, and proapoptotic mechanisms, the functional consequences of B cell depletion and the in vivo factors that regulate B
cell immunotherapy are not well understood (reviewed in Ref. 9).
A recent report using a panel of anti-mouse CD20 mAbs demonstrated a role for monocytes as the dominant effector cell through
Fc␥RI- and Fc␥RIII-dependent pathways (10). In this study, we
demonstrate a greater complexity involving the circulatory dynam*Department of Immunology, †Antibody Engineering, ‡Assay and Automated Technology, and §Department of Pathology, Genentech, One DNA Way, South San Francisco, CA 94080; and ¶Department of Immunology, The Scripps Research Institute,
Institute for Childhood and Neglected Diseases 118, 10550 North Torrey Pines Road,
La Jolla, CA 92037
Received for publication August 6, 2004. Accepted for publication October 5, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work is supported by National Institutes of Health Grant RO1AI055509 (to
H.R.). The authors, with the exception of H.R., are employees of Genentech.
2
Address correspondence and reprint requests to Dr. Andrew C. Chan, Departments
of Immunology and Antibody Engineering, Genentech, One DNA Way, South San
Francisco, CA 94080. E-mail address: [email protected]
Copyright © 2005 by The American Association of Immunologists, Inc.
ics of B cells and survival factors from the microenvironment as
critical regulators that determine the mechanistic basis for immunodepletion through complement or the reticuloendothelial system
(RES)3. These data provide insights into the effectiveness of B cell
immunotherapy and provide additional strategies by which to enhance therapeutic efficacy.
Materials and Methods
Generation of human CD20⫹ mouse
Generation of human CD20 (hCD20)⫹ mice was accomplished through the
use bacterial artificial chromosomes (BAC) incorporating the hCD20 locus.
Two independent BACs were injected into blastocytes derived from FVB
mice to generate multiple transgenic (Tg) founder lines that expressed
hCD20. Two founder mice that transmitted hCD20 expression were subjected to more detailed analysis. Both founder lines demonstrated identical
patterns of hCD20 expression and hence data from only one founder line
will be presented in this report. The expression level on hCD20 Tg⫹/⫹
mice, as determined by mean fluorescence intensity, was ⬃50% in level
when compared with human circulating B cells (data not shown). This
difference could be due to differences in transcriptional compatibility between species, the presence of the mouse CD20 gene, enhancer elements
required for efficient hCD20 mRNA transcription not found within the
BAC or the integration site of the hCD20 BAC. The latter was less likely
because all founder lines expressed identical mean fluorescence intensity
levels of hCD20 expression (data not shown).
Abs and FACS staining
Two anti-hCD20 mAbs, rituximab and 2H7, were used for immunodepletion studies. All other Abs were purchased from Becton Dickinson and BD
Pharmingen. Cell surface expression of molecules was determined using
FACScan and FACSCalibur machines.
Mobilization of marginal zone (MZ) compartment B cells
Mice were pretreated with control IgG2a 3 days before the initiation of the
study (day 3) to minimize nonspecific effects of IgG on cellular trafficking.
At day 0, mice were treated with 0.2 mg control IgG2a or anti-hCD20
mAb. Mice were injected i.v. on day 2 with 0.1 mg anti-CD11a (M17) and
3
Abbreviations used in this paper: RES, reticuloendothelial system; hCD20, human
CD20; BAC, bacterial artificial chromosome; Tg, transgenic; GC, germinal center;
MZ, marginal zone; CVF, cobra venom factor; FO, follicular; NHL, non-Hodgkin’s
lymphoma.
0022-1767/05/$02.00
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B cell immunotherapy has emerged as a mainstay in the treatment of lymphomas and autoimmune diseases. Although the
microenvironment has recently been demonstrated to play critical roles in B cell homeostasis, its contribution to immunotherapy
is unknown. To analyze the in vivo factors that regulate mechanisms involved in B cell immunotherapy, we used a murine model
for human CD20 (hCD20) expression in which treatment of hCD20ⴙ mice with anti-hCD20 mAbs mimics B cell depletion observed
in humans. We demonstrate in this study that factors derived from the microenvironment, including signals from the B cellactivating factor belonging to the TNF family/BLyS survival factor, integrin-regulated homeostasis, and circulatory dynamics of
B cells define distinct in vivo mechanism(s) and sensitivities of cells in anti-hCD20 mAb-directed therapies. These findings provide
new insights into the mechanisms of immunotherapy and define new opportunities in the treatment of cancers and autoimmune
diseases. The Journal of Immunology, 2005, 174: 817– 826.
818
anti-␣4 integrin (PS/2) mAbs and blood analyzed 1.5 and 6 h following the
administration of the anti-integrin mAbs. For studies with LPS, the antiintegrin mixture was substituted with 25 ␮g of LPS treatment and mice
analyzed 6 h following LPS treatment.
Inhibition of lymphocyte egress
Human CD20 Tg⫹ mice were treated by oral gavage with vehicle control
or compound SEW2871 (10 mg/kg every 6 h). A single dose of control or
anti-hCD20 mAb (0.5 mg i.p.) was administered 2 h after the first dose of
compound SEW2871. B and T lymphocytes isolated from lymph nodes
20 h following treatment.
Generation of chimeric mice
Chimeras were generated by transferring 15 ⫻ 106 bone marrow-derived
cells from hCD20⫹ (100:0 chimeras) or 7.5 ⫻ 106 bone marrow-derived
cells each from hCD20⫹ and hCD20⫺ littermates (50:50 chimeras) into
irradiated hCD20⫺ littermates (2 doses of 525 rads). The latter mixture
resulted in 35– 60% hCD20 Tg⫹ B cells in three independent experiments.
Five weeks following transfer, mice were treated with 0.2 mg of control
IgG2a or anti-hCD20 mAb and analyzed 7 days following treatment.
Inhibition of complement by cobra venom factor (CVF)
Depletion of NK, polymorphonuclear neutrophils, and
macrophages
Mice were treated with anti-NK1.1 (0.2 mg/mouse, i.p.) or anti-Gr-1 (0.25
mg/mouse, i.v.) mAbs to deplete NK and neutrophils, respectively. Treatment with control IgG2a (0.1 mg) or anti-hCD20 mAb (0.1 mg) on day 2
was followed by FACS analysis of circulating or splenic B220⫹, MZ or
follicular (FO) B cells on day 4.
For macrophage depletion, mice were pretreated with buffer or clodronate-loaded liposomes. One day later, mice were then treated with either
0.1 mg of control IgG2a or anti-hCD20 mAb.
Results
Hierarchy of B cell subset susceptibilities
To understand the in vivo mechanisms by which therapeutic mAbs
eliminate cells by targeting cell surface Ags, we used a murine
model with an integrated BAC encoding hCD20 that recapitulates
the developmental expression of hCD20 in the mouse B cell lineage under its own genetic regulatory elements. Expression of
hCD20 was readily detected at the immature B cell stage in the
bone marrow and was highly regulated during differentiation (Fig.
1A). Only B220⫹ peripheral blood cells expressed hCD20 (Fig.
1B). Immunohistochemical analysis of splenic tissue derived from
these hCD20 Tg⫹ mice revealed colocalization of hCD20 staining
with IgM among the B cell zones (Fig. 1C). Paralleling human B
lineage cells, hCD20 was not colocalized with IgMhigh staining
plasma cells by immunohistochemical analysis (Fig. 1C, white arrows) nor on syndecan-1⫹ plasma cells by FACS analysis (data not
shown). This model also preserved the Ag epitopes of hCD20 and
Ab specificities of two anti-hCD20 mAbs, rituximab and 2H7. Rituximab is a chimeric anti-hCD20 mAb approved in the treatment
of non-Hodgkin’s lymphoma (NHL) and 2H7 is a humanized antihCD20 mAb in clinical development in autoimmune disorders (reviewed in Ref. 9).
Similar to the depletion of peripheral B cells observed in patients treated with rituximab or 2H7, administration of a mouse
anti-hCD20 (2H7) mAb resulted in complete and reversible depletion of peripheral blood B cells (Fig. 2A, top row, Fig. 2B, and data
not shown). Because hIgG1 demonstrates substantially different
binding to human and murine Fc␥Rs, the remaining studies used
an anti-hCD20 mAb (2H7) with a murine IgG2a Fc backbone,
which best recapitulates hIgG1-hFc␥RI/hFc␥RIII interactions.
Consistent with the lack of expression of hCD20 in the mouse
early B cell progenitor population, only CD20⫹ immature and
recirculating mature B cells in the bone marrow were depleted
(Fig. 2C).
In addition to the depletion of circulating B cells, treatment also
resulted in depletion of B220⫹ cells from lymph nodes and peritoneal cavity of hCD20 Tg⫹ mice (Fig. 2A, middle and bottom
row). Intriguingly, the kinetics of depletion differed among these
three compartments. Although ⬎90% of circulating B cells were
depleted within 3 h following i.v. administration of anti-hCD20
mAbs, lymph node B cells were depleted within 2 days, and peritoneal B cells required ⬃21 days for ⬎90% depletion despite i.p.
administration of the anti-hCD20 mAb. Because peritoneal B cells
recirculate more slowly than lymph node B cells, the distinct kinetics of depletion paralleled the kinetics of lymphocyte circulation (11).
Although circulating mature B cells were completely depleted,
⬃33% of B220⫹ splenocytes remained following anti-hCD20
mAb treatment (Fig. 3A). Analysis of splenic B cell subsets revealed that FO B cells were significantly depleted (⬎90% depletion). Additional analysis of 2-wk-old mice, in which immature B
cells are more abundantly represented, demonstrated a similar
⬎90% depletion of both T1 and T2 immature B cell subsets following anti-hCD20 mAb treatment (data not shown). In contrast,
the CD21highCD23low MZ B cell compartment exhibited greater
apparent resistance to anti-hCD20 mAb depletion. Approximately
50% of these cells remained following anti-hCD20 mAb therapy
(Fig. 3B). Resistance was not due to the lack of hCD20 expression
in MZ B cells as hCD20 was expressed at a higher level in MZ as
compared with FO B cells (Fig. 1A), nor due to the lack of accessibility of the therapeutic mAb as CD20 on resistant splenic B cells
were nearly saturated with the in vivo administered anti-hCD20
mAb (Fig. 3C). Neither administration of anti-hCD20 mAb up to
10 mg/mouse (⬃15-fold greater than the clinical dose of rituximab
for treatment of NHL) nor continued treatment of mice every other
week for 4 mo with anti-hCD20 mAb resulted in any greater depletion of the MZ B cell compartment (data not shown).
In contrast to the relative resistance of the MZ compartment,
germinal center (GC) B cells resident within Peyer’s patches demonstrated greatest resistance to anti-hCD20 mAb treatment. Although mature B220⫹CD38high B cells were readily depleted, the
B220⫹CD38low GC B cells were resistant to anti-hCD20 mAb
therapy (Fig. 3D). To extend our observations on Peyer’s patch
resident GC B cells, we tested whether splenic GC B cells, generated through immunization with SRBC, were similarly resistant.
As GCs are maximally formed by day 8 following immunization,
mice were treated on day 8 with anti-hCD20 or control IgG mAbs.
Although unimmunized mice did not develop B220⫹PNA⫹ GC B
cells, SRBC immunized mice developed PNA⫹ GC B cells that
were resistant to anti-hCD20 mAb killing (Fig. 3E). Resistance
was independent of: 1) hCD20 expression, as both Peyer’s patch
resident or splenic GC B cells expressed higher levels of hCD20
than the sensitive mature circulating B cells (Fig. 1A and data not
shown); 2) binding of mAb to GC cells, as in vivo recovered GC
B cells were saturated with the administered mAb; 3) treatment
dose; and 4) duration of treatment (data not shown). Hence, a
hierarchy of sensitivity to anti-hCD20 mAb treatment exists for B
cells within different splenic microenvironments with FO (most
sensitive) ⬎ MZ ⬎ GC (most resistant) compartments.
The residual B cells in treated mice were functional as antihCD20 mAb-treated mice were capable of mounting substantial,
albeit reduced, immune responses to immunogens and bacteria
(data not shown). Conversely, baseline serum IgM levels were not
decreased following 4 wk of anti-hCD20 mAb treatment (Fig. 2D).
Because the serum half-life of IgG is longer than IgM, we assessed
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Mice were treated with daily doses of CVF (2 ␮g/mouse, i.p.) for three
consecutive days. One hour following the second dose of CVF, mice were
treated with either 0.1 mg of control IgG or anti-hCD20 mAb. Serum C3
levels were monitored using a cell-based FACS assay (Sigma-Aldrich).
IN VIVO MECHANISMS OF ANTI-CD20 mAb THERAPY
The Journal of Immunology
819
the effects of chronic administration of anti-hCD20 mAb (⬎12 mo
of anti-hCD20 mAb every other week) on serum IgG levels. Serum
IgG1, IgG2b, and IgG3 were not altered with long-term chronic
anti-hCD20 mAb treatment (Fig. 2E). Interpretation of the serum
IgG2a levels was not possible because the administered antihCD20 IgG2a mAb interfered with measurement of the serum
IgG2a levels. These data are consistent with the differential effects
observed on self- and non-self-reactive Abs in patients treated with
rituximab (reviewed in Ref. 12).
Intravascular access as a major determinant of susceptibility
To explore factors that contribute to the relative resistance of the
MZ B cell compartment, we first evaluated the intrinsic sensitivity
of the MZ B cells to anti-hCD20 mAb-mediated depletion. MZ B
cells were mobilized into the vasculature with the coadministration
of anti-␣L and anti-␣4 integrin mAbs (Fig. 4A, panels 1–3) (13).
Mobilization of CD21highCD23low MZ B cells rendered them sen-
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FIGURE 1. Generation of a mouse
model of hCD20 Tg expression. A, Surface expression of hCD20 during B cell
ontogeny. B cell progenitors and subsets
in the bone marrow (top row), spleen
(middle row), and other lymphoid organs
(bottom row) were analyzed for hCD20
expression. The thick red lines represent
staining for hCD20 from a hCD20 Tg⫹
mice and the thin blue lines represent
hCD20 staining from a control hCD20
Tg⫺ littermate. Mean fluorescence intensity MN is represented numerically in
each plot. These data are representative
of over 20 independent analyses. B, Expression of hCD20 in circulating lymphocytes. Peripheral lymphocytes were
analyzed for surface expression of B220
and CD3 (upper left panel). B220⫹ (upper right), CD3⫹ (lower right), and
CD3⫺B220⫺ (lower left) cells were analyzed for hCD20 expression. Expression of hCD20 was not detectable on
Tg⫺ littermates (shaded). C, Immunohistochemical analysis of hCD20 expression in Tg⫹ mice. Spleens from Tg⫹ (left
column) or Tg⫺ (right column) mice
were stained for IgM (green), hCD20
(red), or CD5 (blue). Single staining for
IgM (top panels) and hCD20 (middle
panels) is shown. The bottom panel represents merging of all three markers.
sitive to anti-CD20 mAb-mediated depletion (compare Fig. 4A,
panel 2 vs panel 5 and panel 3 vs panel 6, and Fig. 4B). Histologic
analysis of the spleen confirmed the preferential depletion of MZ
B cells outside of the MOMA-1 staining marginal sinus with the
combined treatment of anti-integrins and anti-hCD20 mAbs as
compared with anti-hCD20 mAb alone (Fig. 4D, panel 2 and panel
3). In contrast, mobilization of cells from the MZ into the follicle
with administration of LPS did not result in depletion of the
CD21highCD23low MZ splenic B cells (Fig. 4C, and D, panel 4)
(14). Together, these data indicate that MZ B cells are not intrinsically resistant to anti-hCD20 mAb-mediated killing and that trafficking of MZ B cells into the vasculature can render them susceptible to depletion.
Conversely, we tested whether retaining mature B cells within
the lymph node would interfere with B cell depletion. Mice,
treated with vehicle control or a sphingosine 1-phosphate receptor
ligand (compound SEW2871), were treated with anti-hCD20
820
IN VIVO MECHANISMS OF ANTI-CD20 mAb THERAPY
mAbs (15). Consistent with the inhibitory effects of sphingosine
1-phosphate receptor ligands on lymphocyte egress from lymph
node to circulation (16), both B and T cells were significantly
decreased in the blood of mice treated with compound SEW2871
(Fig. 4F). Although lymph node B cells were readily depleted by
anti-hCD20 mAbs in vehicle-treated mice, they were minimally
affected by anti-hCD20 mAbs in the presence of compound
SEW2871 (Fig. 4E, left panel). The effect was specific to B cells
as no effects were observed on T cells (Fig. 4E, right panel). Together, these data support the requirement for B cells to access the
circulation for efficient depletion.
Contributions of microenvironment to B cell susceptibility
Although access to the intravascular compartment was important for efficient B cell killing, we further analyzed the roles of
the microenvironment to anti-hCD20 mAb-mediated depletion.
We first used cellular competition experiments to evaluate the
importance of the microenvironment. Chimeric mice were gen-
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FIGURE 2. Depletion of B cells with antihCD20 mAbs. A, Distinct kinetics of B cell
depletion in blood, lymph node, and peritoneal cavity. Mice were treated with 0.2 mg of
anti-CD20 mAb and blood (top), lymph node
(middle) and peritoneal cavity (bottom) analyzed for the presence of B cells at 3 h, 2 days,
or 21 days following treatment (n ⱖ 5 mice/
group). B, Depletion and repletion of B cells
following anti-hCD20 mAb treatment. Mice
were treated with a single dose IgG2a or antihCD20 mAb (0.1 mg, mouse 2H7). Peripheral
blood was analyzed on day 6, week 6, or week
14 following anti-hCD20 mAb administration
as described in A. Depletion correlated with
the circulating serum half-life of the therapeutic mAb (data not shown). Six weeks following
treatment, when the serum concentration of antihCD20 mAb was ⬍1 ␮g/ml, B cells were again
detected within the circulation and subsequently
normalized to pretreatment levels (n ⫽ 5 mice/
group). C, Depletion of bone marrow CD20⫹
cells. Mice were treated with the 2H7 antihCD20 mAb as described in B. Cells derived
from bone marrows of control IgG2a (䡺) or
anti-hCD20 mAb (f) treated mice were analyzed by FACS analysis and subsets quantified.
B cell subsets were defined as follows: Pro-B:
B220⫹CD43⫹; Pre-B: B220⫹CD43lowIgM⫺;
Immature: B220⫹CD43lowIgM⫹IgD⫺; and Mature: B220highCD43⫺IgM⫹IgD⫹. D, Effects of
anti-hCD20 mAb on serum IgM levels. Mice
were treated with control IgG or 2H7 antihCD20 mAb (0.5 mg on days 1 and 15). Serum
was measured for IgM levels at day 28. E,
Effects of long-term anti-hCD20 mAb treatment on serum Ig levels. Mice were treated
with control IgG or 2H7 anti-hCD20 mAb
(0.1 mg every other week for 12 mo). Serum
was measured for IgG1, IgG3, and IgG2b levels at 13 mo.
erated from 100% hCD20⫹ or a mixture of hCD20⫹:hCD20⫺
bone marrow-derived cells. Similar to the hCD20 Tg⫹ mice
(Fig. 3A), treatment of chimeric mice generated from 100%
hCD20⫹ bone marrow resulted in 50% depletion of the MZ B
cell compartment (Fig. 5A, top panels). In contrast, treatment of
chimeric mice derived from the mixture of hCD20⫹:hCD20⫺
bone marrow with anti-hCD20 mAbs resulted in ⬎90% depletion of the hCD20⫹ MZ B cell compartment (Fig. 5A, bottom
panels). Conversely, ⬎99% of FO B cells were depleted from
both groups of chimeric mice (data not shown). Hence, cellular
competition, potentially reflected by B cell survival factors
within the MZ microenvironment, can alter the threshold of
anti-hCD20 mAb-mediated killing.
As an example of such survival factors, we analyzed the contribution of the B cell-activating factor belonging to the TNF family (BAFF)/BLyS/TALL-1 member of the TNF superfamily (17).
BAFF/BLyS/TALL-1 plays an important role in the survival and
maturation of immature T2, FO, and MZ B cells and enhances
The Journal of Immunology
821
competitive survival of autoreactive B cells (18 –20). Overexpression of a soluble form of BAFF/BLyS/TALL-1 in mice results in
B cell hyperplasia, hypergammaglobulinemia, and autoimmune lupus-like syndrome (21). Conversely, treatment of lupus-prone
mice with a BAFFR/BR3-Fc fusion protein, which neutralizes
BAFF/BLyS, results in improved autoimmune serologies, renal
pathology, and mortality (22). As expected, treatment of hCD20⫹
mice with BAFFR/BR3-Fc for ⬃2 wk resulted in a marked decrease in MZ and T2/FO B cells (Fig. 5B, panel 3). Combined
treatment of BAFFR/BR3-Fc and anti-hCD20 mAb, surprisingly,
resulted in the depletion of all splenic B cell subsets (Fig. 5B,
panel 4). To further explore the potential synergy of BAFF neutralization and anti-hCD20 mAb, we quantified the extent of B cell
loss 4 days following treatment with single doses of anti-hCD20
mAb and BAFFR/BR3-Fc. Although treatment with single doses
of anti-hCD20 mAb or BAFFR/BR3-Fc resulted in ⬃40 –50% loss
of MZ B cells and ⬃33–70% loss of FO B cells, the combination
anti-hCD20 mAb and BAFFR/BR3-Fc resulted in ⬎90% loss of
MZ and FO B cells (Fig. 5C). Hence, survival factors also play an
important role in determining susceptibility to anti-hCD20 mAbmediated B cell depletion.
Effector mechanisms required for depletion
To analyze the effector mechanisms important for in vivo depletion, we assessed the contributions of complement and Fc receptor
effector function. In vivo depletion of C3 by administration of
CVF (23) did not affect the ability of wild-type anti-hCD20 mAbs
to deplete circulating, lymph node, or splenic FO B cells (Fig. 6A,
panels 1, 2, and 5). In contrast, the absence of C3 abrogated the
ability of anti-CD20 mAbs to deplete MZ B cells (Fig. 6A, panel
4). A similar requirement of C3 for MZ B cell depletion was also
observed in hCD20⫹ C3⫺/⫺ mice (data not shown). Hence, the
MZ B cell compartment exhibits a selective dependence on complement for anti-hCD20 mAb killing.
As Fc receptor binding has been demonstrated to be required for
the efficacy of rituximab in the elimination of CD20⫹ B cell xenografts (24), we assessed the contributions of Fc effector function
through the use of an anti-hCD20 mAb with two mutations within
the Fc effector domain (D265A, N297A). This mutant exhibited no
binding to Fc␥R (RI, RII, and RIII) but also a partial reduction in
complement activation (data not shown). Treatment of mice with
the anti-hCD20 (D265A, N297A) mutant resulted in the loss of
ability of the mutant anti-CD20 mAb to deplete circulating, lymph
node, and splenic FO B cells (Fig. 6B, panels 1, 2, and 5). This was
not due to altered pharmacodynamics as recovery of these B cells
demonstrated saturation of surface CD20 with the in vivo administered mutant anti-hCD20 mAb (data not shown). In contrast, MZ
B cells were still depleted by the mutant anti-hCD20 mAb though
to a lesser degree than wild type anti-CD20 mAb (Fig. 6B, panel
4). The intermediate depletion of MZ B cells observed with the
D265A, N297A mutant was likely due to the partial loss of complement activation. Consistent with this idea, administration of
CVF reversed the partial depletion by the mutant anti-CD20 mAb
(Fig. 6C). Hence, different mechanisms appear important for depletion of distinct splenic B cell compartments with complement
dependent mechanisms playing a dominant role in the MZ B cell
compartment and Fc receptor-mediated mechanisms in the elimination of circulating, lymph node, and splenic FO B cells.
We next assessed the cellular components (neutrophils, macrophages, and NK cells) required for depletion of circulating, lymph
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FIGURE 3. Hierarchy of B cell sensitivities to anti-hCD20 mAb depletion. A, Mice were treated with 0.5 mg of anti-hCD20 mAb (bottom) or control
IgG2a mAb (top). Spleens were harvested at day 4 following treatment and analyzed for B220 and IgM staining (left panels) or CD21, CD23, and B220
(right panels). Enumeration of FO and MZ B cells depletion are represented in B (n ⫽ 5 mice/group). These data are representative of over 50 independent
experiments. C, Saturation of CD20 with anti-hCD20 mAbs on resistant splenic B cells. B220⫹ splenocytes isolated from anti-hCD20 mAb-treated mice
were analyzed ex vivo with either a FITC anti-mouse IgG2a mAb (to detect bound anti-hCD20 mAb, blue) or with additional anti-hCD20 mAb followed
by FITC anti-mouse IgG2a mAb (to detect the total amount of CD20 expressed, red) on resistant splenic B cells. D, Resistance of Peyer’s patch GC B cells
to anti-hCD20 mAb-mediated cell depletion. Peyer’s patches were isolated from control IgG2a-treated (top) or anti-hCD20 mAb-treated (bottom) mice as
described in A and analyzed for B220 and CD38 staining. Mature and GC B cells were quantified on the right (n ⫽ 5 mice/group). These data are
representative of over 10 independent experiments. E, Resistance of splenic GC B cells. Unimmunized and SRBC-immunized mice that generate splenic
GCs were treated with control IgG2a (top) or anti-hCD20 (bottom) mAbs. Splenic GC B cells were quantified by B220 and peanut agglutinin staining (n ⫽
5 mice/group). These data are representative of two independent experiments.
822
IN VIVO MECHANISMS OF ANTI-CD20 mAb THERAPY
node, and FO B cells. Depletion of macrophages using clodronateloaded liposomes resulted in loss of B cell killing (Fig. 7A, panel
3 and panel 4) (25). Greater than 90% of B cells remained in
circulation despite anti-hCD20 mAb treatment and these cells were
saturated with the in vivo administered mAb (Fig. 7A, panel 4 and
data not shown). In contrast, depletion of NK cells and neutrophils
did not affect B cell killing (data not shown). Hence, macrophages
play important roles as effectors in anti-hCD20 mAb-mediated B
cell depletion.
Because macrophages within the RES represent a major modality for clearance of apoptotic cells and immune complexes (26), we
examined the contributions of the spleen and liver to B cell depletion. Surprisingly, splenectomy mice did not compromise the
ability of anti-hCD20 mAbs to deplete B cells, but actually resulted
in accelerated B cell depletion (Fig. 7B), an effect that was likely
secondary to reduced B cell numbers in splenectomized mice. Compromise of blood flow to the liver through ligation of both portal vein
and celiac artery resulted in a significant loss in the depleting ability
of anti-hCD20 mAbs (Fig. 7C). Histological examination of livers 15
min following anti-hCD20 mAb administration demonstrated colocalization of B220⫹ staining B cells within F4/80⫹ staining macrophages in anti-hCD20 mAb-treated, but not control IgG-treated mice
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FIGURE 4. Intravascular access of B cells is required for efficient B cell depletion. A, Mobilization of MZ B cells enhances their sensitivity to
anti-hCD20 mAb depletion. Mice pretreated with control IgG2a (top row) or anti-hCD20 (bottom row) mAbs were additionally treated with control IgG2a
(panel 1 and panel 4) or a mixture of anti-integrin mAbs (panels 2, 3, 5, and 6) as described in Materials and Methods. Peripheral B cells were analyzed
by FACS analysis at 1.5 or 6 h following mobilization. Absolute numbers of MZ B cells (CD21highCD23low) in the blood were quantified in B. Analysis
of splenic B cells revealed a concomitant decrease in MZ B cells (data not shown) (n ⫽ 5 mice/group). These data are representative of three independent
experiments. N.D., Not detectable. C, Splenocytes were isolated and analyzed from mice treated with saline or LPS as described in Materials and Methods
(n ⫽ 5 mice/group). D, Immunohistochemistry of splenic tissue from mice treated with control IgG2a, anti-hCD20 mAb, anti-hCD20, and anti-integrin
mAbs, or anti-hCD20 mAb and LPS. E, hCD20 Tg⫹ mice pretreated with vehicle control or compound SEW2871 were subsequently treated with control
IgG2a or anti-hCD20 mAb as described in Materials and Methods. Lymph node T cells and B cells were quantified and expressed as mean (⫾ SE; n ⫽
4). F, Inhibition of lymphocyte egress by compound SEW2871. Circulating T cells and B cells from mice treated with vehicle (䡺) or compound SEW2871
(f), as described in Materials and Methods, were analyzed and quantified by FACS analysis (n ⫽ 5 mice/group). These data are representative of two
independent experiments.
The Journal of Immunology
823
(Fig. 7D). Hence, consistent with the function of the RES, the liver
and, to a lesser degree, the spleen represents the major site of depletion of circulating B cells (data not shown).
Discussion
These data identify two in vivo mechanisms by which anti-hCD20
mAbs eliminate B cells. Upon administration of anti-hCD20
mAbs, the mAb rapidly binds CD20⫹ B cells and circulating B
cells are cleared through the RES. B cells residing within lymphoid
tissues, including splenic FO B cells, Peyer’s patch mature B cells,
lymph node B cells, and peritoneal cavity B cells require access to
the vasculature to deliver the targeted B cells to effector cells
within the RES. This mechanistic model accounts for the longer
durations required for depletion of slower recirculating peritoneal
and lymph node B cells as compared with the circulating B cells.
Accordingly, compromise of RES function, as has been described
in some patients with systemic lupus erythematosus, may contribute to observations of a lower efficiency of peripheral B cell clearance by rituxan in systemic lupus erythematosus patients as compared with patients with rheumatoid arthritis (5, 27, 28).
FIGURE 6. Effector mechanisms of
anti-hCD20 mAb treatment. A, Control
(䡺) or complement-depleted (f) mice
were treated with control IgG2a or antihCD20 mAbs. Blood (panel 1), lymph
node (panel 2), and spleen (panels 3–5)
were analyzed by FACS and enumerated (n ⫽ 5 mice/group). These data are
representative of five independent experiments. B, Mice were treated with
0.1 mg of control IgG2a (䡺), wild
type anti-hCD20 (f), or an Fc mutant
anti-hCD20 (u) mAbs. Three days
following mAb treatment, blood
(panel 1), lymph node (panel 2),
splenic B220⫹ (panel 3), splenic
CD21highCD23low MZ (panel 4), and
splenic CD21lowCD23high FO (panel
5) were analyzed by FACS and enumerated (n ⫽ 20 mice/group). C,
Mice were treated as described in B in
the presence and absence of CVF as
described in A (n ⫽ 5 mice/group).
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FIGURE 5. Contributions of BAFF/BLyS to antihCD20 mAb-mediated cell killing. A, Chimeras generated by adoptive transfer of hCD20 Tg⫹:hCD20 Tg⫺ B
cells (100:0 or 50:50 chimeras) were treated with control IgG2a or anti-hCD20 mAb and analyzed 7 days
following treatment. FACS analysis of splenic
CD21highCD23low MZ B cells is depicted. Although the
percentage of MZ B cells recovered from the 100:0 chimeras were similar between control IgG2a and antihCD20 mAb-treated mice, the absolute numbers of MZ
B cells recovered were 6.9 ⫾ 1.3 and 3.5 ⫾ 0.4 ⫻ 106
(mean ⫾ SE) splenic MZ B cells, respectively (n ⫽ 4
control and 6 anti-CD20 mAb-treated mice). B, hCD20
Tg⫹ mice were treated with control IgG2a, BAFFR/
BR3-Fc (100 ␮g/mouse i.p. daily for 12 days), antihCD20 mAb (100 ␮g/mouse i.p. on day 9), or the combination of BAFFR/BR3-Fc and anti-hCD20 mAb
(same dosing as single treatment groups). B220⫹
splenocytes were isolated on day 13 and stained for
CD21 and CD23 (n ⫽ 5 mice/group). C, hCD20 Tg⫹
mice were treated with single doses of 0.1 mg control
IgG2a, BAFF/BR3-Fc, or anti-hCD20 mAb. Splenocytes were analyzed on day 4 (n ⫽ 5 mice/group).
824
IN VIVO MECHANISMS OF ANTI-CD20 mAb THERAPY
In contrast to the mechanism used for circulatory B cells, B cells
within the MZ compartment, with reduced circulatory capacity,
exhibit a greater dependence on complement-dependent, rather
than Fc receptor, mediated mechanisms for depletion. This notwithstanding, not all B cells within the MZ compartment are depleted even with longer duration of anti-hCD20 mAb treatment
and the extent of depletion of the MZ compartment varies among
different mouse genetic backgrounds (data not shown). Hence,
other factors, including cellular competition, microenvironment,
and differential expression of inhibitory proteins likely contribute
to additional mechanistic determinants of sensitivity. As an example of the latter, both CD55 and CD59 complement regulatory
proteins are highly regulated during B cell differentiation and ac-
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FIGURE 7. Effector cells and organs required for anti-hCD20 mAb treatment. A, Mice
were pretreated with buffer (panel 1 and panel
3) or clodronate (panel 2 and panel 4) loaded
liposomes and subsequently treated with either
control IgG2a (panel 1 and panel 2) or antihCD20 mAb (panel 3 and panel 4). Enumeration of circulating B cell number is shown on the
right. Depletion of splenic macrophages was
confirmed by FACS analysis of F4/80⫹ cells
(n ⫽ 5 mice/group). These data are representative of over 5 independent experiments. B, The
spleen is not required for B cell depletion. Mice
underwent either sham splenectomy (top row) or
splenectomy (bottom row) and analyzed for B
cell depletion (n ⫽ 5 mice/group). These data
are representative of three independent experiments. The percentage of circulating B cells
from splenectomized mice treated with control
IgG2a or anti-hCD20 mAb at various time
points are quantified on the right. C, The liver is
required for B cell depletion. Mice underwent
sham surgery (left) or clamping of the portal
vein and celiac artery (right) within 1 min after
i.v. injection of control IgG2a or anti-hCD20
(0.2 mg) mAbs. Following 10 min anti-hCD20
mAb administration, peripheral blood was analyzed for B220⫹IgM⫹ B cells. All cells isolated
from anti-hCD20 mAb-treated mice were saturated with the in vivo administered mAb (data
not shown) (n ⫽ 4 mice/group). These data are
representative of three independent experiments.
D, F4/80⫹ Kupffer cells colocalize with B220⫹
B cells. Mice were treated with 0.1 mg of control IgG2a (left) or anti-hCD20 (middle) mAbs.
At 15 min following administration, livers were
harvested and analyzed for B220 and F4/80
staining for B cells and macrophages, respectively. Arrows indicate B220⫹ B cells colocalized within F4/80⫹ macrophages. Colocalized
B220⫹ and F4/80⫹ cells from four control and
anti-hCD20 mAb-treated mice are quantified at
the right. LPF, low power field.
tivation that, in turn, may alter the thresholds for complement mediated lysis (data not shown). Expression of CD59 on leukemic
cells and NHL-derived cell lines has been reported to be associated
with resistance; conversely, neutralization by an anti-CD59 blocking mAb restores sensitivity to anti-CD20 mAb-mediated killing
(29, 30). Our studies also illustrate the importance of the BAFF/
BLyS survival factor in defining the threshold for anti-hCD20
mAb-mediated killing. As this survival factor has been recently
demonstrated to promote NHL B cell survival (31), elucidation of
factors contributing to resistance of MZ (e.g., BAFF/BLyS) and
GC (e.g., CD40/CD40L) B cell compartments may parallel those
used by lymphomas that are not responsive or relapse following
rituximab treatment (32, 33).
The Journal of Immunology
Acknowledgments
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
We thank Henry Lowman, Sean Bohen, Karin Reif and Melissa
Starovasnik for helpful discussions.
28.
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Similar to our study, the in vivo importance of the Fc␥RI and
Fc␥RIII pathways in B cell depletion by anti-mouse CD20 has
been recently recognized (8). However, in contrast to our study,
the effect of anti-mouse CD20 mAb depletion in the splenic compartment was more dramatic. Depletion of B220⫹ lymphocytes
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NHL patients that express the high affinity allele of Fc␥RIIIA
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