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A Novel IL-10−Independent Regulatory Role
for B Cells in Suppressing Autoimmunity by
Maintenance of Regulatory T Cells via GITR
Ligand
Avijit Ray, Sreemanti Basu, Calvin B. Williams, Nita H.
Salzman and Bonnie N. Dittel
J Immunol published online 24 February 2012
http://www.jimmunol.org/content/early/2012/02/24/jimmun
ol.1103354
http://www.jimmunol.org/content/suppl/2012/02/24/jimmunol.110335
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Copyright © 2012 by The American Association of
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Supplementary
Material
Published February 24, 2012, doi:10.4049/jimmunol.1103354
The Journal of Immunology
A Novel IL-10–Independent Regulatory Role for B Cells in
Suppressing Autoimmunity by Maintenance of Regulatory
T Cells via GITR Ligand
Avijit Ray,* Sreemanti Basu,*,† Calvin B. Williams,†,‡ Nita H. Salzman,†,x and
Bonnie N. Dittel*,†
B
cells functionally contribute to both innate and adaptive
immune responses by contributing to Ag presentation and
through Ab production. The first evidence for the existence
of regulatory B cells in autoimmunity was obtained using the
mouse model of multiple sclerosis (MS), experimental autoimmune
encephalomyelitis (EAE). We showed that after EAE induction by
immunization with the myelin basic protein (MBP) peptide Ac111, B10.PL mice deficient in peripheral B cells (mMT) failed to
undergo spontaneous recovery and exhibited chronic disease (1,
2). These studies were replicated in C57BL/6 mMT mice immunized with a myelin oligodendrocyte glycoprotein peptide containing residues 35–55 (MOG35–55) (3). This same study showed
that B cell production of IL-10 was required for their regulatory
function (3). A role for B cell-derived IL-10 in suppressing autoimmunity has also been reported in models of arthritis and lupus
(4). Regulatory B cells have also been reported in humans (4).
*Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53201;
†
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53201; ‡Section of Rheumatology, Department of Pediatrics,
Medical College of Wisconsin, Milwaukee, WI 53201; and xDivision of Gastroenterology, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI
53201
Received for publication November 22, 2011. Accepted for publication January 23,
2012.
This work was supported by National Institutes of Health Grant R01 AI069358 and
the BloodCenter Research Foundation.
Address correspondence and reprint requests to Dr. Bonnie Dittel, BloodCenter
of Wisconsin, P.O. Box 2178, Milwaukee, WI 53201-2178. E-mail address: bonnie.
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: BM, bone marrow; EAE, experimental autoimmune encephalomyelitis; EGFP, enhanced GFP; GITR, glucocorticoid-induced
TNFR family-related protein; GITRL, glucocorticoid-induced TNFR ligand; MBP,
myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; MOG35–55, MOG
peptide containing residues 35–55; MS, multiple sclerosis; nTreg, natural T regulatory cells; Treg, T regulatory cell.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103354
Recently, the role of B cells in autoimmune diseases has been
further studied by their depletion using anti-CD20, which targets
B cells from the pre-B cell to memory stages. Plasma cells, which
do not express CD20, are not eliminated. In EAE, anti-CD20
depletion of B cells before induction of EAE with MOG35–55 recapitulated chronic disease observed in mMT mice (5). However,
in humans, treatment of MS with anti-CD20 (rituximab) resulted
in a significant reduction in the number of gadolinium-enhancing
lesions, providing evidence that B cells play a pathogenic role in
MS (6, 7). Evidence that rituximab also depletes regulatory B cells
are reports that its treatment for autoimmunity has led to severe
exacerbation of colitis and the spontaneous onset of colitis and
psoriasis soon after the start of treatment (2, 8–10). Treatment of
non-Hodgkin’s lymphoma with rituximab has also been associated
with the onset of autoimmunity (11).
Although the mechanism whereby B cell depletion results in
spontaneous autoimmunity is not known, a link with CD4+Foxp3+
T regulatory cells (Treg) is possible. Both humans and mice with
mutations in Foxp3 spontaneously develop autoimmune disorders
at a young age, which is now known to be due to a deficiency in
Treg (12). In addition, the adoptive transfer of Treg has been
shown to significantly reduce the severity of EAE (13). Also in
EAE, we showed that mMT mice had a reduction in the percentage
of Treg in the CNS (14). A subsequent study further demonstrated
a critical role for Treg in inhibiting late-phase EAE disease (15).
Although the later study did not investigate B cell–Treg interactions, both mMT and anti-CD20–depleted mice have been
shown to have reduced percentages of peripheral Foxp3+ Treg (16,
17). However, the absolute number of Treg was not determined.
Other studies have provided strong evidence that B cells regulate
Treg numbers by the induction of Foxp3 expression using both
in vitro and in vivo models by several mechanisms including
B cell production of IL-10 and TGF-b (18–20). However, it is not
known whether B cells can also regulate the number of natural
Treg (nTreg), which develop within the thymus (21).
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B cells are important for the regulation of autoimmune responses. In experimental autoimmune encephalomyelitis (EAE), B cells are
required for spontaneous recovery in acute models. Production of IL-10 by regulatory B cells has been shown to modulate the
severity EAE and other autoimmune diseases. Previously, we suggested that B cells regulated the number of CD4+Foxp3+ T
regulatory cells (Treg) in the CNS during EAE. Because Treg suppress autoimmune responses, we asked whether B cells control
autoimmunity by maintenance of Treg numbers. B cell deficiency achieved either genetically (mMT) or by depletion with antiCD20 resulted in a significant reduction in the number of peripheral but not thymic Treg. Adoptive transfer of WT B cells into
mMT mice restored both Treg numbers and recovery from EAE. When we investigated the mechanism whereby B cells induce the
proliferation of Treg and EAE recovery, we found that glucocorticoid-induced TNF ligand, but not IL-10, expression by B cells
was required. Of clinical significance is the finding that anti-CD20 depletion of B cells accelerated spontaneous EAE and colitis.
Our results demonstrate that B cells play a major role in immune tolerance required for the prevention of autoimmunity by
maintenance of Treg via their expression of glucocorticoid-induced TNFR ligand. The Journal of Immunology, 2012, 188: 000–000.
2
Materials and Methods
Mice
B10.PL (H-2u) WT, B6.129P2-Il10tm1Cgn/J (IL-102/2), and B6.SJL-Ptprca
Pep3b/BoyJ (CD45.1+) mice on the C57BL/6 (H-2b) background were
purchased from The Jackson Laboratory (Bar Harbor, ME). B6.CgFoxp3tm2Tch/J mice (Foxp3EGFP) were generated as described previously
(25). B cell-deficient B10.PL mice (mMT), MBP-TCR transgenic mice,
and generation of mixed bone marrow (BM) chimera mice were previously
described (1, 14). Foxp3EGFP and knockout mice were backcrossed to B10.
PL for three generations and then intercrossed, and were housed and bred
within the animal facility of the Medical College of Wisconsin. All animal
protocols were approved by the Medical College of Wisconsin Institutional
Animal Care and Use Committee.
B cell depletion in vivo
B cell-depleting anti-mouse CD20 mAb (18B12IgG2a) and its corresponding isotype control (2B8 msIgG2a) were provided by Biogen Idec. A
total of 250 mg Ab was i.v. injected into mice once or twice, 14 d apart.
B cell depletion kinetics was as previously reported (26).
Peptide and Abs
MBP Ac1–11 peptide (Ac-ASQKRPSQRSK) was generated by the Protein
Core laboratory of the Blood Research Institute, BloodCenter of Wisconsin. The 2.4G2 and Y19 hybridomas were obtained from American
Tissue Culture Collection. Mouse-specific CD4-allophycocyanin-eFluor
780, CD25-Alexa Fluor 700, TCR-b–FITC, CD11b-PE, and IL-17–
Alexa Fluor 647 were purchased from eBioscience (San Diego, CA).
Mouse-specific B220-PE-Texas Red, IFN-g–PE, Vb8.1,8.2-FITC, and
anti-human Ki-67–FITC and anti–BrdU-allophycocyanin were purchased
from BD Biosciences (San Diego, CA). Anti-mouse CD25-PE-Cy7 and
anti-mouse GITRL-PE were purchased from BioLegend (San Diego, CA).
Anti-mouse GITRL (YGL 386) was provided by BioLegend. F(ab9)2
fragment of goat anti-mouse IgM was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
EAE induction
EAE was induced by the i.v. adoptive transfer of 1 3 106 MBP-specific
encephalitogenic T cells activated in vitro with MBP Ac1–11 peptide into
sublethally irradiated (360–380 rad) mice as described previously (14).
Clinical symptoms of EAE were scored daily as follows: 0, no disease; 1,
limp tail; 1.5, hind-limb ataxia; 2, hind-limb paresis; 2.5, partial hind-limb
paralysis; 3, total hind-limb paralysis; 4, hind- and fore-limb paralysis; and
5, death.
Adoptive transfer of B cells
Splenic B cells from 8- to 10-wk-old mice were purified by complement
depletion of T cells using anti-Thy 1 and rabbit complement (Pel-Freez
Biological, Rogers, AR) (27) followed by removal of adherent cells by
incubating in serum-coated petri dishes at 37˚C for 30 min. B cell purities
were ∼95% as determined by flow cytometry. GITRL was blocked by
incubating cells with anti-mouse GITRL (10 mg/ml) at 4˚C for 60 min.
After washing in PBS, 15–25 3 106 cells were i.v. injected into each recipient mouse. EAE was induced 3 d later or splenic CD4+Foxp3+ cells
were enumerated on day 10. To determine proliferation of CD4+Foxp3+
cells, BrdU (0.8 mg/ml; Sigma, St. Louis, MO) was added to the drinking
water for 7 d.
B cell-mediated maintenance and proliferation of Treg in vivo
Splenic enhanced GFP (EGFP)+ Treg were sorted from WT Foxp3EGFP mice
and labeled with 3 mM Cell Proliferation Dye eFluor 670 (eBioscience,
San Diego, CA). A total of 0.2 3 106 Treg with or without 20 3 106 purified B cells were i.v. transferred into mMT recipient mice. Seven days
later, absolute numbers and proliferation of splenic CD4+EGFP+ cells were
determined.
Cell isolation, flow cytometry, and cell sorting
Mice were perfused with PBS and the brain and spinal cord were dissected
and homogenized. Mononuclear cells were isolated using 40/70% discontinuous Percoll gradients (Sigma-Aldrich, St. Louis, MO). Single-cell
suspensions from lymph nodes and spleen were obtained, counted, and
0.2–1 3 106 cells were incubated with anti-CD16/CD32 (clone 2.4G2; Fc
block) for 15 min followed by cell surface staining. Intracellular Foxp3
staining was performed using an anti-mouse/rat Foxp3-PE staining kit
from eBioscience, as per manufacturer’s instructions. Cells were acquired
on an LSRII flow cytometer (BD Biosciences, San Diego, CA), and data
were analyzed using FlowJo software (Tree Star, Ashland, OR).
Fluorochrome-labeled and/or EGFP-expressing cells were sorted using
a FACSAria cell sorter (BD Biosciences).
Detection of intracellular cytokines
Isolated cells were stimulated in vitro with PMA (50 ng/ml) and ionomycin
(600 ng/ml) (both from Sigma-Aldrich) for 4 h in the presence of monensin
(BD Biosciences). Cells were surface stained, fixed, and permeabilized, and
stained for intracellular cytokines using the Foxp3 staining buffer set from
eBioscience.
Histological analysis
Small and large intestine were fixed, paraffin embedded, and 5-mm sections
were stained with H&E.
T cell suppression assay
Splenic CD4+CD252 cells from WT (CD45.1) mice were sorted and
labeled with 3 mM CFSE (Molecular Probes, Invitrogen). EGFP+ Treg
(CD45.2) were sorted from WT Foxp3EGFP and mMT Foxp3EGFP mice.
CD4+CD252 cells (1 3 105) were cultured alone or with Treg from WT or
mMT mice in different ratios for 96 h. Anti-CD3 (2 mg/ml) and irradiated
(3000 rad) T cell-depleted syngeneic splenocytes (1 3 105) were added
into each well. Percentages of proliferating CD4+CD45.1+ cells were determined by CFSE dye dilution by flow cytometry.
In vitro coculture of B cells and Treg
Purified splenic B cells were activated in vitro with 5 mg/ml anti-IgM for 48
h. CD4+EGFP+ Treg from Foxp3EGFP mice were sorted and labeled with 3
mM Cell Proliferation Dye eFluor 670 (eBioscience, San Diego, CA). Treg
(0.5 3 105) alone or Treg mixed with naive or anti-IgM activated B cells
(1 3 105) were cultured in the presence of soluble anti-CD3 (2 mg/ml) and
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The homeostasis of Treg in the periphery has been shown to be
dependent on the presence of dendritic cells (22). TCR ligation fails
to induce Treg expansion in a similar manner as observed in conventional naive T cells (12). However, a role for glucocorticoidinduced TNFR family-related protein (GITR) in Treg expansion
has been described (23). Glucocorticoid-induced TNFR ligand
(GITRL)-expressing cells include dendritic cells, macrophages,
and B cells (24). Although it is not known whether GITRLexpressing cells play a role in nTreg homeostasis in WT mice,
transgenic mice bearing a B cell-specific GITRL transgene had
significantly increased numbers of peripheral Foxp3+ Treg (23),
suggesting that B cells could play a role in nTreg homeostasis.
In this study, we asked whether B cells regulate autoimmunity
via interactions with Treg. To address this question, we asked
whether mice genetically susceptible to spontaneous autoimmunity
would succumb to disease after B cell depletion with anti-CD20.
We found that B cell depletion resulted in the rapid onset of both
EAE and colitis, which was accompanied by a significant reduction in the number of peripheral Treg. From these data, we further
hypothesized that B cells control autoimmunity through the
maintenance of Treg numbers. In support of this hypothesis, we
found that mMT mice had a significant reduction in the absolute
number of Treg that was recapitulated by anti-CD20 B cell depletion in WT mice. Consistent with a reduction in Treg numbers,
anti-CD20–depleted mice exhibited chronic EAE, similar to mMT
mice. We found that B cell expression of GITRL, but not IL-10,
induced Treg proliferation, allowing the homeostatic maintenance
of peripheral Treg numbers. Furthermore, we showed that restoration of Treg numbers in mMT mice with WT or IL-102/2 B cells
resulted in spontaneous recovery, whereas mice that received
GITRL-blocked B cells exhibited chronic EAE similar to mMT
mice. Our results demonstrate that B cells via expression of
GITRL play an essential role in nTreg homeostasis by maintaining
their numbers above a threshold required for the prevention of
autoimmunity.
B CELLS CONTRIBUTE TO Treg HOMEOSTASIS VIA GITRL
The Journal of Immunology
irradiated (3000 rad) APC (1–2 3 105) for 96 h. After culture, the cells
were stained with CD4, and percentages of EGFP+ cells gated on CD4+
cells were determined by flow cytometry.
Statistical analysis
Data were analyzed using GraphPad prism software and were presented as
mean 6 SEM. Statistical significance was determined using the nonparametric Mann–Whitney U test or unpaired t test. The p values ,0.05 were
considered significant. The cumulative disease score was calculated by
adding the daily EAE scores from day 7 to the end of the experiment on
day 25 (Fig. 3B) or 30 (Fig. 7C) after EAE induction.
Results
3
To further demonstrate that B cells play a fundamental role in
maintaining autoimmune tolerance, we asked whether B cell depletion would accelerate the onset of colitis in IL-102/2 mice (31).
We found that a single injection of anti-CD20 resulted in severe
colitis by day 15, as evidenced by significant weight loss (Fig. 2A)
and histopathology of the colon (Fig. 2B). Anti-CD20, but not
isotype control-treated, mice also developed rectal prolapse by
day 15 (data not shown) and had significantly reduced numbers of
Treg in the Peyer’s patch (Fig. 2C). These data demonstrate that
B cells play a gatekeeping role in the prevention of autoimmunity
by controlling the number of Treg.
B cell depletion with anti-CD20 results in chronic EAE and
reduced numbers of Treg
The depletion of B cells with anti-CD20 (rituximab) is being increasingly used for the treatment of autoimmunity. Because a small
subset of patients treated with anti-CD20 spontaneously developed
autoimmunity (2, 8–10), we asked whether B cell depletion put
a mouse susceptible to spontaneous autoimmunity at increased
risk for development of disease. TCR transgenic mice specific for
MBP (MBP-TCR) have been shown to develop spontaneous EAE,
especially when housed under non-SPF conditions (28–30). In our
colony with strict SPF conditions, spontaneous EAE is rare and
does not occur under 12 wk of age. The treatment of 6-wk-old
MBP-TCR transgenic mice with anti-CD20 treatment exhibited
signs of EAE as early as 7 d later that continued to progress (Fig.
1A). Consistent with EAE induction, encephalitogenic T cells
were present in the CNS (Fig. 1B), displayed an activated
CD25+ phenotype (Fig. 1C), and produced IL-17 and IFN-g (Fig.
1D). Because the onset of spontaneous autoimmunity is consistent
with a deficiency in Treg, we next determined that anti-CD20
treatment resulted in a reduction in the percentage of CD4+
cells coexpressing Foxp3 (Fig. 1E) and in the absolute number of
splenic Treg numbers (Fig. 1F).
If B cell regulation of Treg numbers is a critical factor in the
regulation of EAE, then anti-CD20 B cell-depleted mice should
exhibit chronic EAE. For these studies, we induced EAE by
adoptive transfer instead of active immunization to avoid stimulation of immune cell subsets, including B cells, with TLR ligands
present in CFA. As shown in Fig. 3A, when EAE was induced
in anti-CD20–treated mice, disease onset and peak severity were
similar to the isotype control group and, like mMT mice, were
unable to resolve EAE (1, 3, 14). In addition, the anti-CD20 group
had a significant increase in both the cumulative (Fig. 3B) and
final EAE disease score (Fig. 3C), which was accompanied by
a significant increase in the absolute number of encephalitogenic
T cells within the CNS (Fig. 3D) and a significant reduction in the
number of Treg in the spleen (Fig. 3E), but not the CNS (Fig. 3F).
Because the ongoing EAE could have influenced the number of
Treg, we next demonstrated that B cell depletion with anti-CD20
resulted in a subsequent significant reduction in the number of
Treg in the spleen (Fig. 3G), but not the thymus (data not shown).
The absolute number of splenic CD4+ and CD8+ T cells was not
reduced in the anti-CD20–treated group (data not shown). To
FIGURE 1. B cell depletion leads to spontaneous EAE in mice susceptible to autoimmunity. Groups of three to four MBP-TCR transgenic mice at 6 wk
of age were administered anti-CD20 (18B12IgG2a) or its isotype control (2B8msIgG2a) on day 0. Clinical signs of EAE were scored daily (A). On days
17–21 post-Ab injection, the absolute number of CNS encephalitogenic T cells (CD4+Vb8.2+) (B) and those expressing CD25 (C) and IL-17 (x-axis) and
IFN-g (y-axis) (D) was determined by flow cytometry. The percentage of cells producing each cytokine is indicated. A representative dot plot showing the
percentage of CD4-gated cells expressing Foxp3 is shown (E). The absolute number of splenic Treg was also determined at the same time point (F). Pooled
or representative data from two to four independent experiments with three to nine mice in each group are shown. *p , 0.05, ***p , 0.001.
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Anti-CD20 depletion of B cells results in accelerated onset of
spontaneous autoimmunity
4
B CELLS CONTRIBUTE TO Treg HOMEOSTASIS VIA GITRL
further demonstrate that B cells regulate Treg numbers, we analyzed mMT mice and found that Treg numbers were reduced in the
spleen and lymph node (Fig. 3H), but not the thymus (data not
shown) compared with WT mice. We obtained identical results
with C57BL/6 mMT mice purchased from The Jackson Laboratory (data not shown).
During our analysis, we found that splenic Treg from mMT mice
exhibited a lower CD25 mean fluorescence intensity (Fig. 3I).
Because CD25 expression has been linked to Treg suppressive
capacity (32), we compared the ability of WT and mMT Treg to
suppress the proliferation of CD3 activated naive T cells and
found no difference (Fig. 3J). These data indicate that B cells are
important for Treg homeostasis, and that loss of Treg numbers,
and not function, is likely a contributing factor in B cell regulation
of autoimmunity.
B cells regulate Treg numbers in an IL-10– and B7-independent
manner
The finding that mMT mice and anti-CD20 B cell–depleted mice
exhibit largely identical EAE disease curves demonstrates that
chronic disease in mMT mice is likely not due to alterations in the
development or function of the peripheral immune system. This
question is of importance because mMT mice have been reported
to have T cell deficiencies (33, 34). Thus, we asked whether B cell
reconstitution in mMT mice would restore Treg numbers. As
shown in Fig. 4A, the adoptive transfer of naive WT splenic
B cells resulted in a significant increase in the number of Treg
cells as compared with mMT mice within the short timeframe of
10 d. To confirm that the transferred B cells are retained for at
least 10 d, we first determined that the splenocyte population in
WT mice contained 56% B220+ B cells and 19% CD4 T cells
(Fig. 4B). As expected, mature B cells were not detected in mMT
mice; thus, the percentage of CD4 T cells was subsequently increased to 35% (Fig. 4B). After B cell transfer into mMT mice,
B cells composed 12% of splenocytes, slightly reducing the percentage of CD4+ T cells to 30% (Fig. 4B). Because B cell pro-
duction of IL-10 has been implicated in their regulatory function
in EAE (3, 5), we asked whether this cytokine was also required
for B cell-mediated Treg homeostasis. Interestingly, the adoptive
transfer of IL-10–deficient B cells also led to a significant increase
in Treg similar to transfer of WT B cells (Fig. 4A). Because the B7
molecules have been implicated in Treg development and because
we have previously shown their importance on B cells in the
promotion of recovery from EAE (14), we next asked whether
CD80 and CD86 were critical mediators of B cell-mediated
Treg homeostasis. The adoptive transfer of CD80/CD86 doubledeficient (B71.22/2) B cells resulted in a significant increase in
the number of Treg as compared with mMT mice (Fig. 4C). Although the Treg increase was lower than that observed with WT
B cells, the difference between the two groups was not statistically
significant (Fig. 4C). When CD80 and CD86 single-deficient
B cells were transferred, the partial reduction in Treg numbers
was due to a loss of CD86 (Fig. 4C). Because MHC class II has
also been implicated, at least in part, in Treg homeostasis (35), we
transferred B cells rendered deficient in MHC class II via disruption of the C2ta gene (36) into mMT mice and found that they
functioned similar to their WT counterparts in driving an increase
in Treg numbers (Fig. 4D).
B cells contribute to Treg homeostasis by inducing their
proliferation
We next determined whether B cells maintained Treg homeostasis
by inducing their proliferation. First using an in vitro approach, we
cocultured B cells with Treg isolated from Foxp3EGFP reporter
mice (25) in the presence of anti-CD3 and measured cell proliferation using a fluorescent dye dilution assay. In the absence of
B cells, Treg underwent minimal proliferation (∼12%; Fig. 5A).
However, in the presence of resting B cells, ∼28% of Treg underwent proliferation (Fig. 5A). B cells deficient in IL-10 or
CD80/CD86 induced the proliferation of Treg similarly to WT
(data not shown). To determine whether B cells drive Treg proliferation in vivo, we adoptively transferred B cells into mMT
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FIGURE 2. B cell depletion leads to accelerated colitis in IL-102/2 mice. Groups of three to four IL-102/2 mice at 6 wk of age were administered antiCD20 (18B12IgG2a) or its isotype control (2B8msIgG2a) on day 0. Mice were weighed daily and the percentage of the starting weight is shown (A).
Representative histopathology of the colon from anti-CD20–treated IL-102/2 mice (original magnification 3200) is shown (B). The absolute number of
CD4+Foxp3+ Treg in the Peyer’s patches was determined by flow cytometry (C). Pooled or representative data from two to four independent experiments
with three to nine mice in each group are shown. *p , 0.05, ***p , 0.001.
The Journal of Immunology
5
mice and found that the percentage of proliferating Ki-67+ Treg
was increased by 1.5-fold as compared with mMT mice that received PBS (Fig. 5B). To further confirm that B cells promote
Treg expansion in vivo, we performed a continuous BrdU labeling
study and again found that the percentage of Treg that had proliferated and accumulated over 7 d was increased by 1.5-fold in
the presence of B cells (Fig. 5C). These data indicate that B cells
either directly or indirectly contribute to the homeostasis of Treg
by inducing their proliferation.
B cells regulate Treg homeostasis via GITRL
Because GITR has been implicated in the Treg proliferation and
B cells have been reported to express GITRL (23, 24), we asked
whether this receptor–ligand pair was responsible for Treg expansion. First, we confirmed previous reports that resting splenic
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FIGURE 3. Reduced Treg caused by B
cell depletion with anti-CD20 leads to unresolved EAE. (A–F) B10.PL mice in groups
of three to four were i.v. administered antiCD20 (18B12IgG2a) or its isotype control
(2B8msIgG2a) twice, 14 d apart (A, black
arrows). EAE was induced 3 d after the first
Ab treatment by adoptive transfer of 1 3 106
encephalitogenic T cells. Clinical signs of
EAE were evaluated daily (A). The cumulative disease score (B) and disease score on
day 25 of EAE (C) are shown. (D–F) At the
peak of EAE disease, the absolute number of
CNS CD4+Vb8.2+ T cells (D) and splenic
(E) and CNS (F) CD4+Foxp3+ Treg was
determined by flow cytometry. Pooled data
from 2–3 independent experiments with 6–
10 mice in each group are shown. (G) B10.
PL mice in groups of three to four were i.v.
administered anti-CD20 (18B12IgG2a) or
its isotype control (2B8msIgG2a) twice,
14 d apart. Five days after the second Ab
treatment, the absolute number of CD4+
Foxp3+ Treg in the spleen was determined
by flow cytometry. (H) The absolute number
of CD4+Foxp3+ Treg in the spleen and inguinal lymph node of WT and mMT mice
was determined by flow cytometry. Pooled
data from two to three independent experiments are shown (G, H). n = 6–10 (G) or 3–5
mice (H) in each group. (I) The average
(6SEM) mean fluorescence intensity (MFI)
of CD25 on splenic Treg (CD4+Foxp3+)
from WT and mMT mice is shown. (J) The
suppressive capacity of WT and mMT Treg
was measured in an in vitro assay using
CD4+CD252 responder (Tresp) cells from
CD45.1 mice (labeled with CFSE) and
EGFP+ Treg sorted from the spleens of
WT Foxp3EGFP and mMT Foxp3EGFP mice
(CD45.2). Proliferation of responder cells
stimulated with anti-CD3 in the presence or
absence of Treg was determined by CFSE
dye dilution by flow cytometry. Percentage
suppression at different Tresp/Treg ratios is
shown. Pooled data from two to three independent experiments (I) or an average of
two independent experiments (J) are shown.
*p , 0.05, **p , 0.01.
B cells express a low level of GITRL (Supplemental Fig. 1) (37).
When GITRL Ab-blocked B cells were transferred into mMT mice,
the absolute number of splenic Treg was significantly reduced as
compared with WT B cells (Fig. 6A). To further confirm a role for
GITRL in B cell-induced Treg expansion, we cotransferred EGFP+
Treg with B cells with or without blocked GITRL into mMT mice
and measured Treg recovery and proliferation. As compared with
Treg transfer alone, the number of recovered Treg was increased 4fold in the presence of B cells (Fig. 6B) with an increase in the
number of proliferating Treg from 10 to 35%, respectively (Fig.
6C). Ab blocking of GITRL on B cells before cotransfer with
EGFP+ Treg resulted in a significant reduction in the number of
recovered (Fig. 6B) and proliferating (Fig. 6C) Treg. These data
indicate that a primary mechanism whereby B cells regulate Treg
homeostasis is via their expression of GITRL.
6
B CELLS CONTRIBUTE TO Treg HOMEOSTASIS VIA GITRL
B cells require GITRL, but not IL-10, to promote recovery from
EAE
Using an active immunization model, it was previously reported
that B cell production of IL-10 was required for the recovery from
EAE (3). Because the CFA used for those studies contained TLR
FIGURE 5. B cells induce Treg proliferation
in vitro and in vivo. (A) Splenic Treg (CD4+EGFP+)
were sorted and labeled with cell proliferation dye
and cultured with soluble anti-CD3 and irradiated
splenic APC in the presence or absence of WT
splenic B cells. Four days postculture, proliferation
of EGFP+ cells gated on CD4+ T cells was determined by flow cytometry. (B and C) A total of 20 3
106 splenic B cells from WT mice were i.v. transferred into groups of mMT mice. BrdU was added to
the drinking water 3 d later and continued for 7 d.
Ten days posttransfer, the percentages of Ki-67+ (B)
and BrdU+ (C) splenic CD4+Foxp3+ Treg was determined by flow cytometry. Representative contour
plots of CD4Foxp3-gated cells that had incorporated
BrdU in mMT mice in the absence and presence of
adoptively transferred B cells are shown (C, left
panels). Pooled data from two to four independent
experiments (A) or pooled data from two independent experiments with six mice per group are shown
(B, C). *p , 0.05, **p , 0.01.
ligands with the potent ability to induce IL-10 production by
B cells, we asked whether in the absence of such external stimuli,
B cell production of IL-10 was required for recovery from EAE.
As was have previously reported, EAE induced by adoptive
transfer results in chronic disease in B10.PLmMT as compared
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FIGURE 4. B cell-mediated Treg homeostasis is not dependent on B cell production of IL-10 or expression of B7 or MHC class II. A total of 20 3 106
splenic B cells from WT (A–D), IL-102/2 (A), CD80/CD86 double-deficient (B71.22/2) (C), CD802/2 (C), CD862/2 (C), or C2ta2/2 (D) mice were i.v.
transferred into groups of mMT mice. Ten days posttransfer, the absolute number (mean 6 SEM) of splenic CD4+Foxp3+ Treg (A, C, D) and the percentage
of B220+ and CD4+ cells (B) was determined by flow cytometry. (A and C) Pooled data from 2–3 independent experiments with 6–10 mice per group are
shown. (B) Data are one representative experiment. (D) Pooled data from two independent experiments with three to four mice per group are shown. *p ,
0.05, **p , 0.01, ***p , 0.001.
The Journal of Immunology
7
with WT mice that undergo spontaneous remission and complete
recovery (Fig. 7A, 7B) (14). The adoptive transfer of WT B cells
once 3 d before EAE induction was sufficient to induce EAE recovery (Fig. 7A). Interestingly, neither B cell production of IL-10
nor expression of B7 was required for their ability to drive EAE
recovery (Fig. 7A). In contrast, when GITRL-blocked B cells were
adoptively transferred, the mMT mice exhibited chronic EAE (Fig.
7B). The cumulative disease score of mMT mice that received
GITRL-blocked B cells (39.4 6 1.5) was significantly more severe
than both WT (21.7 6 2.2) and mMT mice that received WT
B cells (23.7 6 1.2; Fig. 7C). An identical statistical result was
obtained for the final disease score on day 30 (Fig. 7C). As expected, mMT mice exhibited a chronic cumulative disease course
(44.8 6 4.7; Fig. 7C). On day 30, when we determined the absolute number of Treg in the spleen, we found that mice that received GITRL-blocked B cells had 46% fewer Treg as compared
with those that received WT B cells (Fig. 7D). Because of the
direct correlation between the number of B cells and Treg, we
confirmed that GITRL-blocked B cells were not deleted after
transfer. As shown in Fig. 7E, at day 30 after EAE induction, the
absolute number and percentage of GITRL-blocked B220+ cells
was similar to mice receiving WT B cells (Fig. 7E). These cumulative data demonstrate that B cells play an essential role in
immune tolerance to autoimmunity by maintaining a critical
number of Treg cells by promoting their expansion through
GITRL.
Discussion
In this study, we investigated the mechanism whereby B cells
regulate autoimmunity. We found that in the absence of B cells,
the absolute number of Treg was reduced rendering mice vulnerable to the rapid onset of spontaneous autoimmunity and the
inability to resolve EAE. Furthermore, we discovered that B cell
expression of GITRL controlled the number of Treg by inducing
their proliferation. Thus, we conclude that B cells play an essential
role in controlling the onset and severity of autoimmunity by
controlling Treg homeostasis and maintaining their numbers at
a critical threshold required for optimal inhibition and downregulation of autoimmune responses.
The clinical relevance of our study is that the loss of B cells and
the subsequent reduction in Treg resulted in the rapid onset of
spontaneous EAE and colitis (Figs. 1, 2). These data are highly
significant because one adverse effect of rituximab (anti-CD20)
treatment of autoimmunity has been the spontaneous onset of
a different autoimmune disease, often with a putative T cellmediated cause, with colitis and psoriasis being the most prominent (2, 8–10). Rituximab was developed as a treatment for nonHodgkin’s lymphoma and incidences of autoimmunity in these
patients are rare (11). One prominent reason for the increased
incidence of autoimmunity after rituximab treatment of autoimmune disorders in a subset of patients is likely due to their genetic
susceptibility to multiple autoimmune disorders (38). It is unlikely
that non-Hodgkin’s lymphoma patients would have increased risk
for the development of autoimmunity because cancer and autoimmunity are at opposite ends of the immune spectrum, with
cancer being associated with an underactive and autoimmunity
with an overactive immune response. Similar to our studies in
mice, the onset or exacerbation of autoimmunity after rituximab
treatment in several cases occurred within days (9, 10), which is
consistent with a rapid decline in Treg numbers (Fig. 3).
Although rituximab results in dramatic reduction in the number
of peripheral blood B cells, little is known about its effects on Treg
numbers. In this regard, in immune thrombocytopenia patients, it
was recently reported that the ratio of circulating CD4+CD25hi
Foxp3+ to CD4+ blood cells was not altered 1 mo after the first
infusion of rituximab, whereas B cells were essentially eliminated
(39). After splenectomy therapy, a similar analysis was conducted
using the spleen of healthy control subjects and patients with and
without rituximab therapy (39). The percentage of splenic B cells
was similar between healthy control subjects and untreated immune thrombocytopenia patients (39). As with peripheral blood,
patients treated with rituximab had a significant reduction in
the percentage of B cells (39). When the ratio of Treg/CD4+ cells
was examined, untreated immune thrombocytopenia patients had
a significant reduction in the ratio as compared with healthy
control subjects, consistent with their autoimmune status (39). Of
interest was the finding that rituximab-treated patients exhibited
a further reduction in the Treg/CD4+ ratio, although the reduction
did not reach significance (39). When the Th1/Treg ratio was
examined in CD4+ splenocytes, patients treated with rituximab
had a significant increase in the ratio compared with both control
subjects and nontreated patients (39). To our knowledge, this is the
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FIGURE 6. GITRL expressed by B cells promotes the maintenance and proliferation of Treg. (A) A total of 20 3 106 splenic WT or GITRL-blocked WT
B cells were i.v. transferred into groups of mMT mice. Ten days posttransfer, the absolute number (mean 6 SEM) of splenic CD4+Foxp3+ Treg was
determined by flow cytometry. (B and C) Splenic Treg (CD4+EGFP+) were sorted and labeled with a cell proliferation dye and were i.v. transferred into
mMT mice with or without purified B cells. Seven days later, the absolute number (mean 6 SEM) (B) and proliferation (C) of the labeled EGFP+ Treg in the
spleen of the mMT mice was determined by flow cytometry. Representative data (C, left panel) and pooled data from 2–4 independent experiments (B, C) or
pooled data from 2–3 independent experiments (A) with 6–10 mice per group are shown. **p , 0.01, ***p , 0.001.
8
B CELLS CONTRIBUTE TO Treg HOMEOSTASIS VIA GITRL
only human study that examined splenic Treg after rituximab
therapy. The cumulative data demonstrate that the findings in immune thrombocytopenia patients are consistent with our studies in
the mouse. Of particular interest is that we also did not detect
a difference in CD4+Foxp3+ Treg in the blood after anti-CD20
treatment (data not shown), indicating that changes in Treg cells in
the circulation cannot be used as a therapeutic marker of whether
a particular patient would be susceptible to the onset of spontaneous autoimmunity.
Although blocking of GITRL on B cells led to a significant
reduction in the number of Treg, the affect was not complete (Fig.
6). One reason is likely due to an incomplete block in B cell–Treg
interactions because of Ab shedding and/or turnover of GITRL.
We also cannot exclude the possibility that blocking of GITRL on
B cells alters Treg function. Another possibility is that other cell
surface molecules are involved. Because of the importance of B7
in Treg development and homeostasis (33, 40), we examined
whether CD80 and/or CD86 were also required. The transfer of
CD86-deficient B cells into mMT mice resulted in partial recovery
of the Treg as compared with WT B cells (Fig. 4). CD80-deficient
B cells were equivalent to WT. This difference is likely due to
resting B cell expression of CD86, but not CD80 (data not shown).
Although CTLA-4 has been implicated in Treg function (41–43),
using a blocking Ab, we found no evidence that it plays a role in
B cell-induced Treg homeostasis (data not shown). Because the
homeostatic expansion of CD4+CD25+ T cells was shown to require MHC class II (44), we determined whether its expression by
B cells promoted Treg expansion. Using both in vitro and in vivo
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FIGURE 7. B cell expression of GITRL, but not IL-10 or B7, is required for the resolution of EAE. A total of 20 3 106 splenic B cells from WT (A–E),
IL-102/2 (A), B71.22/2 (A) mice, or GITRL-blocked WT B cells (B–E) were i.v. transferred into groups of mMT mice. Three days posttransfer, EAE was
induced and the mice were scored daily for clinical signs of disease (A, B). (C) The cumulative disease score (mean 6 SEM, left panel) and the final disease
score on day 30 post-EAE induction (mean 6 SEM, right panel) are shown. (D, E) The absolute number (mean 6 SEM) of splenic CD4+Foxp3+ Treg (D)
and the absolute number (mean 6 SEM) and percentage of B220+ cells in the spleen (E) on day 30 post-EAE induction were determined by flow cytometry.
Pooled data from 2–3 independent experiments with 6–10 mice per group (A–C) or data from 1 experiment with 3 mice per group (D, E) are shown. *p ,
0.05, ***p , 0.001.
The Journal of Immunology
TLR9 ligands have been shown to induce IL-10 production by
B cells (51) (A. R. and B.N. D., unpublished observations). In
regard to TLR ligands, the TLR-signaling adaptor protein MyD88
was found to be essential for induction of EAE when globally
knocked out, but not when deficient in B cells (52). However,
MyD88 expression by B cells was required for their ability to
mediate recovery from EAE (52). In earlier studies using a similar
approach, this same group also demonstrated that B cell production of IL-10 was required for their regulatory function (3). This
leads to the speculation that TLR signaling in B cells in response
to CFA induces IL-10 production and subsequent immune regulation leading to recovery from EAE. However, more recently it
was shown that MyD88-deficient mice were also resistant to EAE
induction by adoptive transfer, indicating that exogenous TLR
ligands are not required for EAE induction (53). Interestingly, the
transferred encephalitogenic T cells proliferated and migrated to
the CNS in MyD882/2 mice in the absence of lesion progression
(53). A role for suppression by IL-10 in MyD882/2 mice was
demonstrated by the susceptibility to EAE in MyD88/IL-10
double-knockout mice (53). Interestingly, the source of the IL10 was determined to be from T cells, not B cells. Because in
this later study the EAE disease was very severe (score 4), the
ability of the mice to recover could not be assessed. Nevertheless,
in our adoptive transfer model using a less severe EAE disease
(score 2), the mice are able to recover without the need for TLR
ligands. These data indicate that engagement of TLR on B cells
and their production of IL-10 is not an absolute requirement for
their regulation of EAE. Rather, we propose that TLR ligands in
CFA produce a bystander effect by inducing B cell production
of IL-10 that then, in turn, downregulates the immune response,
allowing recovery from EAE. Indeed, IL-10 production by T cells
and its forced expression within the CNS also alleviates EAE
clinical signs (53, 54). Because all B cell subpopulations produce
IL-10 upon TLR engagement, bystander production of IL-10 by
B cells will be common to all autoimmune models that require
CFA, such as arthritis, or when TLR ligands are naturally present,
such as in colitis.
We previously reported that mice bearing B7-deficient B cells
had a delay in the production of IL-10 and presence of Treg
cells in the CNS during EAE (14). From these data we hypothesized that B cells regulate Treg via B7 and found in this study that
CD86 plays only a minor role in Treg homeostasis. However, one
major difference between our previous and current studies is that
when mMT mice were reconstituted with B7-deficient B cells by
BM transplantation, the mice exhibited chronic EAE (14). Because neither study used CFA, the difference is not attributed to
B cell activation via TLR ligands. We do not think that the irradiation required for the transplant resulted in dysfunctional immune responses because mMT mice receiving WT BM and WT
mice that received B72/2 BM were both able to recover from
EAE. In addition, we found that mMT mice transplanted with WT
and B72/2 BM had similar numbers of Treg (data not shown),
ruling out a Treg deficiency in our previous study. We think that
the most likely explanation resides in differences in the balance of
B cell subpopulations present in the two systems. BM transplantation will reconstitute all populations of B cells, whereas in the
transfer studies, only splenic B cells will be present. B cell transfer
into the partially lymphopenic mMT host will drive homeostatic
expansion of the transferred cells, likely resulting in changes in
cell surface expression. We speculate that this process, which does
not occur in the transplantation system, upregulates a currently
unknown molecule that compensates for the loss of B7. Both the
B7 and TNF families contain members that could contribute to
B cell–Treg interactions. In addition, we speculate that a unique
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approaches, we found no evidence for its role in Treg expansion
induced by B cells (Fig. 4D and data not shown). Several experimental differences could account for this result including the use
of CD4+CD25+ T cells in the original study (44). Because Foxp3
expression could not be measured at the time, it is possible that
CD4+CD25+ cells that were Foxp32 expanded in the lymphopenic
host (Rag-12/2). In our studies, we used Foxp3+ cells and an
animal that was only partially lymphopenic (mMT). A later study
using conditional ablation demonstrated an indirect role for MHC
class II in Treg maintenance (35). Finally, we showed that B cell
production of IL-10 was not required for the maintenance of Treg
(Fig. 4). Additional supporting evidence is that IL-102/2 mice do
not have altered numbers of Treg (A.R., S.B., and B.N.D., unpublished observations).
Although in our study, we determined that MHC class II expression was not required for their interactions with Treg, a role for
self-Ag–specific B cells in either promoting or inhibiting EAE has
been reported using genetically modified mice. An elegant study
by Wekerle and colleagues (45) demonstrated that T cells bearing
a TCR specific for myelin oligodendrocyte glycoprotein (MOG)
were able to recruit endogenous MOG-specific B cells, which
expanded and produced pathogenic Igs contributing to the onset
of spontaneous EAE. Using transgenic mice in which B cells
expressed MOG-MHC class II complexes, Frommer and Waisman (46) showed these Ag-specific B cells to contribute to the
negative selection of MOG-TCR transgenic T cells. In a second
study using the same mice, Frommer and colleagues (47) showed
the Ag-specific B cells to contribute to peripheral tolerance of
MOG-specific CD4 T cells. No effect on Treg was noted in either
study, which supports our data that MHC class II is not required
for B cell-mediated Treg homeostasis. Although Ag-specific
B cells can induce tolerance in genetically modified mice,
whether a similar mechanism exists in humans is not known.
Evidence that immune tolerance to myelin self-antigens is at least
incomplete in humans is the finding that healthy donors harbor
T cells with specificity for a variety of myelin Ags, including
MOG (48). In addition, rituximab studies in which treated MS
patients had significant reductions in the formation of new lesions
supports a role for B cells in Ag presentation (2, 6, 7). Rituximab
does not deplete plasma cells nor does it reduce serum Ig levels,
further supporting a role for Ag presentation (2). Thus, Ag presentation by B cells likely has dual functions contributing to both
tolerance and disease onset/progression.
When we determined whether adoptively transferred B cells
could function therapeutically in EAE, we found that WT B
cells were able to drive the resolution of EAE in mMT mice (Fig.
7). B cells deficient in IL-10 or B7 also promoted EAE recovery.
This result was surprising because both have been implicated in
regulatory B cell functions in EAE (3, 14). In contrast, mMT mice
that received GITRL-blocked B cells were unable to resolve EAE
and had significantly fewer Treg cells 33 d after B cell transfer as
compared with WT B cells. The finding that equal numbers of
B cells were present in the anti-GITRL and WT groups suggests
that the Ab blocking had a long-term effect (Fig. 7).
Given that IL-10 production by B cells has been implicated in
their regulatory potential in a number of autoimmune disorders,
including as studied in this work in EAE and colitis (3, 5, 49), our
finding that B cell maintenance of Treg is IL-10 independent is
particularly interesting. One major difference between our study
and others in EAE is that our adoptive transfer model does not
require the use of CFA, which can contain TLR2, TLR4, and
TRL9 ligands. Because we generate our T cell lines from MBPTCR transgenic mice, we also avoid the use of CFA in their
generation by using an in vitro approach (50). TLR2, TLR4, and
9
10
without affecting their function via GITR–GITRL interactions
opens a therapeutic window for strategies whereby B cells or
GITR-based drugs could be used to quickly increase or decrease
the number of Treg. We envision the ability to decrease Treg
numbers in cancer and during immunotherapies or increase their
numbers for the treatment of autoimmunity and other inflammatory diseases. These studies also illustrate that, as a lineage, B cells
are very complex and contain a large arsenal of mechanisms
whereby they control the onset and extent of immune responses.
Acknowledgments
We thank Robert Dunn and Biogen Idec for providing the anti-CD20 Ab,
BioLegend for providing the anti-GITRL Ab, Shelley Morris and Nichole
Miller for technical support, and Dr. Jeffrey Woodliff and Hope Campbell
for cell sorting assistance.
Disclosures
The authors have no financial conflicts of interest.
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numbers are limiting.
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B CELLS CONTRIBUTE TO Treg HOMEOSTASIS VIA GITRL
The Journal of Immunology
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