*Li Qian,1,2*Cheng Qian,1Yongjian Chen,1Yi Bai,1Yan Bao,1Liwei Lu,3and Xuetao Cao1,4
1National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China;2Laboratory of Immunology,
Yangzhou University School of Medicine, Yangzhou, China;3Department of Pathology, University of Hong Kong, Hong Kong, China; and4National Key
Laboratory of Medical Molecular Biology, ChineseAcademy of Medical Sciences, Beijing, China
Regulatory dendritic cells (DCs) play im-
portant roles in the induction of periph-
eral tolerance and control of adaptive
immune response. Our previous studies
mature DCs to proliferate and further dif-
CD11bhiIalowregulatory DCs, which could
inhibit T-cell response, program genera-
tion of immunosuppressive memory CD4
T cells. However, the effect of regulatory
DCs on B-cell function remains unclear.
Here, we report that regulatory DCs can
induce splenic B cells to differentiate into
a distinct subtype of IL-10–producing
regulatory B cells with unique phenotype
CD19hiFc?IIbhi. CD19hiFc?IIbhiB cells in-
hibit CD4 T-cell response via IL-10.
CD19hiFc?IIbhiB cells have enhanced
phagocytic capacity compared with con-
ventional CD19?B cells, and Fc?RIIb
mediates the uptake of immune complex
by CD19hiFc?IIbhiB cells. We found that
regulatory DC-derived IFN-? and CD40
ligand are responsible for the differentia-
tion of CD19hiFc?IIbhiB cells. Further-
CD19hiFc?IIbhiB cells in the spleen and
lymph nodes with similar phenotype and
regulatory function has been identified.
Our results demonstrate a new manner
for regulatory DCs to down-regulate im-
mune response by, at least partially, pro-
gramming B cells into regulatory B cells.
B cells are generally considered to induce immune responses
through antibody production and optimal CD4 T-cell activation.1
Now, more subsets of B cells with nonclassic functions have been
identified.2-4For example, innate effector B cells have been shown
to be important in the initiation and effector phase of innate
response against infections.5-7B cells with regulatory functions
exert the immune regulatory functions either through the secreted
antibodies and/or cytokines with a suppressive effect or directly by
cellular interactions.2,4,8These B cells with regulatory functions,
independently of secreted antibodies, are designated as regulatory
B cells. Regulatory B cells have been identified to be involved in
the regulation of several immune-mediated pathologic processes in
both mice and humans, including autoimmune diseases and
infections.9-12The regulatory function of regulatory B cells may be
mediated directly by the production of regulatory cytokines IL-10
and TGF-? and/or by the ability of B cells to interact with
pathogenic T cells to inhibit harmful immune responses.2,13Our
previous study suggested that B cell–activating factor can induce
marginal zone (MZ) B-cell differentiation into regulatory B cells,
which could suppress autoimmunity in an IL-10–dependent fash-
ion.14So, more and more evidence shows the importance of
regulatory B cells in the maintenance of immune homeostasis and
the pathogenesis of immune disorders. However, the characteristics
of regulatory B cells and the factors programming regulatory B-cell
generation remain to be further identified.
Dendritic cells (DCs) are at the crossroads of innate and
adaptive immunity and essential mediators of immunity and
tolerance.15The functional versatility of DCs is enabled in part by
the various DC subsets with heterogeneous cell surface markers,
distinct cytokine profiles, and different developmental stages.16
DCs with regulatory function, designated as regulatory DCs, have
attracted much attention because they play important roles in
maintaining immune homeostasis.17,18Regulatory DCs negatively
regulate immune response by inducing regulatory T cells, inhibit-
ing T-cell proliferation, and inducing T-cell anergy.19-21Distinct
subsets of DCs have been found to regulate the function of B cells.
lar (FO) B cells to differentiate into IFN-? and IL-12–producing
effector B cells.22In addition, plasmacytoid DCs regulate B-cell
growth, differentiation, and immunoglobulin (Ig) secretion.23How-
ever, there is no report about the effect of regulatory DCs on B-cell
function to date.
Our previous studies show that splenic stromal cells, mimicking
the secondary lymph organ microenvironment, can drive mature
DCs (maDCs) to proliferate and differentiate into a novel subset of
regulatory DCs (diffDCs, DCs differentiated from maDCs), with
unique phenotype (CD11bhiIalow) and cytokine profile (higher
IL-10 but minimal IL-12p70 production), which can inhibit
tion between regulatory DCs and other immune cells in physiology
and pathology conditions. For example, we showed that regulatory
DCs can selectively recruit Th1 cells and inhibit Th1 proliferation,
and program generation of IL-4–producing alternative memory
CD4 T cells with suppressive activity.25,26We have identified an
in vivo counterpart of regulatory DCs in the spleen with similar
phenotype and functions. Considering that B-cell development
Submitted August 31, 2011; accepted June 6, 2012. Prepublished online as
Blood First Edition paper, June 12, 2012; DOI 10.1182/blood-2011-08-377242.
*L.Q. and C.Q. contributed equally to this study.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2012 by TheAmerican Society of Hematology
581BLOOD, 19 JULY 2012?VOLUME 120, NUMBER 3
For personal use only.on October 18, 2015. by guest
encompasses a continuum of stages that begin in primary lymphoid
tissue, with subsequent functional maturation in secondary lym-
phoid tissue, such as spleen,1these findings suggest that secondary
lymphoid tissue may be potential locations for regulatory DC–
B-cell interaction. However, whether regulatory DC and B cells
interact in these locations and what the consequence of these
interactions have not been elucidated.
In this study, we show that regulatory DCs can induce splenic
T1,T2, MZ, and B1 B cells to differentiate into a distinct regulatory
B subset, CD19hiFc?IIbhiB cells, which preferentially secret IL-10
and exert regulatory functions both in vitro and in vivo. We further
investigated how the regulatory DCs program the generation of
regulatory CD19hiFc?IIbhiB cells and how CD19hiFc?IIbhiB cells
negatively regulate T-cell response. Our results provide a new
manner of regulatory DCs for negative feedback control of T-cell
immune response and maintenance of immune homeostasis by, at
least partially, inducing differentiation of B cells into regulatory
B cells. In addition, one new subset of regulatory B cells with
CD19hiFc?IIbhiphenotype has been discovered, providing clues for
investigating the roles of B-cell populations in the health and
C57BL/6J mice were obtained from JointVentures Sipper BK Experimental
Animal Co. OVA323-339-specific TCR-transgenic DO11.10 mice or OT-2
mice, B6.SJL-PtprcaPep3b/BoyJ mice (CD45.1 mice), Fc?IIb?/?mice, and
CD40?/?mice were obtained from The Jackson Laboratory. CD40L?/?
mice were generated on C57BL/6J ? 129SV/J (H-2b) mice as previously
reported.27The mice were bred in specific pathogen-free conditions.
Animal experiments were performed in accordance with the National
Institutes of Health Guide for the Care and Use of LaboratoryAnimals, with
the approval of the Scientific Investigation Board of the Second Military
Medical University, Shanghai, China.
Recombinant mouse GM-CSF and IL-4 were from PeproTech. Neutralizing
antimouseVEGF, IL-6, IP-10 antibodies, isotype control mAbs, and soluble
CD40L (sCD40L) were from R&D Systems. The rabbit polyclonal
antimouse IFN-? and recombinant mouse IFN-? were from PBL Biomedi-
cal Laboratories. Fluorescence-conjugated mAbs to CD1d, CD4, CD5,
CD11c, B220, IgM, CD21, CD23, CD93, TIM-1, CD19, CD62L, CD16/
CD32, and IL-10 were from BD PharMingen or BioLegend. GolgiStop and
Cytofix/Cytoperm kit were from BD PharMingen. Phorbol myristate
acetate, ionomycin, polyriboinosinic acid/polyribocytidylic acid (poly I:C),
lipopolysaccharide (LPS), 7-amino-actinomycin D (7-AAD), OVA stock,
and anti-OVAmAb were from Sigma-Aldrich.
Preparation of mouse imDCs, maDCs, and regulatory DCs
Bone marrow–derived immature DCs (imDCs), maDCs, and regulatory
DCs (designated as diffDCs in our previous studies) from C57BL/6J mice
were generated as described previously by us.20,21
Purification of mouse splenic B cells
Splenic B cells were enriched using CD19-conjugated microbeads as
previously described.14Splenic transitional 1 (T1), T2, T3, FO, MZ, or B1
B cells were sorted respectively with a MOFLO High Speed Flow
Cytometer (Beckman Coulter).
Coculture of regulatory DC and B cells
Splenic B cells were cocultured with DCs from wild-type mice. In some
experiments, Fc?IIb?/?B cells or CD40?/?B cells were cocultured with
regulatory DCs or CD40L?/?regulatory DCs. First, purified splenic B cells
were plated in 96-well U-bottom plates at a density of 2.0 ? 105per well.
Then, purified imDCs, maDCs, or regulatory DCs were added at the
indicated ratios (DC/B). In some cases, DCs were incubated with anti–IL-6,
anti-VEGF, anti–IFN-?, or anti–IP-10 neutralizing Abs for 1 hour before
coculture with B cells. After coculture, the supernatants were collected for
ELISAor/and cells were harvested for flow cytometry analysis.
Mouse IL-6, IL-10, IL-12, IP-10, TGF-?, PGE2and IFN-? levels were
assayed by ELISA kits. For intracellular cytokine staining, splenic B cells
or specific B-cell subsets were cocultured with regulatory DCs for 48 hours
and then stimulated with cytosine-phosphate-guanosine oligodeoxynucle-
otide(CpGODN;2 ?g/mL),phorbolmyristateacetate(50 ng/mL),ionomy-
cin (500 ng/mL), and GolgiStop for additional 6 hours. Then cells were
stained for surface markers and then fixed and permeabilized with the
Cytofix/Cytoperm kit. Permeabilized cells were incubated with anti–IL-10
mAb. The cells were acquired with the BD LSRII flow cytometer (BD
Biosciences) and analyzed by FlowJo Version 5.7.2 software (TreeStar).
Total RNA was extracted with TRIzol (Invitrogen). LightCycler (Roche)
and SYBR RT-PCR kits (Takara) were used for quantitative RT-PCR
analysis. Date were normalized to ?-actin expression.
Sorting of CD19hiFc?IIbhiB cells or CD19hiFc?IIb?/?B cells
Freshly purified splenic B cells or Fc?IIb?/?B cells were cocultured with
regulatory DCs at a ratio of 1:10 (DC/B) for 48 hours, and then the cells
were labeled with anti-CD19 and anti-CD16/CD32 mAbs. The
CD19hiFc?IIbhiand CD19hiFc?IIb?/?B cells were sorted, respectively.
Regulatory DCs were seeded at a density of 6 ? 104per 600 ?L/well in
24-well plates. In addition, 6 ? 105B cells were either directly added or
placed in transwell chambers (Millicell, 1.0 ?m; Millipore) in the same
well. IL-10 production was detected at 48 hours by ELISA.
Analysis of phagocytic ability of CD19hiFc?IIbhiB cells
FITC-OVA–containing immune complex (FITC-OVA-IC) were prepared as
previously described.28Purified B cells, sorted CD19hiFc?IIbhiB cells, or
CD19hiFc?IIb?/?B cells were incubated with FITC-OVA-IC at 37°C for
2 hours. The cells were then washed and resuspended in chilled PBS for
immediate flow cytometry. Cells incubated with FITC-OVA-IC at 4°C were
used as a negative control. For confocal microscopy, cells were fixed with
4% formaldehyde and then washed with PBS. Nuclei were stained with
Hoechst. Coverslips were mounted on slides using mounting media and
read using Leica TCS SP2 confocal laser microscope (Wetzlar).
Assays for CD4 T-cell proliferation in vitro and in vivo
The in vitro and in vivo CD4T-cell proliferation was measured as described
previously.19,20For in vitro assay, purified CD4 T cells from DO11.10
OVA323-339–specific TCR transgenic ? C57BL/6J F1 hybrid mice cocul-
tured with maDCs in the presence of OVA323-339peptide for 24 hours; then
the cells were washed. The collected activated CD4 T cells were cocultured
with 1 ? 105CD19hiFc?IIbhiB cells or CD19?B cells (1 ? 105activated
CD4Tcells/200 ?L/well).After 5 days of culture, cells stained for CD4 and
7-AAD were resuspended in exactly 200 ?LPBS and cellular data acquired
for 56 seconds by flow cytometry. The number of CD4?7-AAD?live cells
was calculated to represent the altitude of CD4 T-cell proliferation. For in
vivo assay, CFSE labeled-CD4 T cells from OT-2 ? CD45.1 F1 hybrid
mice were injected intravenously into C57BL/6J mice (5 ? 105cells/
mouse) on day ?1. On day 0, 1 ? 106OVA323-339peptide-loaded maDCs
were injected subcutaneously into the left footpad of C57BL/6J mice
adoptively transferred with CD4 T cells. On day 1, CD19hiFc?IIbhiB cells
or CD19loFc?IIbloB cells were also injected subcutaneously into the
582 QIAN et alBLOOD, 19 JULY 2012?VOLUME 120, NUMBER 3
For personal use only.on October 18, 2015. by guest
Contribution: L.Q., C.Q., Y.C., Y. Bai, and Y. Bao performed the
experiments; and X.C., L.Q., C.Q., and L.L. designed the experi-
ments, analyzed data, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Xuetao Cao, National Key Laboratory of
Medical Immunology & Institute of Immunology, Second Military
Medical University, 800 Xiangyin Road, Shanghai 200433, China;
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online June 12, 2012
2012 120: 581-591
Li Qian, Cheng Qian, Yongjian Chen, Yi Bai, Yan Bao, Liwei Lu and Xuetao Cao
regulatory B cells through IFN-
Regulatory dendritic cells program B cells to differentiate into CD19
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