*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
immunized mice’s left footpad (5 ? 105cells/mouse). On day 5, single-cell
suspensions prepared from the left popliteal lymph nodes were stained with
anti-CD45.1 and anti-CD4 mAbs and were analyzed for CFSE dilution.
Identification of a natural counterpart of CD19hiFc?IIbhiB cells
DO11.10 OVA323-339–specific TCR transgenic ? C57BL/6J F1 hybrid mice
injected intraperitoneally with or without 40 ?g OVAplus 10 ?g CpG ODN
were killed on days 3, 6, and 9. CD19?cells enriched by CD19-conjugated
microbeads from spleen and lymph nodes were double-stained with
anti-CD19 and anti-CD16/CD32, and then CD19hiFc?IIbhiB cells and
CD19loFc?IIbloB cells were sorted. In addition, the cytokine profile,
phagocyotic capacity, and inhibitory function of the sorted CD19hiFc?IIbhi
B cells and CD19loFc?IIbloB cells were analyzed.
Data are shown as mean ? SD for separate experiments. Statistical
significance was determined by Student t test with a value of P less than .05
considered as statistically significant.
Regulatory DCs induce splenic B cells to differentiate into
IL-10–producing B cells
Our previous studies showed that regulatory DCs secrete higher
levels of IL-10 but lower levels of IL-12p70 than imDCs and
maDCs.21,24DCs have been shown to be able to regulate B-cell
functions.22,29,30Therefore, we analyzed the proliferation, apopto-
sis, antibody secretion, and cytokine production by splenic CD19?
B cells after their interaction with various DCs. As shown in
Supplemental Materials link at the top of the online article), the
proliferation and apoptosis of splenic B cells were not significantly
affected after interaction with imDCs, maDCs, or regulatory DCs.
Moreover, no difference in IgM and IgG2b production was
detected among B cells cocultured with imDCs, maDCs, or
regulatory DCs (supplemental Figure 1C).There was no significant
difference in production of IL-12, IFN-?, IL-6, IP-10, PGE2, and
TGF-? by B cells cocultured with or without regulatory DCs
(Figure 1A). Interestingly, B cells cocultured with regulatory DCs
could produce higher levels of IL-10 than B cells cocultured with
imDCs or maDCs (Figure 1B). IL-10 production by B cells
cocultured with regulatory DCs increased gradually and reached a
maximum after 48 hours of coculture (Figure 1C).To verify the cell
source of IL-10, the IL-10 production by B220?CD11c?regulatory
DCs and CD19?B220?CD11c?B cells in coculture system was
first confirmed to be positive by intracellular staining (Figure 1D).
However, quantitative RT-PCR results showed that regulatory
B cells expressed more IL-10 than diffDCs per cell (Figure 1E).
These results demonstrated that more IL-10–producing B cells
were generated in the B-cell/regulatory DC coculture system.
Considering that regulatory DCs are purified from coculture
system of endothelial-like splenic stromal cells (ESSCs)/DCs, we
analyzed IL-10 production by splenic B cells after interaction with
Figure 1. Regulatory DCs induce the differentiation of splenic B cells into IL-10–producing B cells. (A) Freshly purified splenic naive B cells were cocultured with
regulatory DCs at a ratio of 1:1 for 24 hours. Culture supernatants were collected and assayed for cytokines (IL-10, IP-10, IL-6, IL-12p70, PGE2, TGF-?, and IFN-?) by ELISA.
(B) Splenic naive B cells were cocultured with various DCs at the indicated ratios (DC/B). Supernatants were collected at 24 hours and assayed for IL-10. (C) Splenic naive
B cells were cocultured with various DCs at a ratio of 1:10 (DC/B), and supernatants were collected at different times for assay of IL-10. (D) Intracellular staining for IL-10 in
splenic CD19?B220?CD11c?B cells and B220?CD11c?regulatory DCs in coculture system. The percentages of each population are indicated in the plots. (E) B220?CD11c?
B cells and B220?CD11c?regulatory DCs were sorted after coculture, and IL-10 expression in the purified regulatory DCs and B cells was checked by quantitative RT-PCR.
IL-10 expression in diffDCs was designated as 1 for the comparison of IL-10 expression between regulatory B cells and diffDCs. Results are representative of 3 independent
experiments. Data are mean ? SD. **P ? .01. NS indicates not significant.
REGULATORY DCs GENERATE REGULATORY B CELLS583 BLOOD, 19 JULY 2012?VOLUME 120, NUMBER 3
For personal use only.on October 18, 2015. by guest
ESSCs. As shown in supplemental Figure 2, ESSCs did not affect
IL-10 secretion of B cells. In addition, regulatory DCs could
promote preferential IL-10 production of mouse splenic B220?
B cells (supplemental Figure 3). Taken together, these results
suggested that regulatory DCs can induce splenic B cells to
differentiate into IL-10–producing B cells.
The regulatory DC-programmed, IL-10–secreting B cells exhibit
unique phenotype (CD19hiFc?RIIbhi)
Next, we investigated the membrane molecule expression by
After the coculture, B cells expressed higher levels of CD19,
CD62L, and CD16/CD32 (Figure 2A; supplemental Figure 4),
whereas the expression of CD40, CD80, CD86, B7H1, and Iab
remained unchanged (data not shown). The available anti-CD16/
CD32 mAb could recognize activating receptors Fc?RIII (CD16)
and inhibitory receptor Fc?RIIb (CD32b). It is reported that
Fc?RIIb is the only Fc receptor expression on B cells.31Fc?RIIb
expression in splenic CD19?B cells and sorted CD19hiCD16/
CD32hiB cells was analyzed at the mRNA level. The Fc?RIIb but
not Fc?RIII (CD16) expression in sorted CD19hiCD16/CD32hi
B cells was up-regulated more significantly than that in splenic
CD19?B cells (Figure 2B). These data suggested that it is Fc?RIIb
that is overexpressed on CD19hiB cells. A previous study showed
that some regulatory DCs in mouse spleen are CD19?.32We found
that the CD19hiFc?RIIbhipopulation was a distinct population from
cocultured B220?CD11c?regulatory DCs (Figure 2C), indicating
that the CD19hiFc?RIIbhipopulation belongs to B cells.
Then we wondered whether the preferential production of IL-10
we observed in Figure 1 was from CD19hiFc?RIIbhiB cells. The
results showed that IL-10–producing B cells after coculture with
regulatory DCs exhibited the CD19hiFc?RIIbhiphenotype (Figure
2D). Furthermore, we sorted the CD19hiFc?RIIbhipopulation and
stimulated them with anti-IgM Ab, sCD40L, or a variety of TLR
ligands, including poly I:C, LPS, and CpG ODN. As shown in
Figure 2E, sCD40L, LPS, and CpG ODN, but not anti-IgMAb and
poly I:C, could stimulate the CD19hiFc?RIIbhipopulation to
produce more IL-10, suggesting that CD40, TLR4, and TLR9
signaling may be critical for CD19hiFc?RIIbhiB-cell function.
CD19?and CD19hiFc?RIIbhiB cells after sCD40L, LPS, or CpG
ODN stimulation (Figure 2F). Together, these results demonstrated
that regulatory DCs can program generation of a new population of
IL-10–secreting CD19hiFc?RIIbhiB cells.
CD19hiFc?RIIbhiB cells have the increased phagocytic capacity
Because Fc?RIIb was identified to be overexpressed on the
IL-10–producing CD19hiFc?RIIbhiB cells in Figure 2Athrough D,
Figure 2. Regulatory DCs program generation of IL-10–producing B population with CD19hiFc?RIIbhiphenotype. (A) Purified splenic naive B cells cocultured with DCs
at a ratio of 1:10 (DC/B) for 48 hours were stained with antibodies to CD19, CD62L, or CD16/CD32 and analyzed by flow cytometry. Numbers in plots indicate percentage of
CD19hicells or CD19hiCD62L?cells or CD19hiCD16/CD32hiin the gated CD19?B cells. (B) Fc?IIb and Fc?RIII mRNA expression by conventional splenic CD19?B cells or
sorted CD19hiCD16/CD32hiB cells was assessed by RT-PCR. The transcript of mouse GAPDH gene was used as the amplification control. (C) Purified splenic naive B cells
were cocultured with regulatory DCs at a ratio of 1:10 (DC/B) for 48 hours. Cells were stained with antibodies to CD19, B220, CD11c, and CD16/CD32 and analyzed by flow
cytometry. Numbers in plots indicate percentage of CD19hiFc?RIIbhiin the gated B220?CD11c?regulatory DCs or B220?CD11c?B cells. (D) Purified splenic B cells were
cocultured with regulatory DCs. CD19?IL-10?B cells or CD19?IL-10?B cells were gated and assessed for Fc?IIb expression. (E) Twenty-four hours after conventional splenic
B cells or sorted CD19hiFc?RIIbhiB cells were stimulated with 20 ?g/mLanti-IgMAb, 2 ?g/mLsCD40L, 200 ng/mLLPS, 2 ?g/mLCpG ODN, or 2 ?g/mLpoly I:C, respectively,
IL-10 production was measured by ELISA. (F) Conventional splenic B cells or sorted CD19hiFc?RIIbhiB cells were labeled with CFSE and cultured with anti-IgMAb, sCD40L,
LPS, or CpG ODN for 5 days. Histograms represent CFSE expression by the CD19?or CD19hiFc?RIIbhiB cells. Gray lines represent CFSE staining of the unstimulated CD19?
or CD19hiFc?RIIbhiB cells. Results are representative of 3 independent experiments. Data are mean ? SD. **P ? .01. NS indicates not significant.
584 QIAN et alBLOOD, 19 JULY 2012?VOLUME 120, NUMBER 3
For personal use only. on October 18, 2015. by guest
we determined whether or not Fc?RIIb is required for IL-10
production. We found that Fc?RIIb?/?B cells were not compro-
mised in their ability of IL-10 production once cocultured with
regulatory DCs (Figure 3A), suggesting that Fc?RIIb is not
involved in preferential IL-10 production by CD19hiFc?RIIbhi
B cells. It has been shown that the Fc receptor mediates effective
internalization of Ag-IgG complexes.33CD19hiFc?RIIbhiB cells
were found to incorporate more FITC-OVA-IC than splenic B cells
(Figure 3B-C), indicating that Fc?RIIb is involved in the phagocy-
tosis of IC by this new B-cell population. To further confirm that
Fc?RIIb mediates potent phagocytosis of FITC-OVA-IC by
CD19hiFc?RIIbhiB cells, B cells from Fc?RIIb?/?mice were
cultured with regulatory DCs and then CD19hiFc?RIIb?/?B cells
were sorted. Asshown in
CD19hiFc?RIIb?/?B cells for the uptake of FITC-OVA-IC signifi-
cantly decreased compared with that of CD19hiFc?RIIbhiB cells.
So, CD19hiFc?RIIbhiB cells have the enhanced phagocytic capac-
ity and Fc?RIIb mediates the uptake of IC by CD19hiFc?IIbhi
IC can polarize macrophages to secrete high levels of IL-10 and
PGE2.28,34We wonder what roles Fc?RIIb plays in the production
of immunoregulatory cytokines by CD19hiFc?RIIbhiB cells once
stimulated with IC. Interestingly, IL-10, TGF-?, PGE2, and IDO
production by CD19hiFc?RIIbhiB cells remained unchanged as
compared with splenic CD19?B cells after stimulation with IC
(Figure 3E; and data not shown). Furthermore, the production of
PGE2by CD19hiFc?RIIbhiB cells and splenic CD19?B cells after
stimulation with IC was independent of Fc?RIIb (Figure 3F).
Therefore, the biologic significance of the enhanced ability of
CD19hiFc?RIIbhiB cells to uptake IC via Fc?RIIb need to be
further elucidated in the future.
IL-10 contributes to the regulatory function of CD19hiFc?RIIbhi
We investigated whether CD19hiFc?RIIbhiB cells have immune
regulatory functions and what are the underlying mechanisms. The
results showed that CD19hiFc?RIIbhiB cells inhibited proliferation
of the activated CD4 T cells more significantly than CD19?B cells
(Figure 4A). To confirm their regulatory effect in vivo, mice
transferred with CD45.1 ? OT-2 naive CD4 T cells 1 day before
being immunized by antigen-loaded maDCs to induce CD4 T-cell
proliferation in vivo, and then CD19hiFc?RIIbhiB cells were
adoptively transferred to the mice. As shown in Figure 4B and C,
CD19hiFc?RIIbhiB cells could also inhibit maDC-induced CD4
T-cell proliferation in vivo. Taken together, CD19hiFc?RIIbhi
B cells have a potent ability to suppress T-cell proliferation both in
vitro and in vivo, demonstrating that CD19hiFc?RIIbhiB cells are a
distinct subset of B cells with regulatory function.
Figure 3. CD19hiFc?RIIbhiB cells exhibit the increased phagocytic capacity. (A) Freshly purified splenic B cells or Fc?RIIb?/?B cells were incubated with regulatory DCs
for 48 hours at a ratio of 1:10 (DC:B). IL-10 in the supernatants was measured. (B) Phagocytic ability of CD19hiFc?RIIbhiB cells and conventional splenic CD19?B cells was
assessed by flow cytometry of FITC-OVA-IC phagocytosis. Ctrl indicates controls (cells incubated with FITC-OVA-IC at 4°C). (C) The confocal analysis of phagocytic ability of
CD19hiFc?RIIbhiB cells and conventional splenic CD19?B cells. Representative images of immunofluorescence were stained with Hoechst staining (nuclei). Images were
acquired by Leica TCS SP2 confocal microscopy under a 20?/0.70 CS objective lens. (D) Phagocytic ability of CD19hiFc?RIIbhiB cells and CD19hiFc?RIIb?/?B cells was
assessed by flow cytometry of FITC-OVA-IC phagocytosis. Ctrl indicates controls (cells incubated with FITC-OVA-IC at 4°C). (E-F) Splenic CD19?B cells, sorted
CD19hiFc?RIIbhiB cells, splenic CD19?Fc?RIIb?/?B cells, and sorted CD19hiFc?RIIb?/?B cells were stimulated with or without IC for 24 hours, respectively, and then assayed
for PGE2production in the supernatants. Data represent 1 of at least 3 experiments with similar results. (B,D) Numbers indicate the mean fluorescence intensity of test
samples. (A,E-F) Data are mean ? SD. **P ? .01. NS indicates not significant.
REGULATORY DCs GENERATE REGULATORY B CELLS 585BLOOD, 19 JULY 2012?VOLUME 120, NUMBER 3
For personal use only.on October 18, 2015. by guest
To determine whether IL-10 could mediate the inhibition of
CD4 T-cell proliferation by CD19hiFc?RIIbhiB cells, we added
anti–IL-10 antibody into the activated-CD4 T/CD19hiFc?RIIbhi
B coculture system and found that IL-10 blockade could signifi-
tion by CD19hiFc?RIIbhiB cells (Figure 4D). In addition, IL-10
was found to be involved in the inhibition of T-cell proliferation by
CD19hiFc?RIIbhiB cells in vivo (supplemental Figure 5). These
results demonstrated that it is IL-10 that mediates the immunosup-
pressive effects of CD19hiFc?RIIbhiB cells.
Regulatory DC-derived IFN-? is involved in the differentiation
of CD19hiFc?RIIbhiB cells
To determine the mechanism of CD19hiFc?RIIbhiB-cell differ-
entiation driven by regulatory DCs, we first tested whether
this differentiation process depends on soluble factors re-
leased by regulatory DCs or direct cell-cell contact with
regulatory DCs. Splenic B cells were separated from regulatory
DCs by a transwell system. We found that IL-10 production was
significantly decreased but not completely abrogated (Figure
5A), suggesting that CD19hiFc?RIIbhiB-cell differentiation
depends on soluble factors from regulatory DCs and cell-cell
To further identify which soluble factor(s) was involved in
CD19hiFc?RIIbhiB cell differentiation driven by regulatory DCs,
IL-6, IP-10, VEGF, and IFN-?, highly secreted by regulatory DCs
we observed before, were blocked, respectively, in the coculture
system. As shown in Figure 5B, blockade of IFN-?, but not
blockade of IL-6, IP-10, and VEGF, led to partial decrease of IL-10
production. Furthermore, neutralizing anti–IFN-? antibody was
added into the B-cell culture system in the presence of 50%
regulatory DCs’ supernatant harvested after culture for 24 hours
Figure 4. CD19hiFc?RIIbhiB cells inhibit CD4 T-cell proliferation both in vitro and in vivo. (A) The effect of CD19hiFc?IIbhiB cells on in vitro maDC-primed proliferation of
activated CD4 T cells was assayed. CD4 T cells were activated by maDCs for 24 hours in the presence of OVA323-339peptide, and then CD19hiFc?IIbhiB cells were added. Five
days later, the cells were collected and double-stained with anti-CD4-allophycocyanin and 7-AAD and counted by flow cytometry. (B-C) The effect of CD19hiFc?IIbhiB cells on
in vivo maDC-initiated proliferation of CD4 T cells was assayed. CD4 T cells from OT-2 ? CD45.1 F1 hybrid mice were labeled with 5?M CFSE and then transferred
intravenously into C57BL/6J mice on day ?1. On day 0, antigen-loaded maDCs were transferred subcutaneously into the left footpad. On day 1, CD19hiFc?IIbhiB cells or
CD19loFc?IIbloB cells were also transferred into the left footpads. On day 5, mononuclear suspensions from the left popliteal lymph nodes were double-stained with
anti–CD4-peridinin chlorophyll protein-Cy5.5 and anti–CD45.1-allophycocyanin for flow cytometry analysis. Representative dot plots showing the percentage of
peptide-specific CD4 T cells (CD45.1?) among total CD4 T cells (B, left), and the percentage of peptide-specific CD4 T cells among total CD4 T cells was calculated (n ? 3;
B, right). Representative histograms showing CFSE expression by the CD4?CD45.1?T cells (C, left), and the proliferation of CD4?CD45.1?T cells was analyzed (n ? 3;
C, right). (D)As described in panelA, neutralizing anti–IL-10 antibody was added into the culture system. Data are mean ? SD. *P ? .05. **P ? .01.
586 QIAN et alBLOOD, 19 JULY 2012?VOLUME 120, NUMBER 3
For personal use only.on October 18, 2015. by guest
(50% diffDC-SN), and the decreased production of IL-10 was
observed (Figure 5C). The results showed that regulatory DC-
CD40L/CD40 interaction is involved in the differentiation of
CD19hiFc?RIIbhiB cells programmed by regulatory DCs
We also wondered what membrane molecules involved in the
CD19hiFc?RIIbhiB cells.The CD40L-CD40 pathway is reported to
be crucial for the differentiation of regulatory B cells.35Regulatory
DCs expressed CD40L (Figure 5D). To further determine whether
expression of CD40L on regulatory DCs is required for the
differentiation of CD19hiFc?RIIbhiB cells, B cells from CD40?/?
mice were prepared to coculture with regulatory DCs or
CD40L?/?regulatory DCs, and then the IL-10 production in the
coculture system was measured (Figure 5E). The absence of
CD40L on regulatory DCs or/and CD40 on B cells impaired
production of IL-10, indicating that the CD40L-CD40 pathway
was involved in the differentiation of CD19hiFc?RIIbhiB cells.
Moreover, we added IFN-? plus sCD40L to imDCs or maDCs
plus splenic B cells, and then found that imDCs or maDCs
plus IFN-?/sCD40L could also induce B cells to differentiate
into IL-10–producing B cells with CD19hiFc?IIbhiphenotype
(Figure 5F-G). Together, these data suggested that direct
regulatory DC-B cell contact in a CD40L-CD40–dependent
way, together with the regulatory DC-derived IFN-?, plays a
critical role in regulatory DC-programmed differentiation of
Regulatory DCs can induce splenic T1, T2, MZ, and B1 B cells
to differentiate into CD19hiFc?IIbhiB cells
Although not defining markers, some markers enriched in regula-
tory B cells have been reported, such as CD5?CD1dhiand
TIM-1?.36,37So we checked these markers on CD19hiFc?RIIbhi
B cells. We found that 16.5% of B cells were CD5?CD1dhiwithin
the CD19hiFc?RIIbhipopulation, 43.5% of B cells were
CD19hiFc?RIIbhiwithin the CD5?CD1dhipopulation (Figure 6A),
and only approximately 1.1% of CD19hiFc?RIIbhiB cells were
CD19hiFc?RIIbhicells are distinguished from TIM-1?regulatory
B cells and not identical with CD5?CD1dhiB cells by their
To identify the B-cell subset(s) from which IL-10–producing
CD19hiFc?RIIbhiB cells were differentiated, sorting-purified
splenic T1, T2, T3, FO, MZ, or B1 B cells38(supplemental
Figure 6) were cultured with regulatory DCs. We found that
IL-10–producing CD19hiFc?RIIbhiB cells were derived from
T1, T2, MZ, and B1 B cells, but not T3 and FO B cells (Figure
6C-D). And CD19?IL-10?T1, T2, MZ, and B1 B cells
expressed higher level of Fc?RIIb compared with that of freshly
sorted T1, T2, MZ, and B1 B cells, respectively (Figure 6E).
Furthermore, these sorted CD19hiFc?RIIbhiT1, T2, MZ, and B1
B cells had enhanced phagocytic capacity (Figure 6F) and
Figure 5. IFN-? and CD40L/CD40 interaction is re-
quired to induce regulatory B-cell generation driven
by regulatory DCs. (A) Splenic naive B cells were
cocultured with regulatory DCs either directly or sepa-
naive B cells were cocultured with regulatory DCs in the
presence of the indicated neutralizing antibodies (5 ?g/
mL). (C) Neutralizing anti–IFN-? antibody was added to
the B-cell culture system in the presence of regulatory
DC supernatants (regulatory DC-SN, collected after
24 hours of culture). And the final concentration of
supernatants used was 50%. (D) CD40L expression on
regulatory DCs and CD40L?/?regulatory DCs was tested
by flow cytometry. (E) Wild-type B cells or CD40?/?
B cells were incubated with regulatory DCs or CD40L?/?
regulatory DCs. Culture supernatants were collected at
48 hours and assayed for IL-10. (F-G) imDCs or
maDCs were cocultured with purified splenic naive
B cells in the presence of IFN-? (2 ?g/mL) and sCD40L
(2 ?g/mL) for 48 hours. Culture supernatants were
collected and assayed for IL-10 (F). Cells were stained
with antibodies to CD19 and CD16/CD32 and analyzed
by flow cytometry (G). The DC/B ratio in all these
experiments was 1:10. Data are mean ? SD of tripli-
cate wells. *P ? .05. **P ? .01.
REGULATORY DCs GENERATE REGULATORY B CELLS587BLOOD, 19 JULY 2012?VOLUME 120, NUMBER 3
For personal use only. on October 18, 2015. by guest
significantly suppressed the activated CD4 T-cell proliferation
via IL-10 (Figure 6G) compared with those of freshly sorted T1,
T2, MZ, and B1 B cells, respectively. Together, these data
indicate that regulatory DCs preferentially induce specific
B-cell subsets, including T1, T2, MZ, and B1 B cells, to
differentiate into IL-10–producing CD19hiFc?IIbhiB cells.
Figure 6. Regulatory DCs induce CD19hiFc?IIbhiregulatory B-cell generation from T1, T2, MZ, and B1 B cells. (A) Splenic B cells cocultured with regulatory DCs were
stained for CD19, Fc?RIIb, CD5, and CD1d. Cells within the CD19hiFc?RIIbhiB-cell gate were assessed for CD1d and CD5 expression. Cells within the CD5?CD1dhiB-cell gate
were assessed for CD19 and Fc?RIIb expression. (B) Splenic B cells cocultured with regulatory DCs were stained for CD19, Fc?RIIb, and TIM-1. Cells within the
CD19hiFc?RIIbhiB-cell gate were assessed for TIM-1 expression. (C) Splenic T1 (CD93?IgMhiCD23lo), T2 (CD93?IgMhiCD23hi), T3 (CD93?IgMloCD23hi), MZ
(CD93?CD21hiCD23lo), FO (CD93?CD21loCD23hi), and B1 (B220?CD5?) B cells were sorting-purified, respectively, and cultured with or without regulatory DCs for 48 hours.
Culture supernatants were collected for IL-10 assay. (D) SplenicT1,T2, MZ, or B1 B cells cultured with regulatory DCs were stained for CD19 and CD16/CD32 and analyzed by
flow cytometry. (E) Splenic T1, T2, MZ, or B1 B cells were cultured with regulatory DCs. CD19?IL-10?B cells were gated and assessed for Fc?IIb expression. Gray lines
represent the expression of Fc?IIb on T1, T2, MZ, and B1 B cells before coculture. (F) Splenic T1, T2, MZ, or B1 B cells were cultured with regulatory DCs for 48 hours.
CD19hiFc?RIIbhiB cells derived fromT1,T2, MZ, and B1 B cells were sorted by flow cytometry, respectively.The phagocytic ability of freshly isolatedT1,T2, MZ, and B1 B cells
and sorted CD19hiFc?IIbhipopulations derived from T1, T2, MZ, and B1 B cells was assessed by flow cytometry of FITC-OVA-IC phagocytosis. Numbers in the histogram indicate the
mean fluorescence intensity of test samples. Ctrl indicates controls (cells incubated with FITC-OVA-IC at 4°C). (G) Freshly isolated T1, T2, MZ, and B1 B cells or sorted CD19hiFc?IIbhi
populations derived from T1, T2, MZ, and B1 B cells were cocultured with activated CD4 T cells (naive peptide-specific CD4 T cells were stimulated by maDCs for 24 hours). In some
cytometry.Resultsarerepresentativeof3independentexperiments.(C,G)Dataaremean ? SDoftriplicatecells.*P ? .05.**P ? .01.NSindicatesnotsignificant.
588QIAN et al BLOOD, 19 JULY 2012?VOLUME 120, NUMBER 3
For personal use only.on October 18, 2015. by guest
Phenotypic and functional identification of a natural
counterpart of CD19hiFc?IIbhiB cells in vivo
B cells, we investigated whether the natural counterpart of
CD19hiFc?RIIbhiB cells exists in vivo. We analyzed CD19?cells
in spleen and lymph nodes of DO11.10 OVA323-339-specific TCR
transgenic ? C57BL/6J F1 hybrid mice after immunization with or
without 40 ?g OVA plus 10 ?g CpG ODN on the basis of the
phenotype of CD19hiFc?RIIbhi.As shown in Figure 7Aand B, cells
with a CD19hiFc?RIIbhiphenotype represented 4.30% of splenic
CD19?B cells and 2.36% of lymph nodes from normal mice; and
they represented 11.15% of splenic CD19?B cells and 6.12% of
lymph nodes from immunized mice. CD19hiFc?RIIbhiB cells,
directly sorted from spleen and lymph nodes of immunized mice,
secreted high IL-10 with or without LPS and CpG ODN stimula-
tion, which resembled the cytokine profile of regulatory
CD19hiFc?RIIbhiB cells induced by regulatory DCs in vitro
CD19hiFc?RIIbhiB cells had enhanced phagocytic capacity com-
pared with that of CD19loFc?IIbloB cells (Figure 7C). Moreover,
these cells significantly suppressed the activated CD4 T-cell
proliferation via IL-10 (Figure 7D), similar to the functional
characteristics of CD19hiFc?RIIbhiB cells we identified in vitro.
We also enriched CD19?cells from the spleen and lymph nodes
at various times after immunization and analyzed the proliferation
of Fc?RIIbhiB and Fc?RIIbloB cells by cell-cycle analysis. In both
spleen and lymph nodes of normal mice, the percentage of
Fc?RIIbhiB cells in the S/M/G2phages of cell cycle was less than
that of Fc?RIIbloB cells (Figure 7E-F). However, in mice that
received OVA and CpG ODN, Fc?RIIbhiB cells underwent
stronger proliferation than Fc?RIIbloB cells, as indicated by the
greater increase in the proportion of cells undergoing S/M/G2
cycling. These results indicated that a subset of regulatory B cells
with similar phenotype and functions to that of CD19hiFc?RIIbhi
7B). Furthermore,wefound thatthese sorted
Figure 7. Identification of the natural counterpart of
CD19hiFc?IIbhiB cells in vivo. Splenocytes and lymph
node cells enriched with anti-CD19 magnetic beads were
B cells were sorted by flow cytometry. (A) Numbers in plot
regions indicate the percentage of CD19hiFc?IIbhiin the
CD19loFc?IIblowere stimulated with 200 ng/mL LPS,
2 ?g/mL CpG ODN, or 2 ?g/mL poly I:C for 24 hours;
then IL-10 production was measured by ELISA. (C) The
phagocytic ability of sorted
CD19loFc?IIbloB cells was assessed by flow cytometry of
FITC-OVA-IC phagocytosis. Numbers in the histogram
indicate the mean fluorescence intensity of test samples.
Ctrl indicates controls (cells incubated with FITC-OVA-IC
at 4°C). (D) CD19hiFc?IIbhior CD19loFc?IIbloB cells were
cocultured with activated CD4 T cells. In some groups,
anti–IL-10 antibody was added into the activated CD4
T/B coculture system.After 5 days, the relative number of
viable CD4 T cells in each well was detected by flow
cytometry. (E-F) DO11.10 OVA323-339-specific TCR trans-
genic ? C57BL/6J F1 hybrid mice were killed on days 3,
6, and 9 after immunization with 40 ?g OVA plus 10 ?g
CpG ODN. CD19?cells were sorted from spleen (E) and
lymph nodes (F). Left: cell-cycle status of Fc?IIbhicells
analyzed by propidium iodide (numbers indicate percent-
age of Fc?IIbhiand Fc?IIblocells, respectively, in S/M/G2).
Right: percentage of proliferating B cells in Fc?IIbhior
Fc?IIblocell population of different phases of immune
response by cycle analysis. (B,D) Data are mean ? SD
of triplicate cells. *P ? .05.
REGULATORY DCs GENERATE REGULATORY B CELLS589BLOOD, 19 JULY 2012?VOLUME 120, NUMBER 3
For personal use only.on October 18, 2015. by guest
B cells identified in vitro do exist in mouse spleen and lymph
nodes. Interestingly, the number of this subset increases after
immunization, suggesting feedback negative regulation of immune
response through induction of regulatory B cells may exist.
Up to now, various regulatory B cells with different phenotypes
have been reported, such as CD1dhiCD5?B cells and TIM-1?
B cells.36,37,39In this study, we identified a new subset of
IL-10-producing regulatory B cells with high CD19 and Fc?IIb
expression. These CD19hiFc?RIIbhiB cells are distinguished from
B cells becauseonly
CD19hiFc?RIIbhiB cells are positive for TIM-1. CD19hiFc?IIbhi
B cells share surface markers with CD1dhiCD5?B cells, such as
high CD1d expression, but several differences exist between these
2 B-cell subsets. In particular, majority of CD19hiFc?IIbhiB cells
are negative for CD5 and cellular origins of these 2 B-cell subsets
were different. CD1dhiCD5?B cells do not express CD93 or CD23,
suggesting that they are unlikely to belong to T1 and T2-MZ
precursor B-cell subsets,37whereas CD19hiFc?RIIbhiB cells can
emerge fromsplenic T1(CD93?IgMhiCD23lo)
(CD93?IgMhiCD23hi) B-cell subsets. All the differences showed
that CD19hiFc?IIbhiB cells and CD1dhiCD5?B cells are not the
same subset of regulatory B cells. Fc?IIb, the well-known unique
inhibitory Fc?R expressed on B cells, has never been reported as a
surface marker of regulatory B cells. Th-2 cytokines, such as IL-10
or TGF-?, can up-regulate the expression of inhibitory Fc receptor
on innate immune effector cells.31,40However, blockade of IL-10,
TGF-?, IFN-?, IP-10, or IL-6 in the regulatory DC/B coculture
system could not reduce Fc?RIIb expression on CD19hiFc?RIIbhi
B cells (data not shown). Moreover, we demonstrate that
CD19hiFc?IIbhiB cells have enhanced phagocytic capacity than
CD19?B cells, and Fc?RIIb mediates the uptake of IC by
CD19hiFc?IIbhiB cells, indicating that CD19hiFc?RIIbhiB cells
may exert its negative regulatory function by attenuating the
IC-mediated activation of immune response and provide potential
new approach for treating IC-mediated autoimmune diseases.
Therefore, which factor(s) is responsible for high expression of
Fc?RIIb on CD19hiFc?RIIbhiB cells and what biologic signifi-
cance of high expression of Fc?RIIb on regulatory B cells need to
be further investigated.
Functionally, regulating T-cell function is the hallmark of
regulatory B cells, but the molecular mechanisms of regulatory
B cells are still poorly defined so far. There are at least 3 kinds of
mechanisms that may account for regulatory properties of B cells,
including the production of IL-10, the ability to suppress the
activated CD4 T-cell proliferation, or inducing Treg cell genera-
tion.2,41,42Although a high level of IL-10 was secreted by
CD19hiFc?RIIbhiB cells in our experiments, IL-10 did not promote
generation of CD4?CD25?Foxp3?Treg cells (data not shown). In
addition, CD19hiFc?RIIbhiB cells did not inhibit Th1 cytokine
secretion of CD4 T cells activated by maDCs (data not shown).
Interestingly, we found that CD19hiFc?RIIbhiB cells could potently
inhibit the proliferation of activated CD4 T cells via IL-10. These
results show that CD19hiFc?RIIbhiB cells display the inhibitory
function by directly secreting IL-10, instead of indirectly inducing
Treg cells, functionally confirming that CD19hiFc?RIIbhiB cells
are a distinct subset of regulatory B cells.
Mechanisms for regulatory B-cell differentiation have not yet
been fully identified. Our results demonstrate that CD40L/CD40
interaction between regulatory DC/B cells is required for optimal IL-10
production by CD19hiFc?RIIbhiB cells. Furthermore, regulatory DC-
derived IFN-? is essential for the differentiation of CD19hiFc?RIIbhi
B cells. Our results that B cells cocultured with imDCs or maDCs in
type further confirm that CD40L and IFN-? are important for the
differentiation of CD19hiFc?RIIbhiB cells. IFN-? is the major drug for
treatment of multiple sclerosis patients and experimental autoimmune
encephalomyelitis (EAE) mouse models. The molecular mechanisms
responsible for the beneficial effect of IFN-? in multiple sclerosis/EAE
are not fully elucidated. Putative mechanisms include alterations of the
Th17 differentiation, suppression of maDC migration, and elimination
of maDCs.43,44Recent data indicate that IFN-? is effective in reducing
IL-10 production by splenocytes.45More recently, induction of IL-10
secretion from human B cells by IFN-? has been reported.46It was also
reported that IFN-? helped to control neonatal acute inflammation by
stimulating the secretion of IL-10 by neonatal B cells.47Our work,
in driving differentiation of IL-10–producing regulatory B cells; thus,
IFN-?–driven differentiation of regulatory B cells might represent a
possible molecular mechanism for the therapeutic effect of IFN-? in
Regulatory IL-10-producing B cells have been shown to
attenuate autoimmune diseases in several mouse models. For
into B cell–deficient mice suppressed T cell–mediated inflamma-
tory response in a contact hypersensitivity mode 1.37Transfer of
regulatory T2-like-B cells is found to be effective in the suppres-
sion of lupus in MRL/lpr mice via an IL-10–dependent mecha-
In addition, a subset of human B cells with
CD19?CD24hiCD38hiphenotype was recently found to possess
regulatory activity in healthy persons but is functionally impaired
in systemic lupus erythematosus patients.9Thus, investigating the
preventive or pathogenic roles of CD19hiFc?IIbhiB cells in
different disease models and identification of CD19hiFc?IIbhi
B cells in humans that similarly regulate inflammatory responses
may add insights to the mechanisms of immune disorders and
provide therapeutic approaches for treating autoimmune diseases.
In conclusion, we demonstrate that regulatory DCs could
program splenic T1, T2, MZ, and B1 B cells to differentiation into
production. Such B cells can regulate immune response by
inhibiting CD4 T-cell proliferation. Regulatory DC-derived IFN-?
and CD40L/CD40 interaction between regulatory DC/B are re-
quired for the differentiation of CD19hiFc?RIIbhiregulatory B cells
programmed by regulatory DCs. So, regulatory DCs may have
strategy to regulate immune responses by, at least partially,
inducing generation of regulatory B cells.
The authors thank Dr Taoyong Chen for critical reading of the
manuscript and Drs Xiongfei Xu, Xingguang Liu, and Yan Gu for
excellent technical assistance.
This work was supported by the National High-Biotech Pro-
gram (grants 2012AA020901 and 2012ZX10002014), the National
Natural Science Foundation of China (grants 30972704, 81001308,
and 30731160623), and a Foundation for the Author of National
Excellent Doctoral Dissertation of China (grant 201180).
590 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;
1. LeBien TW, Tedder TF. B lymphocytes: how they
develop and function. Blood. 2008;112(5):1570-
2. Lund FE, Randall TD. Effector and regulatory
B cells: modulators of CD4? T cell immunity. Nat
Rev Immunol. 2010;10(4):236-247.
3. Pelletier N, McHeyzer-Williams LJ, Wong KA,
Urich E, Fazilleau N, McHeyzer-Williams MG.
Plasma cells negatively regulate the follicular
helper T cell program. Nat Immunol. 2010;11(2):
4. Mauri C, Blair PA. Regulatory B cells in autoim-
munity: developments and controversies. Nat
Rev Rheumatol. 2010;6(11):636-643.
5. Kelly-Scumpia KM, Scumpia PO, Weinstein JS,
et al. B cells enhance early innate immune re-
sponses during bacterial sepsis. J Exp Med.
6. Bao Y, Han Y, Chen Z, Xu S, Cao X. IFN-alpha-
producing PDCA-1? Siglec-H- B cells mediate
innate immune defense by activating NK cells.
Eur J Immunol. 2011;41(3):657-668.
7. Caldero ´n-Go ´mez E, Lampropoulou V, Shen P,
et al. Reprogrammed quiescent B cells provide
an effective cellular therapy against chronic ex-
perimental autoimmune encephalomyelitis. Eur
J Immunol. 2011;41(6):1696-1708.
8. Buettner M, Pabst R, Bode U. Lymph node stro-
mal cells strongly influence immune response
suppression. Eur J Immunol. 2011;41(3):624-633.
9. Blair PA, Noren ˜a LY, Flores-Borja F, et al.
CD19?CD24hiCD38hi B cells exhibit regulatory
capacity in healthy individuals but are functionally
impaired in systemic lupus erythematosus pa-
tients. Immunity. 2010;32(1):129-140.
10. Iwata Y, Matsushita T, Horikawa M, et al. Charac-
terization of a rare IL-10-competent B-cell subset
in man that parallels mouse regulatory B10 cells.
11. Rafei M, Hsieh J, Zehntner S, et al.A
granulocyte-macrophage colony-stimulating fac-
tor and interleukin-15 fusokine induces a regula-
tory B-cell population with immune suppressive
properties. Nat Med. 2009;15(9):1038-1045.
12. Hikada M, Zouali M. Multistoried roles for B lym-
phocytes in autoimmunity. Nat Immunol. 2010;
13. Bouaziz JD, Calbo S, Maho-Vaillant M, et al.
IL-10 produced by activated human B cells regu-
lates CD4(?) T-cell activation in vitro. Eur J Im-
14. Yang M, Sun L, Wang S, et al. Novel function of
B cell-activating factor in the induction of IL-10-
producing regulatory B cells. J Immunol. 2010;
15. Shortman K, Naik SH. Steady-state and inflam-
matory dendritic-cell development. Nat Rev Im-
16. Wu L, Liu YJ. Development of dendritic-cell lin-
eages. Immunity. 2007;26(6):741-750.
17. MorelliAE, ThomsonAW. Tolerogenic dendritic
cells and the quest for transplant tolerance. Nat
Rev Immunol. 2007;7(8):610-621.
18. Pallotta MT, Orabona C, Volpi C, et al. Indoleam-
ine 2,3-dioxygenase is a signaling protein in long-
term tolerance by dendritic cells. Nat Immunol.
19. Xia S, Guo Z, Xu X, Yi H, Wang Q, Cao X. He-
patic microenvironment programs hematopoietic
progenitor differentiation into regulatory dendritic
cells, maintaining liver tolerance. Blood. 2008;
20. Tang H, Guo Z, Zhang M, Wang J, Chen G,
Cao X. Endothelial stroma programs hematopoi-
etic stem cells to differentiate into regulatory den-
dritic cells through IL-10. Blood. 2006;108(4):
21. Zhang M, Tang H, Guo Z, et al. Splenic stroma
drives mature dendritic cells to differentiate into
regulatory dendritic cells. Nat Immunol. 2004;
22. Lund FE. Cytokine-producing B lymphocytes-key
regulators of immunity. Curr Opin Immunol. 2008;
23. Shaw J, Wang YH, Ito T, Arima K, Liu YJ. Plasma-
cytoid dendritic cells regulate B-cell growth and
differentiation via CD70. Blood. 2010;115(15):
24. Qian C, Jiang X, An H, et al. TLR agonists pro-
mote ERK-mediated preferential IL-10 production
of regulatory dendritic cells (diffDCs), leading to
NK-cell activation. Blood. 2006;108(7):2307-
25. Qian C, An H, Yu Y, Liu S, Cao X. TLR agonists
induce regulatory dendritic cells to recruit Th1
cells via preferential IP-10 secretion and inhibit
Th1 proliferation. Blood. 2007;109(8):3308-3315.
26. Xu X, Guo Z, Jiang X, et al. Regulatory dendritic
cells program generation of IL-4-producing alter-
native memory CD4 T cells with suppressive ac-
tivity. Blood. 2011;117(4):1218-1227.
27. Xu J, Foy TM, Laman JD, et al. Mice deficient for
the CD40 ligand. Immunity. 1994;1(5):423-431.
28. Zhang Y, Liu S, Liu J, et al. Immune complex/Ig
negatively regulate TLR4-triggered inflammatory
response in macrophages through FcgammaRIIb-
dependent PGE2 production. J Immunol. 2009;
29. Wykes M, MacPherson G. Dendritic cell-B-cell
interaction: dendritic cells provide B cells with
CD40-independent proliferation signals and
CD40-dependent survival signals. Immunology.
30. Litinskiy MB, Nardelli B, Hilbert DM. DCs induce
CD40-independent immunoglobulin class switch-
ing through BLyS andAPRIL. Nat Immunol. 2002;
31. Smith KG, Clatworthy MR. FcgammaRIIB in auto-
immunity and infection: evolutionary and thera-
peutic implications. Nat Rev Immunol. 2010;
32. Johnson BA3rd, Kahler DJ, Baban B, et al.
B-lymphoid cells with attributes of dendritic cells
regulate T cells via indoleamine 2,3-dioxygenase.
Proc Natl Acad Sci U S A. 2010;107(23):10644-
33. Mousavi SA, Sporstøl M, Fladeby C, Kjeken R,
Barois N, Berg T. Receptor-mediated endocytosis
of immune complexes in rat liver sinusoidal endo-
thelial cells is mediated by FcgammaRIIb2. Hepa-
34. Miles SA, Conrad SM, Alves RG, Jeronimo SM,
Mosser DM.Arole for IgG immune complexes
during infection with the intracellular pathogen
leishmania. J Exp Med. 2005;201(5):747-754.
35. Blair PA, Chavez-Rueda KA, Evans JG, et al. Se-
lective targeting of B cells with agonistic anti-
CD40 is an efficacious strategy for the generation
of induced regulatory T2-Like B cells and for the
suppression of lupus in MRL/lpr mice. J Immunol.
36. Ding Q, Yeung M, Camirand G, et al. Regulatory
B cells are identified by expression of TIM-1 and
can be induced through TIM-1 ligation to promote
tolerance in mice. J Clin Invest. 2011;121(9):
37. Yanaba K, Bouaziz JD, Haas KM, Poe JC,
Fujimoto M, Tedder TF.Aregulatory B-cell subset
with a unique CD1dhiCD5? phenotype controls
T cell-dependent inflammatory responses. Immu-
38. Vigorito E, Gambardella L, Colucci F, McAdam S,
Turner M. Vav proteins regulate peripheral B-cell
survival. Blood. 2005;106(7):2391-2398.
39. Watanabe R, Ishiura N, Nakashima H, et al.
Regulatory B cells (B10 cells) have a suppressive
role in murine lupus: CD19 and B10 cell defi-
ciency exacerbates systemic autoimmunity. J Im-
40. Nimmerjahn F, Ravetch JV. Fcgamma receptors
as regulators of immune responses. Nat Rev Im-
41. Carter NA, Vasconcellos R, Rosser EC, et al.
Mice lacking endogenous IL-10-producing regula-
tory B cells develop exacerbated disease and
present with an increased frequency of Th1/Th17
but a decrease in regulatory T Cells. J Immunol.
42. Matsumoto M, Fujii Y, BabaA, Hikida M,
Kurosaki T, Baba Y. The calcium sensors STIM1
and STIM2 control B cell regulatory function
through interleukin-10 production. Immunity.
43. Yen JH, Ganea D. Interferon beta induces mature
dendritic cell apoptosis through caspase-11/
caspase-3 activation. Blood. 2009;114(7):1344-
44. Yen JH, Kong W, Ganea D. IFN-beta inhibits den-
dritic cell migration through STAT-1-mediated
transcriptional suppression of CCR7 and matrix
metalloproteinase 9. J Immunol. 2010;184(7):
45. Axtell RC, de Jong BA, Boniface K, et al. T helper
type 1 and 17 cells determine efficacy of
interferon-beta in multiple sclerosis and experi-
mental encephalomyelitis. Nat Med. 2010;16(4):
46. Ramgolam VS, Sha Y, Marcus KL, et al. B cells
as a therapeutic target for IFN-beta in relapsing-
remitting multiple sclerosis. J Immunol. 2011;
47. Zhang X, Deriaud E, Jiao X, Braun D, Leclerc C,
Lo-Man R. Type I interferons protect neonates
from acute inflammation through interleukin 10-
producing B cells. J Exp Med. 2007;204(5):1107-
REGULATORY DCs GENERATE REGULATORY B CELLS 591 BLOOD, 19 JULY 2012?VOLUME 120, NUMBER 3
For personal use only.on October 18, 2015. by guest
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
Articles on similar topics can be found in the following Blood collections
Updated information and services can be found at:
(5340 articles) Immunobiology
Information about reproducing this article in parts or in its entirety may be found online at:
Information about ordering reprints may be found online at:
Information about subscriptions and ASH membership may be found online at:
Copyright 2011 by The American Society of Hematology; all rights reserved.
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
For personal use only.on October 18, 2015. by guest