CLINICAL AND VACCINE IMMUNOLOGY, June 2011, p. 1015–1020
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 18, No. 6
Retinoic Acid and ?-Galactosylceramide Differentially Regulate B Cell
Activation In Vitro and Augment Antibody Production In Vivo?
Qiuyan Chen,1Kara L. Mosovsky,2and A. Catharine Ross1*
Department of Nutritional Sciences1and the Immunology and Infectious Diseases Graduate Program,2
Pennsylvania State University, University Park, Pennsylvania 16802
Received 3 January 2011/Returned for modification 13 March 2011/Accepted 28 March 2011
All-trans-retinoic acid (RA) promotes the maturation and differentiation of B cells, which are known as a type
of professional antigen-presenting cells. We show here that CD1d, a major histocompatibility complex class
I-like molecule that presents lipid antigens, is expressed in the mouse spleen B cells and is increased by RA.
Thus, we hypothesized that RA and the CD1d ligand, ?-galactosylceramide (?GalCer), could interact to
promote the differentiation, maturation, and antibody response of antigen-activated B cells. In isolated B cells,
?GalCer alone markedly stimulated, and RA further increased B cell proliferation, synergizing with the B cell
antigen receptor ligation via anti-? antibody (P < 0.05). The significantly increased cell proliferation stimu-
lated by ?GalCer was abrogated in the B cells of CD1d-null mice. RA alone and combined with ?GalCer also
promoted B cell differentiation by the enrichment of sIgG1-, CD138-, and PNA/Fas-positive B cells (P < 0.05),
suggesting a plasmacytic cell differentiation. In vivo, wild-type mice treated with RA and/or ?GalCer during
primary immunization with tetanus toxoid produced a higher serum anti-tetanus IgG response and had more
bone marrow anti-tetanus antibody-secreting cells as determined by enzyme-linked immunospot assay (P <
0.05) in the secondary response, a finding indicative of heightened long-term memory; however, the increased
antibody secretion after ?GalCer treatment was abolished in CD1d-null mice. We provide evidence here that
RA, together with ?GalCer, can effectively regulate B cell proliferation and differentiation, ultimately promot-
ing a more efficient antibody response to protein antigen. The results suggest that the combination of RA and
?GalCer could be a useful adjuvant combination in vaccine strategies.
An adequate supply of vitamin A has been shown to be
life-saving in young children (35), and maintenance of appro-
priate immune functions is widely believed to underlie its ben-
eficial effects. Vitamin A and its active metabolite all-trans-
retinoic acid (RA) are capable of rescuing poor immune
responses in vitamin A-deficient animals and of augmenting
anti-infectious responses in the vitamin A-adequate state (28).
Various processes leading to improved immune response have
been shown to be regulated by RA, including lymphopoiesis,
cell differentiation, T cell proliferation, cytokine production,
and tissue trafficking of B and T cells (7, 24, 25, 30).
Previously, we reported that RA significantly upregulates
the gene expression and activity of CD1d, a major histocom-
patibility complex class I-like molecule that functions by pre-
senting glycolipid antigens to invariant NKT cells (iNKT) (6,
23) and then rapidly secretes both T-helper (Th) type 1 and
type 2 cytokines, such as gamma interferon and interleukin-4,
bridging the innate and adaptive immune systems (9, 14, 16, 31,
32, 34). ?-Galactosylceramide (?GalCer) is a model glycolipid
antigen that binds specifically to CD1d (13). The activation of
iNKT cells by ?GalCer and endogenous glycolipids presented
by CD1d has been shown to be beneficial during several bac-
terial and viral infections, in certain antitumor responses, and
in the regulation of certain autoimmune diseases such as dia-
betes (3, 10, 12, 19, 29, 33).
Our present studies were based on previous findings that: (i)
B cells express CD1d and process CD1d-associated antigen (1,
9, 16); (ii) RA is a strong regulator of CD1d expression, at least
in monocytic cells (6); and (iii) RA serves to promote the
maturation of B cells (4, 26). We hypothesized that RA and
glycolipid (?GalCer) could work together to promote B cell
activation and differentiation, leading to increased antibody
production. In the present study, we have first tested the reg-
ulatory effects of RA on CD1d-mediated splenic B-cell activa-
tion and differentiation in vitro, and then determined whether
RA and ?GalCer combined are able to enhance the antibody
response to tetanus toxoid (TT) in terms of the antigen-in-
duced primary humoral response and long-term memory re-
sponse in vivo.
MATERIALS AND METHODS
Antibody and reagents. CD19-PEcy7, Fas-PE, and CD138-PE antibodies were
purchased from BD Biosciences. IgG1-Alexa 488 was obtained from Invitrogen.
Peanut agglutinin (PNA) conjugated with fluorescein was from Vector Labora-
tories, Inc. (Burlingame, CA). ?GalCer was from Alexis Biochemicals (San
Diego, CA), and ?GalCer was from Sigma. Anti-? antibody used in the B cell
proliferation analyses was from Jackson Laboratory (Bar Harbor, ME). Reagents
and cell culture medium were determined to be endotoxin-free by using a
Limulus amebocyte lysate endotoxin assay kit from GenScrip (Piscataway, NJ).
Animals, splenocytes, and B cell isolation and culture. Animal protocols were
approved by the Institutional Animal Use and Care Committee of Pennsylvania
State University. Adult female BALB/c (?8 weeks old [Charles River Labora-
tories]) were used to obtain spleen B cells for in vitro study as described previ-
ously (5). Female CD1d-null mice (CD1tm1Gru/J) and age-matched control
BALB/cJ mice, 8 weeks old, were from Jackson Laboratory.
Spleen B cells were isolated by using a negative B cell enrichment kit according
to the manufacturer’s instructions (StemCell Technology, Vancouver, British
Columbia, Canada). The purity of isolated B cells was ?94% based on CD19
staining. Cells were cultured in RPMI 1640 medium, which was supplemented
* Corresponding author. Mailing address: Department of Nutri-
tional Sciences and Huck Institute for Life Sciences, 110 Chandlee
Laboratory, University Park, PA 16802. Phone: (814) 865-4721. Fax:
(814) 863-6103. E-mail: email@example.com.
?Published ahead of print on 6 April 2011.
with 10% fetal bovine serum and 5 ? 10?5M ?-mercaptoethanol, all from
In vivo animal experimental design. BALB/c female mice, or CD1d-null and
BALB/cJ control mice, 8 weeks old, were injected subcutaneously with TT (10
?g/mouse ). One dose of ?GalCer (5 ?g/mouse) was injected simultaneously
subcutaneously. ?GalCer was given similarly as ?GalCer to control animals. RA
was given orally (Sigma; 37.5 ?g/mouse/day) in canola oil, with oil only as the
vehicle control, daily for 7 consecutive days (22). Blood was collected from the
retro-orbital sinus prior to and after TT immunization. The treatment and
sampling times in the present study are further described and illustrated with the
results from the study.
Cell proliferation assay. [3H]thymidine incorporation assay was performed to
determine B cell proliferation as described previously (4).
Flow cytometry analysis and sorting. For each experimental condition, 105
isolated B cells were incubated with 0.1 ?g of fluorescence-labeled antibody.
After a washing step, unstained and isotype-control antibody stained cells were
used to set up gates as described previously (4).
Enzyme-linked immunospot (ELISPOT) assay. The procedure was performed
based on a previous report (22). The antigen-specific spots were counted and
calculated as number of spots per 106bone marrow cells.
Enzyme-linked immunosorbent assay (ELISA) for plasma anti-TT antibody.
A plasma anti-tetanus assay was performed as previously described (22). A
standard plasma sample was serially diluted on each assay plate to assure that the
measurements were in a linear dose-response range and that there was compa-
rability across the assays. Titers of antibody (i.e., the fold dilution) were calcu-
lated based on the standard curve developed for each plate.
Statistical methods. Means, standard errors, and P values were determined by
using Prism 5 software (GraphPad Software, Inc). P values were calculated by t
test or analysis of variance, followed by Tukey’s post hoc test. A P value of ?0.05
was considered significant.
RA increases CD1d expression in B cells. Spleen B cells
were isolated that had a purity ca. 94% according to CD19
staining. CD1d mRNA expression level was determined by
quantitative PCR both after and in the absence of treatment
with RA (20 nM, 24 h). We also stimulated B cells with
?GalCer (100 nM), which is known as a ligand for CD1d (2).
Treatment with RA increased CD1d mRNA during the 24-h
experiment (P ? 0.05), which was consistent with the results we
observed in monocytic cells, whereas ?GalCer failed to regu-
late the CD1d mRNA level (Fig. 1A).
RA and ?GalCer differentially regulate B cell proliferation
and differentiation. The CD1d molecule, as a lipid antigen
receptor, could potentially act as an alternative or additional
type of B cell receptor (BCR). Thus, we measured B cell
proliferation by thymidine incorporation after the treatment of
isolated B cells with ?GalCer and RA for 48 and 72 h; more-
over, to test for cooperation or cross talk between ?GalCer
stimulation and the BCR, we incubated cells with anti-? anti-
body in the presence or absence of ?GalCer. We first tested
the doses of ?GalCer used to stimulate B cell and selected
100 nM as an optimal subthreshold dose for the following
experiments (Fig. 1B). As shown in Fig. 1C, ?GalCer ex-
erted a strong mitogenic effect, evident at both 48 and 72 h,
whereas ?GalCer, an isomer that binds to CD1d but fail to
activate iNKT cells (27) and was used in the control group,
did not show any effect. Although RA alone had no stimu-
latory effect on B cell proliferation, it potentiated the mito-
genic effect of ?GalCer (Fig. 1C).
Moreover, both anti-? and ?GalCer were mitogenic to B
cells and, in combination, produced a more than additive in-
crease in B cell proliferation, evident on day 3 (Fig. 2A). In
these experiments, the retinoid antagonist inhibited the effect
of RA (compare results for ?GalCer with RA to ?GalCer with
RA plus antagonist, Fig. 2B). Lipopolysaccharide (LPS), used
as a positive control, strongly stimulated B cell proliferation as
expected, but RA inhibited the stimulatory effect of LPS, as we
have reported previously (4). Thus, the promotion by RA of
?GalCer-induced B cell proliferation is specific for this com-
pound compared to that for LPS.
To test the specificity of the CD1d-mediated B cell activa-
tion, we also conducted the assay in CD1d-null mice. The
deletion of CD1d abrogated the stimulatory effect of ?GalCer
while these cells retained the responsiveness to anti-? stimu-
lation, similar to wild-type cells, in the B cell proliferation assay
(Fig. 2C). These results imply that RA can enhance the sig-
naling of ?GalCer through CD1d, while CD1d- and BCR-
mediated signaling interact in a manner that suggests comple-
RA and ?GalCer help to regulate B cell differentiation into
plasmacytic cells. Next, we tested whether RA and ?GalCer
FIG. 1. Regulation of CD1d expression and cell proliferation by
RA and ?GalCer in mouse splenic B cells. (A) RA increased CD1d
expression in spleen B cells. B cells were cultured in the presence or
absence of RA (20 nM) or ?GalCer (100 nM) for 24 h. Total RNA was
extracted and subjected to quantitative PCR analysis. The data are
presented as the ratio of CD1d to tubulin mRNAs, representing three
independent experiments with treatments in triplicate. a ? b, P ? 0.05.
(B) ?GalCer increases B cell proliferation dose dependently. B cells
were isolated and cultured in the presence of different concentrations
of ?GalCer for 72 h. [3H]thymidine was added for the last 6 h of
culture to monitor cell proliferation activity. a ? b ? c, P ? 0.05. (C) B
cells were isolated and cultured in the presence or absence of ?GalCer
and/or RA or anti-? antibody (0.1 ?g/ml). [3H]thymidine was added on
day 2 or 3 for the last 6 h of culture to monitor cell proliferation
activity. ?GalCer strongly increased B cell proliferation, which was
further enhanced by RA. a ? b ? c (48 h) and a? ? b? ? c? (72 h), P ?
1016 CHEN ET AL.CLIN. VACCINE IMMUNOL.
interact with each other to regulate B cell differentiation into
plasma cells. Surface IgG1 (sIgG1) and CD138 (also known as
syndecan 1, a plasmacytic B cell marker ), as well as Fas
and PNA staining, which serve as biomarkers of germinal cen-
ter B cells (15, 21), were used to determine the B cell pheno-
type after treatment with RA and ?GalCer. B cells were
treated with RA and/or ?GalCer, and anti-? was used as a
positive stimulus to induce B cell activation and differentiation.
After 4 days of culture, cells were subjected to flow cytometry
analysis. As shown in Fig. 3A, RA significantly increased the
percentage of sIgG1?cells (P ? 0.05), while ?GalCer failed to
increase sIgG1 expression. These results are consistent with
the known cell differentiation-inducing effects of RA (4, 5),
suggesting that ?GalCer, while effective in regulating cell pro-
liferation, may be insufficient in promoting B-cell differentia-
tion. It was also observed that RA, or anti-? treatment, in-
creased the proportion of CD138?B cells (Fig. 3B, all P ?
0.05). Although ?GalCer alone had no observable effect, RA
and ?GalCer in combination augmented the increase in
FIG. 3. RA and ?GalCer differentially regulate plasma cell differ-
entiation. B cells were cultured in the presence or absence of RA,
?GalCer, or anti-? for 4 days and then stained with antibodies that
recognize CD19, sIgG1, CD138, and Fas/PNA. (A) RA increased
sIgG1 expression, while ?GalCer did not. Representative flow histo-
grams of B cell sIgG on day 4 of culture are illustrated; the percentage
of sIgG1-positive cells is shown as the mean ? the standard error (n ?
4). a ? b, P ? 0.05. An overlay of the histograms for each condition is
presented below to compare the change in peak positions. (B) RA and
anti-? increased CD138?B cells. a ? b ? c and a? ? b?, P ? 0.05.
(C) RA increased the germinal center B cell population via costaining
of Fas, PNA, and CD19. RA increased Fas?and PNA?B cell popu-
lations. a ? b ? c ? d, P ? 0.05.
FIG. 2. ?GalCer synergized with B cell receptor to stimulate B cell
proliferation. (A) B cells were isolated and cultured in the presence or
absence of ?GalCer and/or anti-? antibody (0.1 ?g/ml). “a ? b ? c”
(48 h) and “a? ? b? ? c? ? d?” (72 h) denote differences of P ? 0.05.
(B) RA increases ?GalCer stimulated B cell proliferation. B cells were
cultured with stimuli in the presence or absence of RA (20 nM) for
72 h. An RAR antagonist (Ro 41-5253, 100 nM) was added to the
culture as indicated in the chart. LPS (100 ng/ml) was used in the
experiment as a positive control. a ? b and a? ? b?, P ? 0.05.
(C) ?GalCer stimulated B cell proliferation via CD1d expression,
whereas anti-? does not.*, P ? 0.05.
VOL. 18, 2011 RETINOID-CERAMIDE AUGMENTATION OF B CELL ACTIVATION 1017
CD138 (syndecan 1)-expressing cells after BCR ligation with
The splenic microenvironment is crucial for B cell plasma-
cytic development. The germinal center B cell markers Fas and
PNA were detected to test whether RA and/or ?GalCer pro-
mote the development of germinal centers. Fas- and PNA-
positive cells were consistently increased by RA (Fig. 3C),
while the number was not affected by either ?GalCer or anti-?.
These data further imply a differential role of RA and ?GalCer
on B cell differentiation. Whereas RA promoted B cell differ-
entiation, as shown by several criteria, ?GalCer may need an
extra signal, such as BCR ligation, and signals initiated by RA
to regulate B cell differentiation.
RA and ?GalCer increase the primary antibody response
and memory B cell formation in vivo after tetanus immuniza-
tion. To explore the role of RA and ?GalCer in the regulation
of immune response, we used a mouse model of TT immuni-
zation, since TT is a clinically relevant antigen that is com-
monly used experimentally as a recall antigen. A single dose of
?GalCer was given simultaneously with the primary TT immu-
nization, followed by treatment with RA orally during the
development of the primary antibody response. A boost injec-
tion was given 4 weeks after the primary immunization (Fig.
4A). Plasma was collected for anti-TT IgG measurement at
both primary and secondary responses. At the end of the study,
bone marrow mononuclear cells were collected to detect mem-
ory B cells by ELISPOT assay. As expected, immunization
elicited a TT-specific IgG response, which was relatively low
during the primary phase (days 10 and 28) but increased prom-
inently after boosting (days 35 and 42) (Fig. 4B). RA and
?GalCer significantly increased the plasma IgG level during
both primary and secondary responses (Fig. 4C and D). Con-
sistent with the ELISA titers measured 14 days after boosting,
a TT-specific ELISPOT assay also showed that ?GalCer sig-
nificantly increased the number of TT-specific antibody-secret-
ing cells (ASC), suggesting the stimulation of memory B cell
formation by ?GalCer given at the time of priming (Fig. 4E).
Although the effect of RA was not statistically significant in the
ELISPOT assay, the trend was consistent with ELISA results
(Fig. 4F) with a strong overall correlation (R ? 0.9263, P ?
0.05). We further tested the antibody response in CD1d-null
mice. Treatment with ?GalCer increased the production of
anti-TT specific IgG in control mice but failed to affect the
antibody response in CD1d-null mice (Fig. 4G), indicating the
importance of CD1d in this immune enhancement. Together,
these data demonstrated that RA and ?GalCer administered,
along with TT antigen could enhance the antibody response
during both the primary and the secondary (memory) B cell
The present studies have shown that RA interacts with
?GalCer to regulate B cell proliferation and differentiation
and that CD1d may be one of the mediators for their effect. B
cells are an important type of antigen-presenting cells that
recognize protein antigen through BCR, as well as lipid anti-
gen via CD1d molecules on its surface. Lang et al. also re-
ported that B cell CD1d expression is required for the NKT
cell activity in the regulation of the antibody response (16).
Moreover, Leadbetter et al. (17) showed that the CD1d-iNKT
arm promotes B cell function in anti-lipid antibody production.
According to several lines of evidence, RA and ?GalCer acted
in a complementary manner, and the combination augmented
B cell differentiation in vitro and the plasma antibody response
First, ?GalCer alone significantly stimulated B cell prolifer-
ation, which was additive with BCR ligation by anti-? (Fig. 1
and 2). The additivity of anti-? with ?GalCer is consistent with
a previous report that the presentation of a CD1d-restricted
antigen such as ?GalCer by the BCR can activate B cells (1).
Whereas we (4) and others (11) have shown that RA typically
FIG. 4. RA and ?GalCer promote B cell differentiation and anti-
tetanus IgG secretion in vivo. (A) BALB/c mice were immunized with
TT antigen, along with ?GalCer, and treated with RA orally (for
details, see Materials and Methods). Blood was collected at the times
indicated for plasma anti-TT IgG ELISA. Bone marrow cells were
collected at the end of experiment for ELISPOT assay. (B) TT-specific
primary and secondary antibody responses. Day 10, primary response;
day 28, immediately before boosting; days 35 and 42, recall response to
TT at days 7 and 14, respectively, after the second immunization. (C
and D) Plasma TT-specific IgG after primary immunization (day 10)
and secondary immunization (day 42). The data are presented as the
fold increase versus the control group (a ? b or a ? b ? c, P ? 0.05).
(E) Number of antibody-secreting cells in bone marrow. The data from
two independent experiments were combined (n ? 8/group; a ? b ?
c, P ? 0.05). (F) Correlation of plasma anti-TT IgG with bone marrow
anti-TT ASC (memory B cells). (G) Serum IgG primary and secondary
responses in wild-type and CD1d-null mice (n ? 4/group).
1018CHEN ET AL.CLIN. VACCINE IMMUNOL.
inhibits B cell proliferation and is a strong inhibitor of anti-?
or LPS stimulated B cell proliferation (4) (Fig. 2B), even while
inducing B cell differentiation, it nonetheless enhanced B cell
proliferation stimulated by ?GalCer. Since B cell expansion is
the critical initial response that increases the pool of antigen-
activated B cells, the interaction of RA and ?GalCer to in-
crease B cell proliferation may contribute to the amplification
of the overall immune response. In spite of the BCR-depen-
dent pathway (1), CD1d may also induce B cell proliferation
through a BCR-independent way.
Since RA works together with ?GalCer to enhance B cell
proliferation, RA and ?GalCer diverge in the regulation of the
B cell differentiation process, a multistep and complex journey.
The processes in which RA and ?GalCer may exert their dif-
ferential effects on B cell activation and differentiation are
summarized in Fig. 5. Mature naive B cells go through expan-
sion and differentiation upon encountering stimulation. The
differential effect of RA and ?GalCer on the expression of
several essential molecules such as sIgG, CD138, and Fas/
PNA, which represent the B cell differentiation and plasmacyte
formation, may suggest an adequate differentiation program
controlling the stages of B cell activation. The differential ef-
fects of RA and ?GalCer are also interesting from the view-
point that they may complement one another, leading to a
heightened response when applied in combination. The dem-
onstration that treatment in vivo with RA and/or ?GalCer at
the time of primary immunization with TT can significantly
increase the specific anti-TT plasma antibody level during both
the primary and the secondary responses indicates the poten-
tial of glycolipid antigen stimulation to affect the response to a
protein antigen, while the similar elevation in the bone marrow
ASC response suggests that the production of a larger pool of
long-lived anti-TT memory cells was initiated when mice were
treated with RA and/or ?GalCer at the time of initial exposure
to antigen (Fig. 4). The absence of the stimulatory affect in the
CD1d-null mice suggests the importance of iNKT cells in the
adaptive immune response.
An interesting question raised here is whether concurrent
stimulation with a glycolipid and a protein antigen activates the
same B cell or whether “bystander” effects on nearby B cells
are in play. Compared to protein and polysaccharide antigens,
glycolipids have received relatively little attention, and it is
only recently, with the understanding that ?GalCer and
endogenous glycolipids such as isoglobosides (37) can activate
iNKT cells, that attention has begun to turn to these molecules
as important antigens. Their natural antigenicity in vivo, how-
ever, is not well understood. The concept that B cells, as
carriers of BCR and CD1d, could be importantly involved in
the response to a protein antigen, while being stimulated by a
glycolipid antigen, seems worthy of further investigation. RA
acted in a complementary manner to ?GalCer to enhance the
B cell differentiation program, leading to increased B cell mat-
uration and increased plasma antibody titers and memory re-
sponse, also as evidenced by increased formation of bone mar-
row ASC indicative of a long-term memory pool.
To conclude, our data provide novel evidence that RA,
which induced CD1d, is involved in ?GalCer-stimulated B cell
activation and differentiation and that both together are effec-
tive in stimulating the humoral B cell antibody response. Sev-
eral recent reports provide evidence that ?GalCer and its an-
alog could be an effective adjuvant in the immunization
protocol (8, 18, 20). With the understanding of the interaction
of RA and ?GalCer, the combination may be promising for
improving vaccine effectiveness in the future.
We thank the Center for Quantitative Cell Analysis, Penn State, for
assistance with flow cytometry.
This study was supported by NIH grant DK-41479 and ARRA sup-
plement R03 DK41479-20S.
We have no financial conflicts or other interests to disclose.
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proliferation and differentiation, leading to the formation of the
plasma cell phenotype, increased B memory cell population, and a
heightened recall response to reimmunization, as shown experimen-
tally in Fig. 4.
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