Oxysterol Gradient Generation by Lymphoid
Stromal Cells Guides Activated B Cell
Movement during Humoral Responses
Tangsheng Yi,1Xiaoming Wang,1Lisa M. Kelly,1Jinping An,1Ying Xu,1Andreas W. Sailer,2Jan-Ake Gustafsson,3
David W. Russell,4and Jason G. Cyster1,*
1HowardHughes Medical Instituteand Department of Microbiologyand Immunology, University of California, San Francisco, CA 94143, USA
2Developmental & Molecular Pathways, Novartis Institutes for BioMedical Research, 4056 Basel, Switzerland
3Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA
4Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
7a,25-dihydroxycholesterol (7a,25-OHC) is a ligand
for the G protein-coupled receptor EBI2; however,
the cellular sources of this oxysterol are undefined.
7a,25-OHC is synthesized from cholesterol by the
stepwise actions of two enzymes, CH25H and
CYP7B1, and is metabolized to a 3-oxo derivative
by HSD3B7. We showed that all three enzymes con-
trol EBI2 ligand concentration in lymphoid tissues.
Lymphoid stromal cells were the main CH25H- and
CYP7B1-expressing cells required for positioning of
B cells, and they also mediated 7a,25-OHC inactiva-
tion. CH25H and CYP7B1 were abundant at the
follicle perimeter, whereas CH25H expression by
follicular dendritic cells was repressed. CYP7B1,
CH25H, and HSD3B7 deficiencies each resulted in
defective T cell-dependent plasma cell responses.
These findings establish that CYP7B1 and HSD3B7,
as well as CH25H, have essential roles in controlling
oxysterol production in lymphoid tissues, and they
suggest that differential enzyme expression in stro-
mal cell subsets establishes 7a,25-OHC gradients
required for B cell responses.
In order to mount a rapid and efficient antibody response, B cells
undergo a series of dynamic movements within secondary
lymphoid organs (Cyster, 2010). Naive B cells express the che-
mokine receptor CXCR5 and are attracted into follicles by this
receptor’s ligand, CXCL13, which is made by stromal cells
distributed throughout the follicle. After encountering antigen,
activated B cells upregulate CCR7 and move within 6 hr to the
B cell zone-T cell zone (B-T) boundary of the follicle in response
to the T zone-expressed CCR7 ligand, CCL21. There, they in-
teract with cognate T helper cells, and subsequently the T cell-
primed B cells downregulate CCR7 and relocate to interfollicular
and outer follicular regions for further clonal expansion prior to
their differentiation into short-lived antibody-secreting plasma
cells or germinal center (GC) B cells (Coffey et al., 2009; Cyster,
2010; Kerfoot et al., 2011; Kitano et al., 2011). EBI2, a G protein-
coupled receptor, guides B cell movement along the B-T
boundary and later to interfollicular and outer follicular regions
(Gatto et al., 2009; Gatto et al., 2011; Kelly et al., 2011; Pereira
et al., 2009). The absence of EBI2 from B cells results in their
premature accumulation in the center of the follicle and dimin-
ished T cell-dependent plasma cell differentiation (Gatto et al.,
2009; Pereira et al., 2009).
7a,25-dihydroxycholesterol (7a,25-OHC) was recently identi-
fied by classic analytical methods as a high-affinity ligand for
EBI2 (Hannedouche et al., 2011; Liu et al., 2011). 7a,25-OHC
was previously identified as an intermediate in the alternate
pathway of hepatic bile acid synthesis (Russell, 2003). The
conversion of cholesterol into bile acids is accomplished in
the liver through two multienzyme pathways, commonly referred
to as the classic and alternate pathways of bile acid syn-
thesis. Studies in gene-deficient mice revealed that the
essential requirement for bile acids can be met through either
of the two pathways, and thus that they serve compensatory
roles in hepatic lipid metabolism (Russell, 2003). 7a,25-OHC
is synthesized from cholesterol by CH25H-mediated hydrox-
ylation at the 25 position, followed by CYP7B1-mediated
hydroxylation at the 7a position (Russell, 2003) (Figure 1A).
Unexpectedly for a protein that carries out a reaction related
to bile acid synthesis, CH25H is poorly expressed in the liver
but is abundant in a number of other tissues, suggesting the
enzyme may function outside the liver (Lund et al., 1998; Russell,
2003). Recent studies have shown that CH25H is highly ex-
pressed in activated macrophages (Bauman et al., 2009; Dicz-
falusy et al., 2009; Park and Scott, 2010; Zou et al., 2011).
Genetic deficiency in CH25H has been shown to cause a loss
of EBI2 ligand generation in lymphoid organs (Hannedouche
et al., 2011). Although macrophages are considered the most
likely cells acting in lymphoid tissues to carry out the 25-hydrox-
ylation reaction needed to generate EBI2 ligand (Hannedouche
et al., 2011; Liu et al., 2011), their role in this process was not
CYP7B1, a member of the cytochrome P450 enzyme family, is
abundant in the liver, but Cyp7b1 transcripts have also been de-
tected in a number of extrahepatic tissues (Stiles et al., 2009). In
Immunity 37, 535–548, September 21, 2012 ª2012 Elsevier Inc. 535
the kidney, CYP7B1 may contribute to de novo sterol synthesis
(Li-Hawkins et al., 2000), and in the reproductive tract, the
enzyme has a role in metabolizing androgens (Omoto et al.,
2005). In a recent report, treatment with the nonspecific cyto-
chrome P450 inhibitor, clotramizole, reduced 7a,25-OHC in
mouse spleen (Liu et al., 2011). This study provides support for
CYP7B1 functioning in 7a,25-OHC generation in the spleen,
but indirect effects of the drug could not be excluded. Moreover,
the cell types involved in 7a,25-OHC generation were not
Figure 1. CYP7B1 Is Required for EBI2 Ligand Generation and Activated B Cell Positioning
(A) Enzymatic steps catalyzed by CH25H, CYP7B1, and HSD3B7.
(B) Bioassay of titrated dilutions of spleen and LN extracts from Cyp7b1–/–or wild-type animals using a reporter cell line (M12) with or without EBI2 (mean ± SEM,
n = 4 mice).
(C)Bioassay oftitrateddilutions ofsupernatantfrom293Tcells transfectedwiththeempty vector(hCD4)orvector(s)encoding CH25H,CYP7B1,andCH25Hplus
CYP7B1 (co-express), or from a mixture of the individually transfected cells (Sep-express) (mean ± SEM, n = 4 mice from two experiments).
(D) EBI2 surface staining of Ebi2–/–or wild-typeMD4 Bcells 10 hr after HEL immunization or 48 hr after HEL-OVA immunization (One experiment representative of
(E) Migration of antigen-specific (MD4) B cells and noncognate B cells from spleens of 10-hr- or 2-day-immunized recipients (as in D) in response to 7a,25-OHC
(mean ± SEM, four mice in each group, one experiment representative of two experiments).
(blue) and endogenous B cells by anti-IgD (brown). Data are representative of at least three independent experiments.
CYP7B1 and HSD3B7 Establish EBI2 Ligand Gradients
536 Immunity 37, 535–548, September 21, 2012 ª2012 Elsevier Inc.
not studied here. Moreover, because both 7a,25-OHC and
7a,27-OHC are substrates for HSD3B7 (Russell, 2003), it is pos-
sible that some of the increase in EBI2 ligand detectable in the
absence of this enzyme is attributable to 7a,27-OHC. Additional
studies will be needed to quantify 7a,25-OHC and 7a,27-OHC
concentrations in mouse lymphoid and peripheral tissues.
Although compromised in EBI2 ligand production, CYP7B1-
deficient mice have elevated circulating 25-OHC and 27-OHC
(Bauman et al., 2009; Li-Hawkins et al., 2000). These oxysterols
have the capacity to modulate some immune cell functions
through actions on nuclear hormone receptors (Bensinger
et al., 2008; Kalaany and Mangelsdorf, 2006; Villablanca et al.,
2010) and to regulate IgA production (Bauman et al., 2009).
Increases in these oxysterols are unlikely to account for the
defective IgM and IgG plasma cell responses we describe
here, because EBI2-deficient B cells are equally compromised
in the generation of plasma cells in both wild-type and
CYP7B1-deficient hosts. That is, the positive influence of
CYP7B1 in promoting IgM and IgG plasma cell responses
appears to depend upon the generation of EBI2 ligand and
signaling via EBI2. Moreover, in contrast to the altered mucosal
IgA responses seen in CH25H- and CYP7B1-deficient mice
(Bauman et al., 2009), we have not detected altered IgA produc-
tion in EBI2-deficient mice (A. Reboldi, L.M.K., and J.G.C.,
unpublished data). Further supporting the conclusion that it is
the amount of EBI2 ligand that is important is the discovery of
similar defects in the plasma cell response in Ch25h?/?and
Hsd3b7?/?mice. An important challenge for future studies will
be elucidating whether EBI2, CH25H, CYP7B1, and HSD3B7
support B cell responses by promoting more efficient interac-
tions between B and T cells at the B-T boundary and in interfol-
licular regions, or by promoting interactions between early
plasmablasts and factors in interfollicular and outer follicular
regions that support their growth and differentiation.
C57BL/6NCr and C57BL/6-cBrd/cBrd/Cr (CD45.1) mice with ages of
7–9 weeks were from the National Cancer Institute (Frederick, MD, USA).
B6.Cg-IghaThy1aGp1a/J mice, B6-Gt (ROSA)26Sortm1(HBEGF)Awai
genic mice (Tg(UBC-GFP)30Scha/J) were from the Jackson Laboratory.
Ebi2?/?mice (Pereira et al., 2009) were backcrossed to C57BL/6J for 11
generations. These mice carry an eGfp gene inserted in place of the Ebi2
open reading frame. Ch25h?/?mice (Bauman et al., 2009) were backcrossed
ten generations to C57BL/6; Cyp7b1?/?mice were of one of two strains (Li-
Hawkins et al., 2000; Rose et al., 2001) and backcrossed to C57BL/6J for
five generations. Hsd3b7?/?mice were backcrossed to C57BL6/J for two
generations and maintained on chow containing 0.5% cholic acid and pan-
vitamin-supplemented water (Shea et al., 2007). CD169DTRmice (Miyake
et al., 2007), B6.Tg(Cr2-Cre)3Cgn (CD21-cre) mice, HEL-specific MD4 Ig-
transgenic and Hy10 mice, and OVA-specific OTII TCR-transgenic mice
were on a C57BL/6J background. Animals were housed in a specific path-
ogen-free environment in the Laboratory Animal Resource Center at the
University of California, San Francisco, and all experiments conformed to
ethical principles and guidelines approved by the Institutional Animal Care
and Use Committee.
Cell Adoptive Transfer and Immunizations
For visualization of in situ B cell position at day 2 of the T cell-dependent
response, mice were adoptively transferred with 1–10 3 106wild-type or
Ebi2?/?MD4 splenocytes and 1–5 3 106wild-type OTII splenocytes. One
day after cell transfer, recipients were immunized intraperitoneally (i.p.) with
50 mg HEL-OVA conjugate in RIBI-based Sigma adjuvant system. To examine
activated B cell distribution at 10 hr after antigen exposure, mice were given
107MD4 splenocytes the day before and were injected i.p. with 5 mg HEL in
the absence of adjuvant. For antibody responses, 1 3 105Hy10 B cells were
adoptively transferred into Cyp7b1?/?, Ch25h?/?, Hsd3b7?/?, or littermate
Cyp7b1+/?recipients. One day after cell transfer, recipients were i.p. immu-
nized with 2 3 108SRBCs conjugated with low-affinity HEL2xas described
(Gatto et al., 2009).
Supplemental Information includes seven figures and Supplemental Experi-
mental Procedures and can be found with this article online at http://dx.doi.
We thank R. Brink for providing HEL2x,M. Tanaka for CD169DTRmice, R. Lathe
for making Cyp7b1+/?mice available, and M. Barnes and A. Reboldi for
comments on the manuscript. T.Y. is an Irvington Institute Postdoctoral Fellow
at the Cancer Research Institute, and J.G.C. in an Investigator at the Howard
Hughes Medical Institute. This work was supported by National Institutes of
Health grants AI40098 and HL20948. A.W.S is a current employee of Novartis
and holds stock and stock options in the Novartis company.
Received: January 6, 2012
Revised: May 24, 2012
Accepted: June 12, 2012
Published online: September 20, 2012
Bauman, D.R., Bitmansour, A.D., McDonald, J.G., Thompson, B.M., Liang, G.,
and Russell, D.W. (2009). 25-Hydroxycholesterol secreted by macrophages in
response to Toll-like receptor activation suppresses immunoglobulin A
production. Proc. Natl. Acad. Sci. USA 106, 16764–16769.
Benned-Jensen, T., Smethurst, C., Holst, P.J., Page, K.R., Sauls, H.,
Sivertsen, B., Schwartz, T.W., Blanchard, A., Jepras, R., and Rosenkilde,
M.M. (2011). Ligand modulation of the Epstein-Barr virus-induced seven-
transmembrane receptor EBI2: identification of a potent and efficacious
inverse agonist. J. Biol. Chem. 286, 29292–29302.
Bensinger, S.J., Bradley, M.N., Joseph, S.B., Zelcer, N., Janssen, E.M.,
Hausner, M.A., Shih, R., Parks, J.S., Edwards, P.A., Jamieson, B.D., and
Tontonoz, P. (2008). LXR signaling couples sterol metabolism to proliferation
in the acquired immune response. Cell 134, 97–111.
Chan, T.D., Gatto, D., Wood, K., Camidge, T., Basten, A., and Brink, R. (2009).
Antigen affinity controls rapid T-dependent antibody production by driving the
expansion rather than the differentiation or extrafollicular migration of early
plasmablasts. J. Immunol. 183, 3139–3149.
Coffey, F., Alabyev, B., and Manser, T. (2009). Initial clonal expansion of
germinal center B cells takes place at the perimeter of follicles. Immunity 30,
Immunol. 11, 989–996.
Cyster, J.G., Ansel, K.M., Reif, K., Ekland, E.H., Hyman, P.L., Tang, H.L.,
Luther, S.A., and Ngo, V.N. (2000). Follicular stromal cells and lymphocyte
homing to follicles. Immunol. Rev. 176, 181–193.
Diczfalusy, U., Olofsson, K.E., Carlsson, A.M., Gong, M., Golenbock, D.T.,
Rooyackers, O., Fla ¨ring, U., and Bjo ¨rkbacka, H. (2009). Marked upregulation
of cholesterol 25-hydroxylase expression by lipopolysaccharide. J. Lipid
Res. 50, 2258–2264.
Gatto, D., Paus, D., Basten, A., Mackay, C.R., and Brink, R. (2009). Guidance
of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral
immune responses. Immunity 31, 259–269.
CYP7B1 and HSD3B7 Establish EBI2 Ligand Gradients
Immunity 37, 535–548, September 21, 2012 ª2012 Elsevier Inc. 547
Gatto, D., Wood, K., and Brink, R. (2011). EBI2 operates independently of but
in cooperation with CXCR5 and CCR7 to direct B cell migration and organiza-
tion in follicles and the germinal center. J. Immunol. 187, 4621–4628.
Hannedouche, S., Zhang, J., Yi, T., Shen, W., Nguyen, D., Pereira, J.P.,
Guerini, D., Baumgarten, B.U., Roggo, S., Wen, B., et al. (2011). Oxysterols
direct immune cell migration via EBI2. Nature 475, 524–527.
Kalaany, N.Y., and Mangelsdorf, D.J. (2006). LXRS and FXR: the yin and yang
of cholesterol and fat metabolism. Annu. Rev. Physiol. 68, 159–191.
Katakai, T., Suto, H., Sugai, M., Gonda, H., Togawa, A., Suematsu, S.,
Ebisuno, Y., Katagiri, K., Kinashi, T., and Shimizu, A. (2008). Organizer-like
reticular stromal cell layer common to adult secondary lymphoid organs.
J. Immunol. 181, 6189–6200.
Kelly, L.M., Pereira, J.P., Yi, T., Xu, Y., and Cyster, J.G. (2011). EBI2 guides
serial movements of activated B cells and ligand activity is detectable in
lymphoid and nonlymphoid tissues. J. Immunol. 187, 3026–3032.
Kerfoot, S.M., Yaari, G., Patel, J.R., Johnson, K.L., Gonzalez, D.G., Kleinstein,
S.H., and Haberman, A.M. (2011). Germinal center B cell and T follicular helper
cell development initiates in the interfollicular zone. Immunity 34, 947–960.
Kitano, M., Moriyama, S., Ando, Y., Hikida, M., Mori, Y., Kurosaki, T., and
Okada, T. (2011). Bcl6 protein expression shapes pre-germinal center B cell
dynamics and follicular helper T cell heterogeneity. Immunity 34, 961–972.
Li-Hawkins,J.,Lund,E.G.,Turley,S.D.,and Russell,D.W. (2000).Disruptionof
the oxysterol 7alpha-hydroxylase gene in mice. J. Biol. Chem. 275, 16536–
J.G., and Luther, S.A. (2007). Fibroblastic reticular cells in lymph nodes regu-
late the homeostasis of naive T cells. Nat. Immunol. 8, 1255–1265.
Liu, C., Yang, X.V., Wu, J., Kuei, C., Mani, N.S., Zhang, L., Yu, J., Sutton, S.W.,
Nature 475, 519–523.
Lund, E.G., Kerr, T.A., Sakai, J., Li, W.P., and Russell, D.W. (1998). cDNA
cloning of mouse and human cholesterol 25-hydroxylases, polytopic
membraneproteinsthat synthesizeapotent oxysterol regulator of lipid metab-
olism. J. Biol. Chem. 273, 34316–34327.
Miyake, Y., Asano, K., Kaise, H., Uemura, M., Nakayama, M., and Tanaka, M.
(2007). Critical role of macrophages in the marginal zone in the suppression of
immune responses to apoptotic cell-associated antigens. J. Clin. Invest. 117,
Muppidi, J.R., Arnon, T.I., Bronevetsky, Y., Veerapen, N., Tanaka, M., Besra,
G.S., and Cyster, J.G. (2011). Cannabinoid receptor 2 positions and retains
marginal zone B cells within the splenic marginal zone. J. Exp. Med. 208,
Okada, T., Miller, M.J., Parker, I., Krummel, M.F., Neighbors, M., Hartley, S.B.,
O’Garra, A., Cahalan, M.D., and Cyster, J.G. (2005). Antigen-engaged B cells
undergochemotaxistowardtheT zoneand formmotileconjugateswithhelper
T cells. PLoS Biol. 3, e150.
Omoto, Y., Lathe, R., Warner, M., and Gustafsson, J.A. (2005). Early onset of
puberty and early ovarian failure in CYP7B1 knockout mice. Proc. Natl. Acad.
Sci. USA 102, 2814–2819.
Park, K., and Scott, A.L. (2010). Cholesterol 25-hydroxylase production
by dendritic cells and macrophages is regulated by type I interferons.
J. Leukoc. Biol. 88, 1081–1087.
Paus, D., Phan, T.G., Chan, T.D., Gardam, S., Basten, A., and Brink, R. (2006).
Antigen recognition strength regulates the choice between extrafollicular
plasma cell and germinal center B cell differentiation. J. Exp. Med. 203,
Pereira, J.P., Kelly, L.M., Xu, Y., and Cyster, J.G. (2009). EBI2 mediates B cell
segregation between the outer and centre follicle. Nature 460, 1122–1126.
Phan, T.G., Green, J.A., Gray, E.E., Xu, Y., and Cyster, J.G. (2009). Immune
complex relay by subcapsular sinus macrophages and noncognate B cells
drives antibody affinity maturation. Nat. Immunol. 10, 786–793.
Reif, K., Ekland, E.H., Ohl, L., Nakano, H., Lipp, M., Fo ¨rster, R., and Cyster,
J.G. (2002). Balanced responsiveness to chemoattractants from adjacent
zones determines B-cell position. Nature 416, 94–99.
Rose, K., Allan, A., Gauldie, S., Stapleton, G., Dobbie, L., Dott, K., Martin, C.,
Wang, L., Hedlund, E., Seckl, J.R., et al. (2001). Neurosteroid hydroxylase
CYP7B: vivid reporter activity in dentate gyrus of gene-targeted mice and
abolition of a widespread pathway of steroid and oxysterol hydroxylation.
J. Biol. Chem. 276, 23937–23944.
Russell, D.W. (2003). The enzymes, regulation, and genetics of bile acid
synthesis. Annu. Rev. Biochem. 72, 137–174.
Schwarz, M., Wright, A.C., Davis, D.L., Nazer, H., Bjo ¨rkhem, I., and Russell,
D.W. (2000). The bile acid synthetic gene 3beta-hydroxy-Delta(5)-C(27)-
steroid oxidoreductase is mutated in progressive intrahepatic cholestasis.
J. Clin. Invest. 106, 1175–1184.
Shea, H.C., Head, D.D., Setchell, K.D., and Russell, D.W. (2007). Analysis of
HSD3B7 knockout mice reveals that a 3alpha-hydroxyl stereochemistry is
required for bile acid function. Proc. Natl. Acad. Sci. USA 104, 11526–11533.
Steinman, R.M., Pack, M., and Inaba, K. (1997). Dendritic cells in the T-cell
areas of lymphoid organs. Immunol. Rev. 156, 25–37.
Stiles, A.R., McDonald, J.G., Bauman, D.R., and Russell, D.W. (2009).
CYP7B1: one cytochrome P450, two human genetic diseases, and multiple
physiological functions. J. Biol. Chem. 284, 28485–28489.
Villablanca, E.J., Raccosta, L., Zhou, D., Fontana, R., Maggioni, D., Negro, A.,
Sanvito, F., Ponzoni, M., Valentinis, B., Bregni, M., et al. (2010). Tumor-medi-
ated liver X receptor-alpha activation inhibits CC chemokine receptor-7
expression on dendritic cells and dampens antitumor responses. Nat. Med.
Wang, X., Cho, B., Suzuki, K., Xu, Y., Green, J.A., An, J., and Cyster, J.G.
(2011). Follicular dendritic cells help establish follicle identity and promote B
cell retention in germinal centers. J. Exp. Med. 208, 2497–2510.
Zou, T., Garifulin, O., Berland, R., and Boyartchuk, V.L. (2011). Listeria mono-
cytogenes infection induces prosurvival metabolic signaling in macrophages.
Infect. Immun. 79, 1526–1535.
CYP7B1 and HSD3B7 Establish EBI2 Ligand Gradients
548 Immunity 37, 535–548, September 21, 2012 ª2012 Elsevier Inc.