cxcr4 expression on activated b cells is downregulated by cd63 and il 21

CXCR4 Expression on Activated B Cells Is Downregulated byCD63 and IL-21 Nobuya Yoshida,*,†Daisuke Kitayama,* Masafumi Arima,* Akemi Sakamoto,* Ayako Inamine,‡ Haruko Watanabe-Takano,xMas

of June 7, 2015.

This information is current as

Downregulated by CD63 and IL-21 CXCR4 Expression on Activated B Cells Is

Takeshi Tokuhisa Watanabe-Takano, Masahiko Hatano, Takao Koike and Akemi Sakamoto, Ayako Inamine, Haruko

Nobuya Yoshida, Daisuke Kitayama, Masafumi Arima,

http://www.jimmunol.org/content/186/5/2800

doi: 10.4049/jimmunol.1003401 January 2011;

2011; 186:2800-2808; Prepublished online 26

J Immunol

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The Journal of Immunology

CXCR4 Expression on Activated B Cells Is Downregulated by

CD63 and IL-21

Nobuya Yoshida,*,†Daisuke Kitayama,* Masafumi Arima,* Akemi Sakamoto,* Ayako Inamine,‡

Haruko Watanabe-Takano,xMasahiko Hatano,xTakao Koike,†and Takeshi Tokuhisa*

CXCR4 expression is critical for localization of centroblasts in the dark zone of germinal centers (GCs), and centrocytes down-regulate CXCR4 and thus leave the dark zone to reside in the light zone However, mechanisms governing CXCR4 downregula-tion on centrocytes are not known In this study, we show that the amount of intracellular CXCR4 in centroblasts was similar to that in centrocytes, suggesting differential control of CXCR4 protein expression in these GC B cells Restimulation of activated

B cells with IL-21, which is a major cytokine produced by T follicular helper cells, accelerated CXCR4 internalization by inducing en-docytosis-related GRK6 expression Although CXCR4 expression was downregulated on GC B cells by IL-21 stimulation, CXCR4low centrocytes developed in the spleens of IL-21R–deficient mice, suggesting other mechanisms for downregulation The level of CD63 (which recruits CXCR4 to late endosome in CD4 T cells) in centrocytes was more than that in centroblasts and was strikingly elevated in activated Bcl6-deficient B cells Bcl6, a transcriptional repressor, was detected on the chromatin of the CD63 gene in resting B cells, therefore CD63 is a molecular target of Bcl6 Downregulation of CD63 mRNA in activated Bcl6-deficient B cells by small interfering RNA upregulated CXCR4 expression on the B cells Furthermore, addition of Bcl6 inhibitor

to activated B cell cultures increased CD63 mRNA expression in (and downregulated CXCR4 expression on) those activated

B cells Thus, CXCR4 can be downregulated on activated B cells by IL-21–induced endocytosis and CD63-mediated endosomal recruitment, and these mechanisms may contribute to downregulation of CXCR4 on centrocytes The Journal of Immunology,

2011, 186: 2800–2808

G erminal centers (GCs) are the site for development of

high-affinity memory B cells and long-lived plasma cells

(1, 2) After Ag-activated B cells collaborate with

acti-vated T follicular helper (Tfh) cells on the follicular border, some

of the activated B cells rapidly proliferate in the follicle to

gen-erate GCs These proliferating B cells, centroblasts, undergo

so-matic hypermutation and form the dark zone of GCs Then, the

centroblasts turn to differentiate to centrocytes with Ig

class-switching to IgG The centrocytes migrate to an area adjacent to

the dark zone of GCs In the area called the light zone of GCs,

centrocytes express high-affinity IgG Abs that competitively bind

to Ags on follicular dendritic cells and also collaborate with

GC–Tfh cells, which produce IL-21 and IL-4 (3) These

acti-vated centrocytes scarcely proliferate and differentiate to memory

B cells or long-lived plasma cells The level of a chemokine

re-ceptor, CXCR4, on centroblasts is significantly more than that on centrocytes (4, 5), and CXCR4 plays an important role in segre-gation of dark and light zones in GCs (4) Thus, CXCR4 ex-pression on centrocytes has to be downregulated to leave the dark zone to migrate to the light zone However, the mechanism of its downregulation on centrocytes is not known

Because CXCR4 is known as a coreceptor for HIV infection and

as a key molecule for cancer metastasis, mechanisms of CXCR4 expression are mainly studied on CD4 T cells and cancer cell lines (6–8) In addition to transcriptional control of the CXCR4 gene (9–12), CXCR4 expression on the cell surface is regulated by its endocytosis and exocytosis The various endocytosis-related molecules including GRK6, b-arrestin2 (Arrb2), and AIP4 are inducible in CD4 T cells for the clathrin-dependent internalization

of CXCR4 (13–16) Of the exocytosis-related molecules, CD63,

a ubiquitously expressed tetraspanin, has been identified as a negative regulator of CXCR4 exocytosis in CD4 T cells (17, 18) CD63 interacts with CXCR4 directly through the N-linked gly-cans portion of CD63 The CD63–CXCR4 complex induces di-rection of CXCR4 trafficking to the endosomes/lysosomes, rather than to the plasma membrane Thus, activation of the endocytosis-related proteins such as GRK6, Arrb2, and AIP4 and/or that of CD63 may be related to the downregulation of CXCR4 on cen-trocytes in the light zone of GCs

We generated GC-like B cells in vitro by sequentially stimulat-ing splenic B cells with anti-IgM Abs and anti-CD40 Abs plus IL-4 and then with IL-21 or IL-4 after a 2-d interval (19) Using the

in vitro stimulation system, we have reported that restimulation

of activated B cells with IL-21 or IL-4 induced proliferation and differentiation of CXCR4low or CXCR4high B cells, re-spectively Thus, IL-21 from Tfh cells may play a role in down-regulation of CXCR4 on centrocytes in the light zone of GCs In this study, we show that restimulation of activated B cells with IL-21 induces GRK6 expression to activate endocytosis of CXCR4

*Department of Developmental Genetics (H2), Graduate School of Medicine, Chiba

University, Chiba 260-8670, Japan; † Department of Medicine II, Hokkaido

Univer-sity Graduate School of Medicine, Sapporo 060-8638, Japan; ‡ Department of

Oto-laryngology (J2), Graduate School of Medicine, Chiba University, Chiba 260-8670,

Japan; andxDepartment of Biomedical Science (M14), Graduate School of Medicine,

Chiba University, Chiba 260-8670, Japan

Received for publication October 13, 2010 Accepted for publication December 22,

2010.

This work was supported in part by Grants-in-Aid from the Ministry of Education,

Culture, Sports, Science and Technology of Japan and by the Global COE Program

(Global Center for Education and Research in Immune System Regulation and

Treat-ment), Ministry of Education, Culture, Sports, Science and Technology of Japan.

Address correspondence and reprint requests to Dr Takeshi Tokuhisa, Department

of Developmental Genetics (H2), Graduate School of Medicine, Chiba University,

1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan E-mail address: tokuhisa@faculty.

chiba-u.jp

Abbreviations used in this article: Ac-H3, acetylation of histone H3 lysine (K) 9 and

K14; Arrb2, b-arrestin2; ChIP, chromatin immunoprecipitation; Ct, threshold cycle;

GC, germinal center; siRNA, small interfering RNA; Tfh, T follicular helper; WT,

wild-type.

Copyright Ó 2011 by The American Association of Immunologists, Inc 0022-1767/11/$16.00

Furthermore, CXCR4 expression was continuously downregulated

in activated B cells from Bcl6-deficient (Bcl62/2) mice with

over-expression of CD63, and the CD63 gene is a molecular target of

Bcl6 We discuss roles of IL-21 and CD63 in maintaining the

downregulation of CXCR4 on centrocytes in the light zone of GCs

Materials and Methods

Mice

C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan).

Bcl62/2 (20) and IL-21R–deficient (IL-21R2/2) (21) mice were as

de-scribed All mice were maintained under specific pathogen-free conditions

in the animal center of the Graduate School of Medicine, Chiba University.

The care of all animals used in the current study was in accordance with

Chiba University Animal Care Guidelines.

Immunization and purification of GC B cells

Mice were immunized intraperitoneally with 50 mg alum-precipitated

(4-hydroxy-3-nitrophenyl) acetyl 25 -chicken gammaglobulin (Biosearch

Tech-nologies) and 1.0 3 10 7

PFU Bordetella pertussis (Nacalai Tesque, Kyoto, Japan) GC B cells were isolated from the spleens of these mice 10 d

after immunization Briefly, spleen cells were first blocked with

un-conjugated anti-CD32/16 mAbs (2.4G2; BD Pharmingen), followed by

incubation with allophycocyanin Cy7–anti-B220 mAbs (BD Pharmingen),

FITC–anti-GL7 mAbs (BD Pharmingen), PE–anti-Fas mAbs (BD

Phar-mingen), and allophycocyanin–anti-CXCR4 mAbs (BD Pharmingen) GC

B cells (B220+GL7+Fas+), centroblasts (B220+GL7+Fas+CXCR4high),

cen-trocytes (B220 + GL7 + Fas + CXCR4 low ), and non-GC B cells (B220 + GL7

-Fas-) were sorted by a FACSAria II (Becton Dickinson) Purity of each

FACS-sorted population was 99%.

Splenic B cell culture

Spleen cell suspensions were treated with lysis buffer (0.155 M ammonium

chloride, 0.1 M disodium EDTA, 0.01 M potassium bicarbonate) to lyse

erythrocytes Single-cell suspensions were prepared in staining solution

(PBS with 3% FCS), and treated with biotinylated anti-CD43 mAbs and

anti-IgG1 mAbs (BD Pharmingen) for 15 min on ice After washing, the

cells were resuspended with streptavidin-Micro Beads (Miltenyi Biotec,

Germany) for 15 min on ice Labeled cells were removed by a MACS

system (Miltenyi Biotec) The resulting B cell fraction contained 95%

B220+B cells Purified B cells were cultured in RPMI 1640 medium

(Sigma Chemical) supplemented with 10% FCS (Intergen), 50 mM

2-mercaptoethanol, 10 mM HEPES, 100 mg/ml streptomycin (Wako

Chemical), and 100 U/ml penicillin G potassium (Banyu Pharmaceutical).

For B cell activation, purified B cells were stimulated with F(ab 9) 2

frag-ments of rabbit anti-mouse IgM Abs (1.0 3 10 25 mg/ml; Alpha

Di-agnostic), anti-CD40 mAbs (1.0 mg/ml; BD Pharmingen), and rIL-4 (2 U/

ml) (22) at the beginning of culture and sequentially stimulated with rIL-4

(20 U/ml) or rIL-21 (30 ng/ml; R&D Systems) at day 2 of culture in

a humidified atmosphere at 37˚C with 5% CO 2 Live cell numbers were

counted under a microscope after staining with trypan blue (Invitrogen).

CXCR4high and CXCR4low cells at day 2 of culture were sorted by

a FACSAria II Bcl6-inhibitor (50–500 nM, pTAT-Bcl6;

YGRKKRR-QRRRGGGGGLVATVKEAGRSIHELPREEL) (23), Dynamin-inhibitor

(80 mM, Dynasore; Ascent Scientific), or human rCXCL12 (1 mg/ml,

SDF-1; Peprotech) was added for the indicated experiments

FACS-isolated GC B cells were stimulated with rIL-21 (30 ng/ml; R&D

Sys-tems) for the indicated experiments.

Nucleofection of small interfering RNA

Predesigned CD63 small interfering RNA (siRNA) (s63677, s63678, and

s63679; Silencer Select Predesigned siRNA; Ambion) and scramble

siRNA (Silencer Select Negative Control #1 siRNA; Ambion) were

pur-chased, and 500 pM of each siRNA was transfected to purified B cells

previously stimulated with anti-IgM Abs and anti-CD40 mAbs plus rIL-4

(2 U/ml), using the nucleofector I device (Amaxa) Nucleofection of each

siRNA was performed according to the manufacturer’s recommendations.

Flow cytometry analysis

Cells were blocked with unconjugated anti-CD32/16 mAbs followed

by incubation with mAbs as indicated: allophycocyanin–anti-IgG1 (BD

Pharmingen), FITC–anti-IgG1 (BD Pharmingen), PE–anti-B220 (BD

Pharmingen), FITC–anti-B220 (BD Pharmingen), allophycocyanin Cy7–

anti-B220 (BD Pharmingen), biotinylated anti-CXCR4 (BD Pharmingen),

allophycocyanin–anti-CXCR4 (BD Pharmingen), FITC–anti-CXCR4 (BD Pharmingen), FITC–rat IgG2b for an isotype control (BD Pharmingen), FITC–anti-GL7 (BD Pharmingen), and PE–anti-Fas (BD Pharmingen) Biotinylated Abs were detected by allophycocyanin–streptavidin (BD Pharmingen) Flow cytometric analysis was performed with a FACSCali-bur (Becton Dickinson) or a FACSCanto II (Becton Dickinson) using CellQuest software (Becton Dickinson) or FlowJo software (TOMY Dig-ital Biology), respectively For intracellular staining, cells were stained with FACS Permeabilizing Solution 2 (Becton Dickinson) according to the manufacturer’s instructions For CFSE staining, purified B cells were la-beled with CFSE (Molecular Probes) as described previously (24).

Real-time quantitative RT-PCR

RT-PCR was performed as described elsewhere (25) Total RNA was extracted from B cells with the TRIzol reagent (Life Technologies) Total RNA was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) and Oligo (dT) (Pharmacia), and the cDNAs were used for PCR After an initial 5-min incubation at 94˚C, 50 cycles of PCR were carried out using the following conditions: denaturation at 94˚C for 60 s, annealing at 60˚C for 60 s, and polymerization at 72˚C for 60 s RT-PCR primers for the cDNA amplification were the following: Bcl6, 5 9-CCG-GCTCAATAATCTCGTGAA-39 and 59-GGTGCATGTAGAGTGGTGAG-TGA-3 9; GRK6, 59-GGAATCGCAAAGGCAAGAGCAAGA-39 and 59-T-CACGAAATAACAGGCGCCCAATG-39; AIP4, 59-AGCGAAGTAAGA-GGAGTAGCAAGG-3 9 and 59-TTGAACTGGATCGCGATGTCCTCT-39; b-arrestin2 (Arrb2), AATTTGCCTTGCTCCGTCACACTG-39 and 59-TGATGATAAGCCGCACAGAGTTCC-3 9; CXCR4, 59-ACGGCTGTAG-AGCGAGTGTT-3 9 and AGGGTTCCTTGTTGGAGTCA-39; CD63, 59-TCAACATAACTGTGGGCTGTGGGA-3 9 and 59-AGCCACCAGCAGT-ATGTTCTTCCT-3 9; CD23, 59-CTAGAAAGCGTTGCTGCTGTGCAA-39 and 59-ATTCTTCTCCGTTTCCCAGTGCCA-39; IRF-4, 59-GTGGAAA-CACGCGGGCAAGC-3 9 and 59-GGCTCCTCTCGACCAATTCCTCA-39; b-actin, 59-CCAGCCTTCCTTCTTGGGTAT-39, and 59-TGGCATAGAG-GTCTTTACGGATGT-3 9.

Immunoblot

Cultured B cells were washed with PBS and lysed with a lysis buffer (1% Nonidet P-40, 5% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mg/

ml leupeptin, 0.1 mM PMSF, 1 mM DTT, 1 mg/ml pepstatin A, 10 mM

Na 3 VO 4 , and 10 mM NaF) Cell lysate was subjected to SDS-PAGE, and immunoblot was carried out as described previously (26) Primary Abs used were goat polyclonal IgG anti-CD63 Abs (R-13, sc-31213; Santa

FIGURE 1 Restimulation of activated B cells with IL-21 and IL-4 in-versely regulates CXCR4 expression on those B cells in vitro Naive B cells were cultured with anti-IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 for 7 d IL-21 or IL-4 was added at day 2 of culture A, IgG1 and CXCR4 expression on activated B220 + B cells at day 4 of culture were analyzed by flow cytometry The numbers in the plot indicate the per-centage of each gate Data are presented as a representative of five in-dependent experiments B, Numbers of CXCR4highand CXCR4lowB cells

in the culture were calculated by the flow cytometry profiles Open and filled circles indicate IL-4 and IL-21 restimulated B cells, respectively.

Cruz Biotechnology) and goat polyclonal IgG anti-actin Abs (C-11,

sc-1615; Santa Cruz Biotechnology).

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assay was performed using a ChIP

assay kit (Upstate Biotechnology) and conducted according to the

manu-facturer’s recommendations as described (27) Briefly, protein and DNA in

B cells (3 3 10 6 /ml) were cross-linked by adding formaldehyde solution

(37%; Fisher Scientific) directly, and then these cells were lysed by SDS

lysis buffer containing protease inhibitors The lysates were subjected to

sonication to reduce DNA length to less than 1 kb Chromatin

immuno-precipitation was performed using a specific Ab to the protein of interest

overnight at 4˚C The amount of the objective DNA region in the

immune-precipitated chromatin was analyzed by quantitative PCR analysis

Real-time PCR was performed in 20- ml reaction volumes containing iQ

SYBR-Green Supermix (Bio-Rad), 200 nM of each primer, and 1 ml each ChIP

DNA fraction using the CFX96 real-time PCR detection system (Bio-Rad).

PCR cycle parameters were 3 min at 95˚C, 45 cycles of 30 s at 95˚C, 30 s

at 60˚C, and 30 s at 72˚C, followed by melting curve analysis The

threshold cycle (Ct) (i.e., the cycle number at which the amount of

am-plified DNA of interest reached a fixed threshold) was determined

sub-sequently Relative quantification of ChIP DNA was calculated by the

comparative Ct method described elsewhere (28) Each ChIP DNA

frac-tions’ Ct value was normalized to the input DNA fraction Ct value for the

same quantitative PCR assay ( DCt [normalized ChIP] = Ct [ChIP] 2 Ct

[Input]) The normalized ChIP fraction Ct value was adjusted for the normalized background [rabbit IgG] fraction Ct value (DDCt [ChIP/IgG] = DCt [normalized ChIP] 2 DCt [normalized IgG]) ChIP fold enrichment above the sample specific background was calculated as 22DDCt [ChIP/IgG] The following three sets of primers were used in the ChIP assays: CD63-1,

5 9-TGTGCCTTAAACAACTTCCCAGCC-39 and 59-AAAGGCGAGAG-TTAGGTCAGATCC-3 9; CD63-2, 59-GAGGGTTAGCCGGGATAGAAG-ATT-3 9 and 59-CCCACAGCCCACAGTTATGTTGAT-39; CD63-3, 59-TA-AAGTGGAGGTTTCCCTCCCATC-3 9 and 59-CCTTGAAATCATTCCC-ACAGCCCA-3 9.

Statistical analysis

Statistical analysis was done using unpaired Student t test, and p values ,0.05 were considered to be significant.

Results

Downregulation of CXCR4 on activated B cells restimulated with IL-21 is due to acceleration of CXCR4 endocytosis When splenic B cells were sequentially stimulated with anti-IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 (which did not induce Ig class-switching) and with IL-21 or IL-4 after a 2-d

FIGURE 3 Restimulation of activated B cells with IL-21 accelerates CXCR4 endocytosis Naive B cells were cultured with anti-IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 for 4 d IL-21 or IL-4 was se-quentially added at day 2 of culture A, Cell surface and intracellular CXCR4 expression of these B cells was analyzed by flow cytometry Filled and open histograms indicate IL-21 and IL-4 restimulated B cells, re-spectively The isotype control of each CXCR4 staining is represented as

an open histogram with a thin broken line The numbers in each histogram indicate the mean fluorescence intensity (isotype controls ,13) Data are presented as a representative of five independent experiments B, Dynasore (Dyn) was added in the culture at day 2 of culture CXCR4 expression on these B cells was analyzed by flow cytometry Filled and open histograms indicate activated B cells cultured with and without Dyn, respectively The numbers in each histogram indicate the mean fluorescence intensity Data are presented as a representative of four independent experiments C, Expression of GRK6 and AIP4 mRNA in these activated B cells was an-alyzed by real-time quantitative RT-PCR Results represent means 6 SD of three independent experiments Bars in figures represent mean values 6

SD *p , 0.05 N.S., not significant.

FIGURE 2 Restimulation of activated B cells with IL-21 downregulates

CXCR4 expression on those B cells Naive B cells were cultured with

anti-IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 for 4 d A,

CFSE-labeled naive B cells were cultured IL-21 (filled histograms), IL-4 (open

histograms), or nothing (open histogram with a broken line) was

sequen-tially added at day 2 of culture B220 + cells in the culture were analyzed by

flow cytometry Data are presented as a representative of three independent

experiments B, CXCR4 low and CXCR4 high B cells at day 2 of culture were

sorted by FACS and restimulated with IL-21 or IL-4 for another 2 d.

CXCR4 expression on these B cells was analyzed by flow cytometry The

number in each histogram indicates the mean fluorescence intensity Data

are presented as a representative of four independent experiments.

interval, these restimulations induced two distinct subsets of IgG1

B cells, CXCR4lowor CXCR4highIgG1 B cells, respectively (Fig

1A) Although the number of the activated B cells restimulated

with IL-21 decreased after day 5 of culture because of the

dif-ferentiation to plasma cells, kinetic studies of activated B cells

clearly demonstrated that restimulation of activated B cells with

IL-21 or IL-4 induced proliferation of CXCR4lowor CXCR4high

activated B cells including IgG1 B cells, respectively (Fig 1B)

To examine the relationship between proliferation and CXCR4

expression on activated B cells, naive B cells labeled with CFSE

were sequentially stimulated for 4 d As shown in Fig 2A, being

associated with cell cycle progression, restimulation of activated

B cells with IL-21 or IL-4 decreased or increased the amount of

CXCR4 on those B cells, respectively To confirm the regulation

of CXCR4 expression on activated B cells by IL-21 or IL-4, we

isolated CXCR4highand CXCR4lowactivated B cells at day 2 of

culture before restimulation by FACS These isolated B cells were

restimulated with IL-21 or IL-4 for 2 d The IL-21 or IL-4

restimulation clearly decreased or increased the amount of

CXCR4 on activated B cells, respectively (Fig 2B)

When we examined the amount of intracellular CXCR4 protein

in sequentially stimulated B cells and compared it with that of

sur-face CXCR4 on these cells at day 4 of culture, the difference in

amounts of intracellular CXCR4 between IgG1 B cells restimulated

with IL-21 or IL-4 was much less than that for amounts of cell

surface CXCR4 (Fig 3A) These results suggested that restimula-tion of activated B cells with IL-21 downregulates CXCR4 expres-sion on IgG1 B cells by endocytosis To examine CXCR4 endocy-tosis in activated B cells restimulated with IL-21, Dynasore, a dynamin inhibitor, was added at day 2 of the culture Dynasore in-creased the amount of CXCR4 on activated B cells restimulated with IL-21 but not with IL-4, indicating an acceleration of CXCR4 endocytosis in activated B cells restimulated with IL-21 (Fig 3B) Then, we measured expression of GRK6, AIP4, and Arrb2 mRNA

in sequentially activated B cells at day 4 of culture by real-time quantitative RT-PCR The amount of GRK6 mRNA but not that

of AIP4 or Arrb2 (data not shown) increased in activated B cells restimulated with IL-21 but not with IL-4 (Fig 3C)

The amount of CD63 mRNA in centrocytes is more than that in centroblasts

In GCs, upregulation and downregulation of CXCR4 allows GC

B cells to localize in dark and light zones, respectively (4) To examine the effect of IL-21 on CXCR4 downregulation in GC

B cells, wild-type (WT) and IL-21R2/2GC B cells were sorted from spleen cells 10 d after immunization and cultured with IL-21 for 48 h CXCR4 expression on WT GC B cells but not on IL-21R2/2GC B cells was downregulated with IL-21 stimulation (Fig 4A) However, when we analyzed CXCR4 expression on GC

B cells from immunized IL-21R2/2mice, CXCR4lowGC B cells

FIGURE 4 CD63 mRNA is upregulated in centrocytes IL-21R2/2and WT mice were immunized with alum-precipitated (4-hydroxy-3-nitrophenyl) acetyl 25 -chicken gammaglobulin Splenic B cells from IL-21R2/2and WT mice 10 d after immunization were analyzed A, GC B cells (Fas + GL7 + ) isolated from splenic B cells of WT and IL-21R2/2(KO) mice were cultured with (filled histograms) or without (open histograms) IL-21 for 48 h CXCR4 ex-pression on these cultured GC B cells was analyzed by flow cytometry The numbers in each histogram indicate the mean fluorescence intensity Data are presented as a representative of three independent experiments B, Cell surface and intracellular CXCR4 expression of GC (Fas + GL7 + ) B cells from spleens

of WT (open histograms) and IL-21R2/2(KO, filled histograms) mice was analyzed by flow cytometry The isotype control of each intracellular CXCR4 staining is represented as an open histogram with a thin broken line The numbers in each histogram indicate the mean fluorescence intensity (isotype controls ,134) Data are presented as a representative of three independent experiments C, Centroblasts (Fas +

GL7+CXCR4high) and centrocytes (Fas+ GL7+CXCR4low) were sorted from splenic B cells of WT (open bars) and IL-21R2/2(filled bars) mice by FACS Expression of Bcl6, IRF-4, CXCR4, GRK6, and CD63 mRNA in these isolated B cells was measured by real-time quantitative RT-PCR Results represent means 6 SD of four independent experiments Bars in figures represent mean values 6 SD *p , 0.05 N.S., not significant.

were detected in the spleens of IL-21R2/2mice, and the amount

of CXCR4 expression on CXCR4lowIL-21R2/2GC B cells was

almost equivalent to that on CXCR4lowWT GC B cells (Fig 4B)

These results suggested that the CXCR4 downregulation on

cen-trocytes in the light zone of GCs could not be explained only by

the acceleration of CXCR4 endocytosis by IL-21

To elucidate the mechanism of CXCR4 downregulation on

centrocytes, we isolated CXCR4highGC B cells (centroblasts) and

CXCR4lowGC B cells (centrocytes) from immunized WT and

IL-21R2/2mice and analyzed expression of Bcl6, IRF-4, and CXCR4

mRNA in those GC B cells by real-time quantitative RT-PCR The

amount of Bcl6 mRNA in centroblasts was more than that in

centrocytes from both WT and IL-21R2/2mice, and the amount

of IRF-4 mRNA was inversely correlated with that of Bcl6 (Fig

4C) Surprisingly, the amount of both CXCR4 mRNA and

in-tracellular CXCR4 protein (Fig 4B) in the centroblasts was

sim-ilar to that in the centrocytes from both WT and IL-21R2/2mice,

suggesting acceleration of endocytosis and/or deceleration of

exo-cytosis of CXCR4 in centrocytes However, amounts of GRK6

mRNA in centrocytes from WT and IL-21R2/2 mice were not

more than those in centroblasts Because CD63 is known to traffic

CXCR4 protein to late endosome in CD4 T cells (17), we

ex-amined CD63 mRNA expression in these GC B cells Indeed,

CD63 mRNA expression was upregulated in the centrocytes of

both WT and IL-21R2/2 mice (Fig 4C), suggesting that CD63

downregulates CXCR4 expression on centrocytes

Bcl6 negatively regulates CD63 mRNA expression in activated

B cells

To elucidate the mechanism of CD63 upregulation in centrocytes,

we first focused on CXCL12 (SDF-1) stimulation Because

CXCL12 can transiently downregulate CXCR4 expression on CD4

T cells (29) and was suggested to be a possible regulator of

CXCR4 expression on GC B cells (5), CXCL12 stimulation might

induce CD63 mRNA to downregulate CXCR4 on centrocytes

Thus, naive B cells stimulated with anti-IgM Abs and anti-CD40

mAbs plus a low dose of IL-4 for 2 d were restimulated with

CXCL12, and the amount of CXCR4 on their cell surfaces was

analyzed by flow cytometry Though the amount of CXCR4 on

activated B cells was downregulated 6 h after CXCL12

restim-ulation, expression completely recovered 24 h after restimulation

(Fig 5A) Moreover, CD63 mRNA expression in these B cells was

not induced after CXCL12 restimulation

Then, we focused on Bcl6, a transcriptional repressor, to

elu-cidate the mechanism of CXCR4 downregulation We noted that

expression of Bcl6 and CD63 mRNA was inversely correlated in

centroblasts and centrocytes, suggesting downregulation of CD63

mRNA in centroblasts by Bcl6 Thus, we examined expression of

CD63 mRNA and protein in activated Bcl62/2B cells

restimu-lated with IL-21 or IL-4 Amounts of CD63 mRNA and protein

were strikingly elevated in activated Bcl62/2B cells restimulated

with IL-21 or IL-4 (Fig 5B, 5C) The amounts of CD63 mRNA

and protein in naive Bcl62/2B cells were also more than those in

naive WT B cells, suggesting CD63 as a molecular target of Bcl6

We looked for Bcl6-binding sequences in the CD63 gene Within

the 4.8-kb sequence spanning 21.0 kb upstream to +1.0 kb

downstream from the CD63 locus (MGI: 99529), four putative

Bcl6-binding sequences (BS1:2846 to 2838,

59-TTCTGTGAA-39; BS2: 1492 to 1500, 59-TTCCTGGAG-59-TTCTGTGAA-39; BS3: +2288 to +2296,

59-TTCCTAGAA-39 and +2409 to +2417, 59-TTCAAGGAA-39

from the CD63 transcription start site) were identified by using

match module of gene regulation (http://www.gene-regulation.com)

(Fig 5D) Then, we investigated Bcl6 binding and acetylation of

histone H3 lysine (K) 9 and K14 (Ac-H3) at each BS site of naive

Bcl62/2and WT B cells by ChIP assay Bcl6 binding was detected

at BS2 and BS3 of WT B cells Levels of Ac-H3 at BS2 and BS3 of Bcl62/2B cells were more than those of WT B cells although those

at BS1 were similar between them When we examined Bcl6

FIGURE 5 Bcl6 negatively regulates CD63 expression in activated

B cells A, Naive WT B cells were cultured with IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 for 4 d CXCL12 was added at day 2

of culture CXCR4 expression on and CD63 mRNA expression in these activated B cells 6, 24, and 48 h after addition were analyzed by flow cytometry and by real-time quantitative RT-PCR, respectively The number

in each histogram indicates the mean fluorescence intensity Data are presented as a representative of three independent experiments Results represent means 6 SD of triplicate culture B and C, Naive B cells from

WT (open bars) and Bcl62/2(KO, filled bars) mice were cultured with anti-IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 for 4 d IL-21

or IL-4 was sequentially added at day 2 of culture B, CD63 mRNA ex-pression in naive and these activated B cells was measured by real-time quantitative RT-PCR Results represent means 6 SD of triplicate culture Bars in figures represent mean values 6 SD *p , 0.05, **p , 0.01 C, CD63 protein in these B cells was detected by Western blot analysis Data are presented as a representative of three independent experiments D, Naive B cells from WT (open bars) and Bcl62/2(KO, filled bars) mice were cultured with anti-IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 for 3 d The top figure indicates the CD63 gene map (boxes; exons) and putative Bcl6/STAT (BS)-binding sites (triangles) Relative amounts of Ac-H3 at and Bcl6 binding to the BS sites were measured by ChIP assay Data are indicated by mean values 6 SD of triplicate real-time quantitative RT-PCR Results are presented as representative of three independent experiments *p , 0.05 N.S., not significant.

binding and Ac-H3 at the CD63 locus of activated Bcl62/2and WT

B cells, Bcl6 binding was diminished in activated WT B cells, and

the level of Ac-H3 increased at the locus in activated WT B cells

but not in activated Bcl62/2 B cells These results strongly

sug-gested that the CD63 gene is a molecular target of Bcl6

Bcl6 upregulates CXCR4 expression on activated B cells by

silencing the CD63 gene

We examined CXCR4 expression on activated B cells from Bcl62/2

mice Naive Bcl62/2 B cells were sequentially stimulated with

anti-IgM Abs and anti-CD40 mAbs plus IL-4 and with IL-21 or

IL-4 after a 2-d interval CXCR4 expression was downregulated

on activated Bcl62/2B cells after restimulation not only with

21 but also with 4 (Fig 6A) Although the number of the

IL-21–restimulated Bcl62/2B cells decreased after day 4 of culture

because of the differentiation to plasma cells and the apoptosis,

kinetic studies of activated Bcl62/2B cells clearly demonstrated

that restimulation with IL-21 or IL-4 induced proliferation of

CXCR4low B cells including IgG1 B cells (Fig 6B) When we

analyzed the amount of intracellular CXCR4 protein in activated

Bcl62/2B cells restimulated with IL-21 or IL-4, these activated

Bcl62/2B cells produced similar amounts of CXCR4 mRNA (Fig

6C) and protein (Fig 6D) compared with those of activated WT

B cells Furthermore, we added Dynasore to the culture to

de-termine whether or not Bcl6 deficiency affects CXCR4 in-ternalization Although CXCR4 expression on activated Bcl62/2

B cells restimulated with IL-21 was slightly increased by the addition of Dynasore, CXCR4 on activated Bcl62/2 B cells restimulated with IL-4 was not increased at all (Fig 6E)

To confirm the CD63-mediated CXCR4 downregulation on activated Bcl62/2B cells, we examined the effect of CD63 siRNA

on CXCR4 expression Naive Bcl62/2B cells were cultured with anti-IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 for 4

d Three types of predesigned CD63 siRNA or scramble siRNA were transfected to activated Bcl62/2B cells at day 2 of culture CXCR4 expression on Bcl62/2 B cells transfected with each CD63 siRNA increased compared with that on Bcl62/2B cells transfected with scramble siRNA at day 4 of culture (Fig 7A) We confirmed the downregulation of CD63 mRNA in CD63 siRNA (s63679)-transfected Bcl62/2B cells (Fig 7B)

Furthermore, we examined the effect of Bcl6 inhibitor on cell surface CXCR4 expression and CD63 mRNA expression in acti-vated WT B cells Naive WT B cells were cultured with anti-IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 for 4 d Bcl6 inhibitor was added to the WT B cell culture twice a day for 4 d CXCR4 expression was partially downregulated on activated

B cells cultured with the Bcl6 inhibitor (Fig 7C) CD63 mRNA expression increased in the activated B cells after addition, and the

FIGURE 6 CXCR4 expression is downregulated on activated Bcl62/2B cells in vitro Naive Bcl62/2(A–E) and WT (C, D) B cells were cultured with anti-IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 for 7 d IL-21 or IL-4 was added at day 2 of culture A, IgG1 and CXCR4 expression on activated B220+Bcl62/2B cells at day 4 of culture was analyzed by flow cytometry The numbers in the flow cytometry profile indicate the percentage of each gate Data are presented as a representative of five independent experiments B, Numbers of CXCR4highand CXCR4lowBcl62/2B cells in the culture were calculated by the flow cytometry profiles Open and filled circles indicate IL-4 and IL-21 restimulated B cells, respectively C, CXCR4 mRNA expression in activated B cells was measured by real-time quantitative RT-PCR Filled and open bars indicate Bcl62/2and WT B cells, respectively Results represent means 6 SD of triplicate culture Bars in figures represent mean values 6 SD N.S., not significant D, Cell surface and intracellular CXCR4 expression of activated B cells was analyzed by flow cytometry Filled and open histograms indicate Bcl62/2and WT B cells, respectively The isotype control of each CXCR4 staining is represented as an open histogram with a thin broken line The numbers in each histogram indicate the mean fluorescence intensity (isotype controls ,12) Data are presented as a representative of five independent experiments E, Dynasore (Dyn) was added in the culture at day

2 of culture CXCR4 expression on these activated Bcl62/2B cells was analyzed by flow cytometry Filled and open histograms indicate Bcl62/2B cells cultured with and without Dyn, respectively The numbers in each histogram indicate the mean fluorescence intensity Data are presented as a representative

of three independent experiments.

amounts of mRNA were correlated with the dose of Bcl6 inhibitor

(Fig 7D) The amount of CD23 mRNA, whose gene is a

molec-ular target of Bcl6 (23), also increased in the activated B cells

cultured with the Bcl6 inhibitor These results strongly suggested

that Bcl6 positively regulates CXCR4 expression on activated

B cells by silencing the CD63 gene

Discussion

CXCR4 expression on GC B cells plays an important role in

controlling dark and light zone segregation (4) CXCR4high

cen-troblasts localized in the dark zone differentiate to centrocytes,

downregulate CXCR4, and move to the light zone in GCs When

centrocytes were cultured in vitro without any stimulation,

CXCR4 was upregulated on centrocytes within 4 h after culture

(5) Thus, CXCR4 is actively downregulated on centrocytes

However, the mechanism of CXCR4 downregulation on

cen-trocytes is not fully understood Although CXCL12 stimulation

transiently downregulates CXCR4 expression on CD4 T cells

(29) and activated B cells, expression was completely recovered

on activated B cells within 24 h after stimulation Thus, it is

im-possible to explain the continuous downregulation of CXCR4 expression on centrocytes by CXCL12 stimulation A previous article reported that CXCR4 expression on GC B cells was reg-ulated by the level of its transcription (5) However, we have shown here that the amount of CXCR4 mRNA in centroblasts was similar to that in centrocytes of WT mice Although it has been proved that CXCR4 mRNA expression is mainly controlled by nuclear respiratory factor-1 and yin-yang 1 in CD4 T cells (9–11), neither nuclear respiratory factor-1 mRNA nor yin-yang 1 mRNA was induced in activated B cells or GC B cells (data not shown) These results strongly suggested that CXCR4 expression on GC

B cells is regulated at the post-transcriptional level

We showed two alternative mechanisms that downregulate CXCR4 expression on activated B cells First, we showed that IL-21 stimulation accelerated CXCR4 endocytosis in activated B cells by increasing GRK6 expression Because IL-21 produced by Tfh cells (3) is required for GC B cells to maintain the size of GCs (30) and for centrocytes to differentiate to long-lived plasma cells (31, 32), the IL-21–induced CXCR4 downregulation may explain the main-tenance of CXCR4 downregulation on centrocytes in the light zone Indeed, IL-21 stimulation downregulated CXCR4 expres-sion on FACS-isolated GC B cells However, the IL-21–mediated CXCR4 downregulation was not clearly detected on GC B cells from WT mice compared with that from IL-21R2/2mice at day 7 (data not shown), day 10, and day 14 (data not shown) after im-munization We tried to detect CXCR4 endocytosis in centrocytes

in vitro, but FACS-isolated GC B cells could not be cultured with Dynasore for the period of time long enough to detect CXCR4 upregulation Although we were not able to show clear results proving IL-21–induced CXCR4 downregulation in centrocytes

in vivo, IL-21 has a potential to control CXCR4 downregulation not only on activated B cells but also on GC B cells

Second, we showed that CD63, which traffics CXCR4 to late endosome in CD4 T cells (17), downregulates CXCR4 expression

on activated B cells The amount of CD63 mRNA was inversely correlated with that of Bcl6 mRNA in GC B cells, and the amount

of CD63 protein was strikingly augmented in activated Bcl62/2

B cells Bcl6 binding was detected on the CD63 gene locus of naive B cells Thus, the CD63 gene is a molecular target of Bcl6

It should be noted, however, that CD63 mRNA was detected in centroblasts, which express a large amount of Bcl6 CD63,

a ubiquitously expressed tetraspanin, may be required in centro-blasts Because the amount of CD63 mRNA in centrocytes was more than that in centroblasts, the larger amount of CD63 can contribute to the CXCR4 downregulation on centrocytes There-fore, IL-21–induced and CD63-mediated CXCR4 downregulation may contribute to maintain CXCR4 downregulation on centro-cytes in the light zone

We showed that IL-21 restimulation induced GRK6 mRNA to accelerate endocytosis of CXCR4 in activated B cells Because IL-21 stimulation does induce Bcl6 in activated B cells (19, 33, 34), CXCR4 expression might be upregulated on activated B cells

by Bcl6-mediated CD63 downregulation However, CD63 was not significantly downregulated in activated B cells restimulated with IL-21 compared with that in activated B cells without re-stimulation IL-21 stimulation also induces Blimp-1 in activated

B cells, and Blimp-1 mutually represses Bcl6 expression in acti-vated B cells (33) Indeed, the restimulation of actiacti-vated B cells with IL-21 induced Blimp-1 (19) and protected against the Bcl6-mediated downregulation of CD63 Therefore, the restimulation of activated B cells with IL-21 downregulates CXCR4 expression on activated B cells by acceleration of the GRK6-mediated endocy-tosis and CD63-induced endosome trafficking IL-21 acts as the inducer of Bcl6 in early phase of GC B cells such as centroblasts

FIGURE 7 Bcl6 positively regulates CXCR4 expression on activated

B cells Naive B cells from Bcl62/2and WT mice were cultured with

anti-IgM Abs and anti-CD40 mAbs plus a low dose of IL-4 for 4 d A and B,

CD63 siRNA (s63677, s63678, and s63679) or scramble siRNA was

transfected to these Bcl62/2 B cells at day 2 of culture A, CXCR4

ex-pression was measured by flow cytometry at day 4 of culture Filled and

open histograms indicate scramble siRNA and CD63 siRNA transfected

B cells, respectively Data are presented as a representative of three

in-dependent experiments B, CD63 mRNA expression in s63679-transfected

(filled bar) and scramble siRNA-transfected (Sc, open bar) B cells at day 3

of culture was measured by real-time quantitative RT-PCR Results

rep-resent means 6 SD of triplicate culture Bars in figures represent mean

values 6 SD *p , 0.05 C, Bcl6 inhibitor (100 nM) was administered in

the WT B cell culture twice a day for 4 d CXCR4 expression was

mea-sured by flow cytometry Filled and open histograms indicate B cells with

and without Bcl6 inhibitor administration, respectively Data are presented

as a representative of three independent experiments D, Bcl6 inhibitor was

added in the WT B cell culture at day 2 and day 2.5 of culture Expression

of CD63 and CD23 mRNA at day 3 of culture was measured by real-time

quantitative RT-PCR Results represent means 6 SD of triplicate culture.

Bars in figures represent mean values 6 SD *p , 0.05, **p , 0.01 N.S.,

not significant.

and as the inducer of Blimp-1 in the late phase of GC B cells such

as centrocytes (34) Thus, these two regulatory mechanisms can

cooperate together to maintain CXCR4 downregulation on

acti-vated B cells and probably on centrocytes

Signal transduction pathways of the IL-21–induced and the

CD63-mediated CXCR4 downregulation on activated B cells may

be new targets for therapy of HIV infection and WHIM syndrome

CXCR4 is a part of the receptor for HIV, and the downregulation

of CXCR4 on CD4 T cells protected against HIV infection (35)

Because these two regulatory mechanisms may work in CD4

T cells, activation of these two mechanisms in CD4 T cells could

protect against HIV infection In WHIM syndrome, heterozygous

truncating mutations in CXCR4 have been proposed to lead to

altered lymphocyte trafficking (36) Leukocytes from WHIM

patients show impaired association of GRK6 with CXCR4 and

delayed recruitment of Arrb2 to CXCR4 leading to slower

in-ternalization of the receptor (15) The impaired CXCR4

signal-ing in WHIM syndrome is thought to result in defective B-cell

functions (36) Thus, IL-21 stimulation induces GRK6 expression,

which may enhance GRK6–CXCR4 association to weaken the

abnormality of B-cell functions in WHIM patients

It has been shown that coengagement of CD3 and CD63 induces

a potent costimulatory signal in T cells (37), and that the

en-gagement of CD63 resulted in rapid translocation of MHC class II

molecules to the endocytic pathway in dendritic cells (38),

sug-gesting that CD63 downregulation participates in Ag presentation

on Ag-activated B cells We found that expression of MHC class II

on activated Bcl62/2B cells was lower than that on activated WT

B cells (data not shown) This observation suggests that the

du-ration of T–B cell contact is impaired in Bcl62/2mice Thus,

CD63 downregulation by Bcl6 may be important not only for

CXCR4 expression on centroblasts but also for Ag presentation on

Ag-activated B cells

In summary, we have found two novel mechanisms of CXCR4

downregulation on activated B cells These two mechanisms

may contribute to the maintenance of CXCR4 downregulation on

centrocytes Further study of these two mechanisms may provide

new insights to development of high-affinity memory B cells and

long-lived plasma cells in GCs

Acknowledgments

We thank Dr M Osawa (Chiba University), Dr A Iwama (Chiba University),

and H Satake for technical assistance, Dr M.J Grusby (Harvard School

of Public Health) for IL-21R2/2mice, Dr D Tumes (Chiba University) for

intensive proofreading, and S Nakamura for secretarial assistance.

Disclosures

The authors have no financial conflicts of interest.

References

1 Manz, R A., A Thiel, and A Radbruch 1997 Lifetime of plasma cells in the

bone marrow Nature 388: 133–134.

2 Han, S., K Hathcock, B Zheng, T B Kepler, R Hodes, and G Kelsoe 1995.

Cellular interaction in germinal centers Roles of CD40 ligand and B7-2 in

established germinal centers J Immunol 155: 556–567.

3 Chtanova, T., S G Tangye, R Newton, N Frank, M R Hodge, M S Rolph,

and C R Mackay 2004 T follicular helper cells express a distinctive

tran-scriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide

help for B cells J Immunol 173: 68–78.

4 Allen, C D., K M Ansel, C Low, R Lesley, H Tamamura, N Fujii, and

J G Cyster 2004 Germinal center dark and light zone organization is mediated

by CXCR4 and CXCR5 Nat Immunol 5: 943–952.

5 Caron, G., S Le Gallou, T Lamy, K Tarte, and T Fest 2009 CXCR4

ex-pression functionally discriminates centroblasts versus centrocytes within human

germinal center B cells J Immunol 182: 7595–7602.

6 Feng, Y., C C Broder, P E Kennedy, and E A Berger 1996 HIV-1 entry

cofactor: functional cDNA cloning of a seven-transmembrane, G

protein-7 Zlotnik, A 2006 Involvement of chemokine receptors in organ-specific me-tastasis Contrib Microbiol 13: 191–199.

8 Tsutsumi, H., T Tanaka, N Ohashi, H Masuno, H Tamamura, K Hiramatsu,

T Araki, S Ueda, S Oishi, and N Fujii 2007 Therapeutic potential of the chemokine receptor CXCR4 antagonists as multifunctional agents Biopolymers 88: 279–289.

9 Moriuchi, M., H Moriuchi, W Turner, and A S Fauci 1997 Cloning and analysis of the promoter region of CXCR4, a coreceptor for HIV-1 entry J Immunol 159: 4322–4329.

10 Wegner, S A., P K Ehrenberg, G Chang, D E Dayhoff, A L Sleeker, and

N L Michael 1998 Genomic organization and functional characterization of the chemokine receptor CXCR4, a major entry co-receptor for human immu-nodeficiency virus type 1 J Biol Chem 273: 4754–4760.

11 Moriuchi, M., H Moriuchi, D M Margolis, and A S Fauci 1999 USF/c-Myc enhances, while Yin-Yang 1 suppresses, the promoter activity of CXCR4,

a coreceptor for HIV-1 entry J Immunol 162: 5986–5992.

12 Jourdan, P., J P Vendrell, M F Huguet, M Segondy, J Bousquet, J Pe`ne, and

H Yssel 2000 Cytokines and cell surface molecules independently induce CXCR4 expression on CD4+ CCR7+ human memory T cells J Immunol 165: 716–724.

13 Fong, A M., R T Premont, R M Richardson, Y R Yu, R J Lefkowitz, and

D D Patel 2002 Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice Proc Natl Acad Sci USA 99: 7478–7483.

14 Woerner, B M., N M Warrington, A L Kung, A Perry, and J B Rubin 2005 Widespread CXCR4 activation in astrocytomas revealed by phospho-CXCR4-specific antibodies Cancer Res 65: 11392–11399.

15 McCormick, P J., M Segarra, P Gasperini, A V Gulino, and G Tosato 2009 Impaired recruitment of Grk6 and b-Arrestin 2 causes delayed internalization and desensitization of a WHIM syndrome-associated CXCR4 mutant receptor PLoS ONE 4: e8102

16 Marchese, A., C Raiborg, F Santini, J H Keen, H Stenmark, and J L Benovic.

2003 The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4 Dev Cell 5: 709–722.

17 Yoshida, T., Y Kawano, K Sato, Y Ando, J Aoki, Y Miura, J Komano,

Y Tanaka, and Y Koyanagi 2008 A CD63 mutant inhibits T-cell tropic human immunodeficiency virus type 1 entry by disrupting CXCR4 trafficking to the plasma membrane Traffic 9: 540–558.

18 Yoshida, T., H Ebina, and Y Koyanagi 2009 N-linked glycan-dependent in-teraction of CD63 with CXCR4 at the Golgi apparatus induces downregulation

of CXCR4 Microbiol Immunol 53: 629–635.

19 Saito, T., D Kitayama, A Sakamoto, N Tsuruoka, M Arima, M Hatano,

M Miyazaki, and T Tokuhisa 2008 Effective collaboration between IL-4 and IL-21 on B cell activation Immunobiology 213: 545–555.

20 Yoshida, T., T Fukuda, M Hatano, H Koseki, S Okabe, K Ishibashi, S Kojima,

M Arima, I Komuro, G Ishii, et al 1999 The role of Bcl6 in mature cardiac myocytes Cardiovasc Res 42: 670–679.

21 Ozaki, K., R Spolski, C G Feng, C F Qi, J Cheng, A Sher, H C Morse, III,

C Liu, P L Schwartzberg, and W J Leonard 2002 A critical role for IL-21 in regulating immunoglobulin production Science 298: 1630–1634.

22 Karasuyama, H., and F Melchers 1988 Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors Eur J Immunol 18: 97–104.

23 Polo, J M., T Dell’Oso, S M Ranuncolo, L Cerchietti, D Beck, G F Da Silva,

G G Prive, J D Licht, and A Melnick 2004 Specific peptide interference reveals BCL6 transcriptional and oncogenic mechanisms in B-cell lymphoma cells Nat Med 10: 1329–1335.

24 Hasbold, J., A B Lyons, M R Kehry, and P D Hodgkin 1998 Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40 ligand and IL-4 Eur J Immunol 28: 1040–1051.

25 Takamori, M., M Hatano, M Arima, A Sakamoto, L Fujimura, T Hartatik,

T Kuriyama, and T Tokuhisa 2004 BAZF is required for activation of naive CD4 T cells by TCR triggering Int Immunol 16: 1439–1449.

26 Watanabe-Takano, H., K Takano, E Keduka, and T Endo 2010 M-Ras is activated by bone morphogenetic protein-2 and participates in osteoblastic de-termination, differentiation, and transdifferentiation Exp Cell Res 316: 477– 490.

27 Arima, M., H Toyama, H Ichii, S Kojima, S Okada, M Hatano, G Cheng,

M Kubo, T Fukuda, and T Tokuhisa 2002 A putative silencer element in the IL-5 gene recognized by Bcl6 J Immunol 169: 829–836.

28 Livak, K J., and T D Schmittgen 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method Methods 25: 402–408.

29 Tarasova, N I., R H Stauber, and C J Michejda 1998 Spontaneous and ligand-induced trafficking of CXC-chemokine receptor 4 J Biol Chem 273: 15883–15886.

30 Linterman, M A., L Beaton, D Yu, R R Ramiscal, M Srivastava, J J Hogan,

N K Verma, M J Smyth, R J Rigby, and C G Vinuesa 2010 IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses J Exp Med 207: 353–363.

31 Zotos, D., J M Coquet, Y Zhang, A Light, K D’Costa, A Kallies,

L M Corcoran, D I Godfrey, K M Toellner, M J Smyth, et al 2010 IL-21 regulates germinal center B cell differentiation and proliferation through

a B cell-intrinsic mechanism J Exp Med 207: 365–378.

32 Ozaki, K., R Spolski, R Ettinger, H P Kim, G Wang, C F Qi, P Hwu,

D J Shaffer, S Akilesh, D C Roopenian, et al 2004 Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1

33 Tsuruoka, N., M Arima, E Arguni, T Saito, D Kitayama, A Sakamoto,

M Hatano, and T Tokuhisa 2007 Bcl6 is required for the IL-4-mediated rescue

of the B cells from apoptosis induced by IL-21 Immunol Lett 110: 145–151.

34 Good-Jacobson, K L., and M J Shlomchik 2010 Plasticity and heterogeneity

in the generation of memory B cells and long-lived plasma cells: the influence of

germinal center interactions and dynamics J Immunol 185: 3117–3125.

35 BouHamdan, M., D S Strayer, D Wei, M Mukhtar, L X Duan, J Hoxie, and

R J Pomerantz 2001 Inhibition of HIV-1 infection by down-regulation of the

CXCR4 co-receptor using an intracellular single chain variable fragment against

CXCR4 Gene Ther 8: 408–418.

36 Mc Guire, P J., C Cunningham-Rundles, H Ochs, and G A Diaz 2010 Oli-goclonality, impaired class switch and B-cell memory responses in WHIM syndrome Clin Immunol 135: 412–421.

37 Pfistershammer, K., O Majdic, J Sto¨ckl, G Zlabinger, S Kirchberger,

P Steinberger, and W Knapp 2004 CD63 as an activation-linked T cell co-stimulatory element J Immunol 173: 6000–6008.

38 Mantegazza, A R., M M Barrio, S Moutel, L Bover, M Weck, P Brossart,

J L Teillaud, and J Mordoh 2004 CD63 tetraspanin slows down cell migration and translocates to the endosomal-lysosomal-MIICs route after extracellular stimuli in human immature dendritic cells Blood 104: 1183–1190.