Inhibition of Terminal Differentiation of B
Cells Mediated by CD27 and CD40 Involves
Signaling through JNK
This information is current as
of June 18, 2017.
Shuchismita Satpathy, Gautam N. Shenoy, Sheetal Kaw,
Tushar Vaidya, Vineeta Bal, Satyajit Rath and Anna George
J Immunol 2010; 185:6499-6507; Prepublished online 25
October 2010;
doi: 10.4049/jimmunol.0903229
http://www.jimmunol.org/content/185/11/6499
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Supplementary
Material
The Journal of Immunology
Inhibition of Terminal Differentiation of B Cells Mediated by
CD27 and CD40 Involves Signaling through JNK
Shuchismita Satpathy,*,1,2 Gautam N. Shenoy,*,1 Sheetal Kaw,*,1 Tushar Vaidya,†
Vineeta Bal,*,3 Satyajit Rath,*,3 and Anna George*,3
T
-dependent B cell responses in peripheral lymphoid organs
are initiated at the border of T and B cell zones, leading to
the establishment of primary foci where clonal expansion
occurs and isotype switching may be initiated (1). Although many
proliferating cells die, some undergo terminal differentiation to
form relatively short-lived plasma cells (2) and a few others
migrate into the B cell follicles and establish germinal centers
(GCs). Within the GC, B cells proliferate extensively, upregulate
activation-induced deaminase (AID), and undergo isotype switching and somatic hypermutation (3–5). Although most mutations
lead to BCRs with unchanged or lower affinity, a few high-affinity
variants are also generated, and they get selected as they compete
*National Institute of Immunology, New Delhi 110067; and †Center for Cellular and
Molecular Biology, Hyderabad 500007, India
1
S.S., G.N.S., and S.K. contributed equally to this work.
2
Current address: Department of Microbiology and Immunology, University of
Louisville Health Sciences Center, Louisville, KY.
3
V.B., S.R., and A.G. contributed equally to this work.
Received for publication October 2, 2009. Accepted for publication September 21,
2010.
This work was supported in part by grants from the Department of Science and
Technology and the Department of Biotechnology, Government of India (to A.G.,
S.R., V.B., and T.V.). The National Institute of Immunology is supported by the
Department of Biotechnology, Government of India. The Center for Cellular and
Molecular Biology is supported by the Council for Scientific and Industrial Research,
Government of India.
Address correspondence and reprint requests to Anna George, National Institute of
Immunology, Aruna Asaf Ali Road, New Delhi 110067, India. E-mail address: anna@
nii.res.in
The online version of this article contains supplemental material.
Abbreviations used in this paper: D, deletion; AID, activation-induced deaminase;
Bach2, BTB and CNC homology1, basic leucine zipper transcription factor 2; BCL6, B cell leukemia/lymphoma-6; BLIMP-1, B lymphocyte induced maturation protein
1; EGFP, enhanced GFP; FL, full-length; GC, germinal center; IRF4, IFN regulatory
factor 4; MFI, mean fluorescence intensity; Mitf, microphthalmia-associated transcription factor; PAX5, paired box gene 5; PI, propidium iodide; pJNK, phosphorylated JNK; TRAF, TNFR-associated factor; WCL, whole-cell lysates; XBP-1, X-box
binding protein 1.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903229
successfully for T cell help and for Ag presented on follicular
dendritic cells (6, 7). Whereas the GC environment is conducive to
rapid and sustained B cell proliferation, some cells manage to
exit cell cycle and to differentiate either into relatively long-lived
plasma cells or into memory cells, and the signaling events that
lead to this exit remain unclear (8–10).
Plasma cells downregulate most surface molecules that are
synonymous with B cell identity but upregulate CD138, CXCR4,
and CD44 [molecules that facilitate migration to and retention in
the bone marrow (11–13)] and the protein synthesis machinery
required for high-level Ab secretion (14). Memory cells, however,
retain high-level expression of B220, BCR, CD19, CD86, and
MHC class II and can recirculate to peripheral lymphoid organs. A
large number of transcription factors, including B cell leukemia/
lymphoma-6 (BCL-6), B lymphocyte induced maturation protein
1 (BLIMP-1), paired box gene 5 (PAX5), X-box binding protein 1
(XBP-1), microphthalmia-associated transcription factor (Mitf),
and IFN regulatory factor 4 (IRF4) have been implicated in
commitment to the plasma cell lineage (15). However, the precise
signaling pathways and transcriptional events that determine cell
fate decision, especially for memory generation in the GC, remain
uncharacterized (16).
We have shown previously that immunization of mice under
cover of anti-CD27 Ab skewed the commitment of activated cells to
the memory pool and inhibited plasma cell generation without
affecting B cell activation, proliferation, or survival (17, 18). CD40
ligation also enhanced commitment of responding B cells to the
memory pool; however, it affected multiple events including proliferation, survival, and isotype switching after B cell activation.
In this study, we have attempted to characterize the signaling
events downstream of CD27 in B cells and to determine whether
inhibition of terminal differentiation by CD27 and CD40 involves
similar pathways. We report that of the two putative TNFRassociated factor (TRAF) binding domains present in the cytoplasmic tail of CD27 (19, 20), the region PIQED and specifically
residues P and D are required for inhibition of plasma cell generation after CD27 ligation. We also report that JNK is an essential
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B cells responding to cognate Ag in vivo undergo clonal expansion that is followed by differentiation into Ab-secreting plasma cells
or into quiescent restimulable memory. Both these events occur in the germinal center and require that cells exit from proliferation,
but the signals that lead to one or the other of these mutually exclusive differentiation pathways have not been definitively characterized. Previous experiments have shown that signals transduced through the TNFRs CD27 and CD40 at the time of B cell
stimulation in vitro or in vivo can influence this cell fate decision by inhibiting terminal differentiation and promoting memory.
In this study, we show that the PIQED domain of the cytoplasmic tail of murine CD27 and the adapter molecule TNFR-associated
factor 2 are involved in this effect. Using pharmacological inhibitors of signaling intermediates, we identify JNK as being necessary
and sufficient for the observed inhibition of terminal differentiation. While JNK is involved downstream of CD40, inhibition of the
MEK pathway can also partially restore plasma cell generation, indicating that both signaling intermediates may be involved. We
also show that inhibition of induction of IFN regulatory factor 4 and B lymphocyte induced maturation protein 1 are downstream
events common to both receptors. The Journal of Immunology, 2010, 185: 6499–6507.
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SIGNALING THROUGH JNK INHIBITS PLASMA CELL GENERATION
signaling intermediate downstream of CD27 and that the transcription factors IRF4 and BLIMP-1, but not BTB and CNC homology 1, basic leucine zipper transcription factor2 (Bach2), are
modulated by CD27 ligation. Yet inhibition of plasma cell generation by CD40 ligation involves JNK as well as MEK; however,
they appear to lead to modulation of the same transcription factors
as are modulated after CD27 ligation. Our data indicate that both
TNFRs influence B cell differentiation via similar signaling
pathways and that CD27 may prove valuable in the further understanding of lineage commitment after B cell activation.
Materials and Methods
Mice
C57BL/6 and BALB/c mice used in the study were obtained from The
Jackson Laboratory (Bar Harbor, ME) and were bred and maintained in the
Small Animal Facility of the National Institute of Immunology (New Delhi,
India). Mice were used for experiments at 8–12 wk of age. Approval from
the institutional animal ethics committee was obtained for all experimental
procedures involving animals.
Cloning of murine CD27 and its C-terminal deletion mutants
Reagents
The following reagents were used: LPS, Triton X-100, sodium orthovanadate, PMSF, KCl, EDTA (Sigma, St. Louis, MO), Tris base (Amresco,
Solon, OH), Blotto (Santa Cruz Biotechnology, Santa Cruz, CA), leupeptin
(Boehringer Ingelheim India, Mumbai, India), aprotinin (Merck India,
Mumbai, India), azide-free anti-myc (clone 9E10; Roche Applied Sciences, Mannheim, Germany), anti-CD40 (clone 1C10; eBioscience, San
Diego, CA), anti-CD8 (clone 53-6.7; BD Biosciences, San Jose, CA),
PE-streptavidin, fluorescein-streptavidin, FITC/PE donkey anti-rabbit IgG
(Jackson ImmunoResearch Laboratories, Westgrove, PA), propidium iodide (PI; Sigma), CFSE (Molecular Probes, Eugene, OR), fluorescein/
biotin/PE-Cy5 anti-B220, PE anti-CD138, CD44, CD27, CD138, BLIMP-1,
Bach2, IRF4 (BD Biosciences or eBioscience), and anti-phospho SAPK/
JNK (Thr183/Tyr185; Cell Signaling Technology, Danvers, MA). ECL reagent and x-ray films were from Amersham Pharmacia Biotech, Little
Chalfont, U.K.
Inhibitors used in culture were obtained from Calbiochem or Sigma and
included JNK-1, JNK-II, JNK-3 (30–3 mM), rottlerin, AG1478, bisindolylmaleimide I (10–0.1 mM), LY294002, AG490, SB203580, genistein
(30–0.3 mM), N-acetylcysteine (30–0.3 mM), olomoucine (100–0.1 mM),
and PD98059 and caffeine (10–1 mM).
Primers
For cloning full-length (FL) CD27: forward 59-CGGAAATTCATGGCATGGCCACCTCCCTA-39, reverse 59-CGGGATCCAGGGTAGAAAGCAGGCTCGG-39. For generating myc-tagged FL CD27: forward 59-GGGGGGCTCGAGCGGATGGAGCAAAAACTCATCTCTGAAGAAGATCTGATGGCATGGCCACCTCCCTA-39, reverse 59-GGGGGGGATCCTCAAGGGTAGAAAGCAGGCTCGG-39; CD27 deletion (D)10: reverse 59GGGGGGGATCCTCAGTCCTCCTGGATAGGGATAG-39; CD27D16: reverse 59-GGGGGGGATCCTCAAGCACTGCCCTCCTCTTCC-39; CD27D23:
reverse 59-GGGGGGGATCCTCAGGGGCAGCTGTAAGGACAAG-39 (all
from Bio Basic). Point mutants in the PIQED domain of the CD27 tail were
generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene,
Cedar Creek, TX) as per the manufacturer’s instructions. Primers used for mutagenesis were as follows: forward 59-GAGGGCAGTGCTATCGCTATCCAGGAGGACTAC-39, reverse 59-GTAGTCCTCCTGGATAGCGATAGCACTGCCCTC-39 for proline to alanine; forward 59-GCAGTGCTATCCCTATCGCGGAGGACTACCGGAAACC-39, reverse 59-GGTTTCCGGTAGTCCTCCGCGATAGGGATAGCACTGC-39 for glutamine to alanine; forward 59-GCTATCCCTATCCAGGAGGCCTACCGGAAACCCGAGCC-39, reverse 59-GGCTCGGGTTTCCGGTAGGCCTCCTGGATAGGGATAGC-39 for aspartic acid to
alanine (Bioserve Biotech, Beltsville, MD). Mutations were confirmed by sequencing with forward 59-AGCTCGGACTGCATCCGGATC-39.
Vectors
pPCR-Script Amp (Stratagene) and pGEM-T Easy (Promega, Madison, WI)
were used for cloning, and pLNCX2-IRES-enhanced GFP (EGFP) was
used for eukaryotic expression. The latter was generated by insertion of the
IRES-EGFP cassette with flanking Xho1 and Not1 sites from pIRES-EGFP
(Clontech, Mountainview, CA) into pLNCX2 (Clontech).
B cells
Single-cell suspensions of cells from spleen were obtained by mechanical
disruption, and erythrocytes in the splenocyte population were lysed by
treatment of the cell pellet with Gey’s solution. B cells were enriched by
treatment of the cell suspension with biotinylated anti-IgD (Southern
Biotechnology, Birmingham, AL) followed by streptavidin microbeads
(Miltenyi Biotec, Auburn, CA) or with anti-B220 magnetic beads (Miltenyi Biotec) and separated on MACS columns (Miltenyi Biotec)
according to the manufacturer’s instructions. Purified cells were routinely
$90% pure by flow cytometric analysis.
Cells were stimulated in 96-well flat-bottom plates or in 24- or 6-well
plates (BD Falcon, Franklin Lakes, NJ) with 10 mg/ml LPS in Click’s
medium (Irvine Scientific, Santa Ana, CA) or RPMI 1640 (Biological
Industries, San Diego, CA) supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mM 2-ME, and penicillin (100 U/ml) and streptomycin (100
mg/ml) (Life Technologies, Carlsbad, CA) in the presence or absence of
the indicated amounts of anti-CD40. Wherever necessary, viable cells were
isolated on a Ficoll-Hypaque gradient (Cedarlane, Hornby, Ontario, Canada) before further analysis. To assess proliferation, cells were labeled
with 10 mM CFSE (Molecular Probes) before stimulation, and 72–96 h
later, CFSE dilution was estimated after excluding dead cells with 1 mg/ml
PI.
For B cell transfection, cells were activated with 10 mg/ml LPS for 48 h,
washed, and seeded at a density of 5 3 105/ml/well of OptiMEM in 6-well
plates and transfected with plasmid DNA using TransIT-LT or TransITExpress (Mirus Bio, Madison, WI) transfection reagent, according to the
manufacturer’s recommendations. OptiMEM was replaced with complete
medium containing LPS 4 h later, and 16 h after transfection, 10 mg/ml
anti-myc Ab was added to ligate CD27 on transfected cells. Cells were
analyzed by flow cytometry after a further 48 h.
For retroviral transduction of primary B cells, the retroviral packaging
cell lines Plat-E (Cell Biolabs, Cambridge, U.K.) or RetroPack PT67
(Clontech) were transfected with 4 mg plasmid DNA using FuGENE 6
(Roche) at a DNA/reagent ratio of 3:2 in OptiMEM in 6-well plates, and
medium replaced 12 h later as described earlier. The efficiency of transfection was checked 24 h later and was routinely .60%. Culture medium
was replaced at this time, the plates transferred to 32 C for virus production, and the supernatant harvested 24 h later and filtered to remove cell
debris. LPS-activated B cells were spin-infected with virus supernatant in
the presence of 8 mg/ml Sequabrene (Sigma) and anti-myc added to ligate
CD27 on transfected cells.
Immunoprecipitation
The B cell line A20 was spin-infected with virus expressing FL CD27 or
various deletion mutants in the presence of 12 mg/ml Sequabrene, and G418
(1 mg/ml; Sigma) was added 24 h later. Two weeks into selection, more
than 95% of cells expressed GFP as determined by flow cytometry. The
stable transductants were either left unligated or ligated with 10 mg/ml
anti-myc for 15 or 30 min. Cells were washed twice with cold PBS after
which they were lysed in TKM buffer (1% Triton X-100, 50 mM Tris
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Splenocytes were stimulated with 4 mg/ml Con A (Sigma) for 48 h, CD27
induction confirmed by flow cytometry, RNA extracted (TRIzol; Sigma),
and 2 mg total RNA used to synthesize cDNA with oligo-deoxythymine
using First Strand cDNA synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. The 752-bp CD27 was
then PCR-amplified from total cDNA using gene-specific primers, with
EcoR1 and BamH1 sites incorporated into the forward and reverse primers,
respectively. The amplicon was resolved on an agarose gel, eluted, and
purified using QIAquick Gel Extraction kit (Qiagen, Valencia, CA), and
blunt-end ligated into an SrfI-digested pPCR-Script vector (PCR-Script
Amp cloning kit; Stratagene) by overnight incubation. Competent cells
(DH5a, Escherichia coli) were transformed with the ligation mixture and
plated on Luria–Bertani agar (Difco Laboratories, Detroit, MI; BD Biosciences) plates containing 100 mg/ml ampicillin (Sigma). Single colonies
were screened by plasmid DNA extraction (Qiagen columns), insert
checked by digestion with EcoR1 and BamH1, and confirmed by sequencing (University of Delhi, South campus facility, New Delhi, India).
Myc-tagged FL CD27 and its mutants were PCR-amplified from pPCRScript Amp containing CD27 DNA using appropriate forward and reverse
primers containing Xho1 and BamH1 sites, respectively. The amplicons
were cloned into pGEM-T Easy, the clones obtained were sequenced
(LabIndia Systems, New Delhi, India), and the constructs subcloned into
the Xho1-BamH1 sites of the LNCX2-IRES-EGFP vector.
The Journal of Immunology
[pH 7.4], 25 mM KCl, 1 mM EDTA) supplemented with protease inhibitors
(10 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mM sodium orthovanadate, 1
mM PMSF) for 30 min on ice. Lysates were immunoprecipitated with antimyc Ab on Protein G beads (Amersham Pharmacia) and proteins resolved
by SDS-PAGE and transferred to nitrocellulose membrane (mdi; Membrane
Technologies, Ambala Cantonment, India). Membranes were blocked with
5% Blotto and probed with Abs specific for TRAFs 2, 3, and 5 (Santa Cruz
Biotechnology). Blots were stripped (2% SDS, 12.5 mM Tris [pH 6.0],
385 mM 2-ME) and probed with anti-myc Ab (BD Biosciences) and bands
visualized by ECL. HRP-linked secondary Abs (anti-rabbit IgG and antimouse IgG) were purchased from Santa Cruz Biotechnology and Cell
Signaling Technology, respectively.
Flow cytometry
Statistical analysis
Data were analyzed by Student t test.
Results
CD27 signaling in primary B cells inhibits plasma cell
generation
We have previously shown that ligation of CD27 on activated B
cells with an Ab inhibits plasma cell generation in vitro and in
vivo (17, 18). To elucidate the mechanisms underlying this inhibition, we cloned myc-tagged FL CD27 into a bicistronic EGFPexpressing retroviral vector for transfection or retroviral transduction of primary B cells (see Materials and Methods). The GFP
tag was included to enable detection of transfected cells by flow
cytometry, and the myc tag was included to enable specific ligation of the transfected molecule with an anti-myc Ab. Expression
of myc and CD27 on GFP+ transfectants was confirmed, and relative levels of CD27 on the transfected cells relative to endogenous levels were found to be high (Fig. 1A, 1B and microscopy
data not shown). The construct was then transfected into primary
B cells stimulated for 48 h with LPS, and 12 h later, anti-myc Ab
was added to ligate transfected CD27 molecules. As shown in Fig.
1C, ligation of transfected FL CD27 led to near-complete abrogation of plasma cell generation, as read out by surface expression
of CD138 and of intracellular BLIMP-1. When FL CD27 was
transfected into cells and left unligated, no effect on plasma cell
generation was seen, indicating that overexpression of CD27 had
no deleterious effects (Supplemental Fig. 1). The transfection data
reproduce what we have reported earlier with anti-CD27 Ab and
are therefore consistent with the physiological possibility indicated in our earlier data of a role for CD27 in skewing cell fate
determination away from terminal differentiation and toward the
memory lineage (17, 18).
The PIQED region of the CD27 tail is involved in inhibition of
terminal differentiation
Signal transduction downstream of CD27 ligation requires the recruitment of TRAF molecules. The cytoplasmic domain of CD27
contains two putative TRAF-binding domains: a PIQED domain,
which could recruit the activating TRAFs 2, 3, and 5, and an EEEG
domain, which could recruit TRAFs 1 and 2 (19, 20). To elucidate
which of these regions was involved in the signaling in B cells,
we next cloned three deletion mutants into the EGFP-expressing
vector (19) (Supplemental Fig. 2). The D-10 mutant contains both
domains, D-16 lacks the PIQED domain, and D-23 lacks both
PIQED and EEEG domains. The myc tag enabled ligation of the
mutant CD27 molecule with an anti-myc Ab, leaving endogenous
CD27 molecules untouched. All transfected cells expressed GFP
and myc (Fig. 2A and microscopy data not shown). The constructs
were then transfected into primary B cells stimulated for 48 h with
LPS, and 12 h later, anti-myc Ab was added to ligate the transfected CD27 molecules. Viable cells were isolated on FicollHypaque (Cedarlane) gradients 48 h later and stained for B220
and CD138. Plasma cell frequencies were then assessed on gated
GFP+ cells. As shown in Fig. 2B, and as expected from Fig. 1B,
ligation of transfected FL CD27 led to near-complete abrogation
of plasma cell generation. No inhibition was seen after ligation of
the D-23 mutant, which lacks both domains, or with the D-16
molecule, which lacks only the PIQED domain, indicating that
this region and not the EEEG domain is involved in signaling. Partial inhibition was seen after ligation of the D-10 molecule, which
contains both domains. These data indicate that the PIQED domain in the cytoplasmic tail of CD27 is likely involved in the
recruitment of factors responsible for signaling events that lead to
inhibition of terminal differentiation. To confirm this, we generated point mutants in the PIQED domain by mutating residues
P, Q, or D to A. The resultant constructs were cloned into a retroviral vector as before and transduced into LPS-stimulated B cells.
Ligation of the Q/A mutant led to abrogation of plasma cell generation, as with the FL molecule (Fig. 3). However, ligation of either the P/A or the D/A mutant had no effect, indicating that these
two residues are critical for recruitment of adapters to the cytosolic
tail of CD27.
TRAF2 is recruited to the CD27 tail
To identify specific TRAFs recruited to the PIQED domain of the
CD27 tail, we transduced the B cell line A20 with the FL CD27
construct and the D-10, the Q/A, and the D/A mutants, and stable
lines expressing GFP were generated by selection in G418. Cells
were stimulated and immunoprecipitated with anti-myc Ab and
blots probed with Abs specific for various TRAFs. We found (Fig.
4A) that TRAF2 was recruited to the FL molecule as well as to the
Q/A mutant. Recruitment to the D-10 mutant was partial, and there
was no recruitment to the D/A mutant.
CD27 signals through JNK in primary B cells
To identify the signaling molecules involved downstream of CD27
ligation, we transfected FL CD27 into primary B cells and ligated
myc in the presence or absence of a range of pharmacological
inhibitors. We reasoned that pharmacological inhibition of a relevant
signaling pathway downstream of CD27 would reverse the effect of
CD27 ligation and lead to equivalent proportions of CD138+ plasma
cells in the GFP2 and GFP+ populations. After testing a wide range
of inhibitor concentrations and identifying a range that had no effect on LPS-mediated cell proliferation or differentiation (data
not shown), we found that addition of JNK-II inhibitor led to a
dose-dependent reversal of the effect of CD27 ligation (Fig. 4B).
Other inhibitors tested (rottlerin, AG1478, bisindolylmaleimide-I,
LY294002, AG490, SB203580, genistein, N-acetyl cysteine, olomoucine, and PD98059) had no effect and failed to restore LPSinduced plasma cell generation at concentrations that did not otherwise affect B cell activation, proliferation, or differentiation
(Supplemental Fig. 3). Ligation of FL CD27 on retrovirally transduced B cells also led to inhibition of induction of IRF4, and the
induction was restored when ligation was done in the presence of
JNK inhibitor (Fig. 4C).
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Cells were incubated with appropriate staining reagents in buffer containing 0.1% sodium azide (Sigma) and 1% FBS for 45 min on ice. Samples
were run on a BD-LSR or FACS Aria (BD Biosciences) flow cytometer. For
determination of cell cycle progression, cells in cold PBS were permeabilized with ice-cold 70% ethanol and treated with 5 mg/ml PI in PBS. For
intracellular staining, cells were stained for surface markers and permeabilized with 100 ml Cytoperm/Cytofix buffer (BD Biosciences) on ice
for 15 min. Abs directed against intracellular markers were then added,
diluted in Permwash buffer (BD Biosciences). Data were analyzed with
FlowJo software (Tree Star, San Carlos, CA).
6501
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SIGNALING THROUGH JNK INHIBITS PLASMA CELL GENERATION
Inhibition of plasma cell generation by CD40 ligation involves
JNK, MEK, and IRF4 but not Bach2
Discussion
FIGURE 1. Expression of cloned myc-tagged, EGFP-expressing FL
CD27. A, Coexpression of CD27 and myc with GFP on PT67 cells or LPSactivated B cells respectively transfected with myc-CD27-EGFP vector
(CD27) or empty vector (vector). Representative of two independent
experiments. B, CD27 levels on ex vivo B cells (shaded histogram) and on
B cells stimulated with 10 mg/ml LPS for 48 h. C, Gating for GFP+ and
GFP2 cells 48 h after retroviral transduction of FL CD27. D, Relative
expression of endogenous CD27 on ex vivo B cells (shaded histogram) and
on B cells 48 h after stimulation with LPS (unbroken line) both gated as in
B, and of retrovirally transduced CD27 96 h into LPS stimulation, but 48 h
Plasma cell differentiation is initiated after activation of B cells by
specific Ags or by LPS. The precise mechanisms leading to plasma
after transduction (hatched line, from the GFP+ gate in C). The dotted line
represents endogenous levels of CD27 at 96 h (from the GFP+ gate in C).
E, Abrogation of plasma cell generation by myc ligation. Primary B cells
were transfected with reagent alone, empty vector, or FL CD27 (as labeled), treated with anti-myc Ab and stained for surface CD138 and intracellular BLIMP-1. One experiment representative of three is shown.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
CD40 is the other TNFR expressed on B cells that is known to
inhibit terminal differentiation and to enhance memory generation
(17, 18, 21). We tested the effect of CD40 ligation on primary
B cells by addition of stimulatory anti-CD40 Ab to LPS-stimulated cultures. As seen in Fig. 5, addition of anti-CD40 leads
to inhibition of terminal differentiation in a dose-dependent
fashion. At the higher concentrations, however, the Ab also induced prolific expansion of B cells (18) (data not shown), and so
we used 4 mg/ml for all experiments. We then tested pharmacological inhibitors of signaling intermediates for their ability to
reverse the CD40-mediated inhibition, and we found that three
different inhibitors of JNK were able to partially restore plasma
cell generation (Fig. 6A). As expected, and as seen with CD27
ligation, abrogation of plasma cell generation was accompanied
by reduction in levels of intracellular BLIMP-1 (Fig. 7). Proliferation, as assessed by CFSE dilution, was similar in the presence or absence of inhibitor (Supplemental Fig. 4), and similar
proportions of live cells were seen in the two groups (45.8 versus
45.2%), indicating that the concentrations of inhibitor used did not
inhibit proliferation or induce apoptosis. Various other inhibitors
were tested, and apart from the MEK inhibitor PD98059, they had
minimal effects on rescue of terminal differentiation in the presence of anti-CD40 (Supplemental Fig. 5A). To show more directly
that JNK is involved downstream of the two receptors, we looked
for phosphorylation of the enzyme after ligation of CD27 or
CD40. As shown in Fig. 6B, JNK is phosphorylated after treatment of primary B cells with anti-CD40 as well as after treatment
of A20 cells stably expressing FL CD27 with anti-myc, and this
phosphorylation is inhibited in the presence JNK-1. We also
confirmed that the inhibitor did not inhibit the phosphorylation of
other MAPKs (Supplemental Fig. 4B, 4C).
As seen with CD27 (Fig. 4C), ligation of CD40 also led to lower
levels of IRF4 (Fig. 7A, 7B), and addition of JNK-II inhibitor to
such cultures partially rescued plasma cell generation as read out
by CD138, BLIMP-1, and IRF4 induction. Together, the data indicate that CD27 and CD40 can both signal through JNK to
suppress induction of IRF4 and BLIMP-1 and thereby to suppress
plasma cell generation. We also looked at intracellular levels of
Bach2, a molecule that has been reported to be involved in plasma cell generation (15, 22). We found that Bach2 was downregulated equivalently by 24 h after LPS stimulation in the presence
or absence of added anti-CD40 Ab and stayed downregulated at
48, 72, and 96 h. (Fig. 7C and data not shown). Hence, it is unlikely to be involved in negatively influencing terminal differentiation after CD40 ligation. It is also unlikely to be involved
downstream of CD27 as the ligation of myc in the transfection and
transduction experiments occurs 60 h after LPS stimulation. Together, the data indicate that CD27 and CD40 can both signal
through JNK to suppress induction of IRF4 and BLIMP-1 and
thereby suppress plasma cell generation.
The Journal of Immunology
6503
FIGURE 2. Inability of CD27 lacking the C-terminal PIQED motif to
abrogate plasma cell generation. A, Coexpression of GFP with CD27 and
myc after transfection of PT67 cells or LPS-activated B cells, respectively,
with the cloned deletion mutants. Representative of two independent
experiments. B, CD138 and BLIMP-1 in gated GFP+ cells after transfection of B cells with empty vector or with the various CD27 constructs
(labeled) followed by myc ligation. No scatter gate has been used for the
analysis. Representative of three independent experiments.
cell commitment remain unclear, but some transcription factors,
especially BLIMP-1, IRF4, and XBP-1, appear to be critical for
plasma cell formation and maintenance (23–26). BLIMP-1 has
been considered a “master switch” for plasma cell generation (23,
27); however, recent data indicate that it is necessary but not
sufficient for the induction (25, 28). IRF4 regulates isotype
switching and terminal differentiation by controlling the expression of AID and BLIMP-1, respectively. Thus, restoration of AID
in IRF42/2 B cells rescues isotype switching, and restoration of
Blimp-1 rescues plasma cell generation (28). XBP-1 expands elements of the secretory pathway and enhances the unfolded protein response (29, 30). Vav may be upstream of some of these
factors, as mature marginal zone B cells from Vav-null mice
show induction of IRF4 but no induction of BLIMP-1 or XBP-1
and fail to undergo terminal differentiation (31). In contrast, the
transcription factors BCL-6 and PAX5, which can repress XBP-1
and BLIMP-1, respectively, are downregulated during plasma cell
differentiation (32–34). BCL-6 is required for GC development
and may be one of the primary molecules that influence differentiation (35–37). There is a considerable amount of cross-talk
between these factors. Thus, upregulation of BLIMP-1 represses
PAX5, a gene product that maintains B cell identity (38, 39) and
also represses BCL-6 (26).
Most of these studies have relied on the use of cell lines or on
B cells from mice deficient for various factors, and they have
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FIGURE 3. The residues P and D in the CD27 tail are required for its
ability to abrogate plasma cell generation. Proportion of B220low
CD138high cells in LPS-activated B cell cultures transduced with virus
expressing CD27 carrying point mutations (P/A, Q/A, or D/A) in the
PIQED domain, or with the D-10 mutant, followed by myc ligation.
Analysis is on live cells gated for GFP. Total CD138+ cells are 49.3
(control), 47.7 (P/A), 24 (Q/A), 48.2 (D/A), and 37.3% (D-10).
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SIGNALING THROUGH JNK INHIBITS PLASMA CELL GENERATION
provided a wealth of information on the complex regulatory interactions between the transcription factors involved. However,
little is known about the cell surface molecules and the signaling
intermediates that are involved in initiating this cascade of transcriptional events in normal B cells. Further, little is known about
signaling events that determine whether a cell exiting from cycle
in the GC will undergo terminal differentiation or become a re-
FIGURE 5. Ligation of CD40 inhibits plasma cell generation. CD138
staining on B cells stimulated with LPS in the presence or absence of titrating doses of anti-CD40 Ab or isotype-matched anti-CD8 Ab. Representative of two independent experiments.
stimulable memory cell. Previous studies have indicated a role for
Ag affinity in controlling this process (40, 41) and also a role for
the TNFRs CD27 and CD40 in this cell fate determination (17, 18,
21). The experiments reported here followed from these earlier
data, which showed that ligation of CD27 or CD40 with an Ab
could specifically inhibit terminal differentiation in vitro (17).
Physiological relevance for the finding came from the observation
that a single dose of anti-CD27, injected at the time of immunization, led to higher memory cell generation in mice by three
independent readouts: limiting dilution assays, adoptive transfer
experiments for secondary responses, and the tracking of Agspecific B cells in vivo over time. As expected, treatment of mice
with anti-CD40 Ab also enhanced memory cell frequencies. Significantly, we also showed that secondary responses were higher to
the T-independent Ag 4-hydroxy-3-nitrophenylacetyl–Ficoll when
immunization was done under cover of anti-CD27 treatment (18).
Thus, CD27, at levels expressed on unmanipulated mouse B cells
can trigger signal modulations leading to altered cell-fate commitment. CD40-mediated signals achieve a plethora of effects in
B cells, whereas CD27-mediated signals appear to be selectively
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FIGURE 4. Inhibition of plasma cell generation by CD27 is mediated
through recruitment of TRAF2, activation of JNK, and inhibition of IRF4.
A, Immunoprecipitation of A20 cells expressing either FL CD27, the D-10
mutant, or point mutations in the PIQED domain after ligation of the
overexpressed molecule. Immunoprecipitation with anti-myc Ab was followed by a Western blot for TRAF2. Blots were stripped and reprobed for
myc. Whole-cell lysates (WCL) served as controls. B, CD138 expression on
gated GFP+ cells after ligation of transfected CD27 on LPS-stimulated B
cells in the presence or absence of JNK-II inhibitor. C, Induction of IRF4
after ligation of transduced CD27 in the presence or absence of JNK inhibitor. B cells transduced with FL CD27 were treated with 10 mg/ml antimyc in the presence or absence of 3 mM or 10 mM JNK-II inhibitor, and 48 h
later, viable cells isolated on a Ficoll-Hypaque gradient were stained for
surface B220, fixed, permeabilized, and stained for intracellular IRF4. Left
panel, Shaded histogram, vector control; thin line, cells transduced with FL
CD27 and treated with anti-myc; thick line, cells transduced with FL CD27
and treated with anti-myc in the presence of 3 mM JNK-II inhibitor; dotted
line, cells transduced with FL CD27 and treated with anti-myc in the
presence of 10 mM JNK-II inhibitor. Right panel, Fold change in mean
fluorescence intensity (MFI) of IRF4 in GFP+ cells from three independent
experiments when transduced CD27 was left unligated, ligated, or was ligated in the presence of 10 mM JNK inhibitor. Rescue with 3 mM inhibitor
did not achieve statistical significance over multiple experiments.
The Journal of Immunology
6505
FIGURE 6. JNK is involved in abrogation of plasma cell generation by
CD40 ligation. A, CD138 expression on cells stimulated with LPS with or
without 4 mg/ml anti-CD40 in the absence or presence of titrating doses of
JNK-1, JNK-II, or JNK-3 inhibitors (as labeled). Cells stimulated with LPS
alone are included as a control. Representative of three independent
experiments. B, JNK is phosphorylated after ligation of CD40 and CD27.
Primary B cells or A20 cells expressing FL CD27 were treated with 10 mM
JNK-1 inhibitor (for 2 h or 1 h, respectively), stimulated with anti-CD40
for 15 min or with anti-myc for 30 min, respectively, in the continued presence of inhibitor, washed, fixed with 2% p-formaldehyde, and stained
for intracellular phosphorylated JNK (pJNK). Histogram overlays for pJNK
expression after CD40 (left panel) or CD27 (right panel) ligation: shaded
histogram, conjugate control; thin line, unstimulated cells; thick line, stimulated cells; dashed line, cells stimulated in the presence of JNK inhibitor.
restricted to the alteration of the plasma cell-memory B cell decision. Thus, characterization of CD27-mediated effects will provide a tool for understanding the signals mediated through TNFR
family members, such as CD40, for the induction of B cell memory.
In pursuit of that aim, we have used a transfection-driven system
to express CD27 mutants to understand the signaling intermediates
involved in the plasma cell-memory B cell decision alteration effect
of CD27 ligation. Identification of the tail regions of CD27 that are
likely to be involved in these effects, most likely TRAF recruitment, required transfection/transduction of CD27 tail mutants
into primary B cells and the ability to ligate selectively the mutant
molecules while leaving the endogenous molecule untouched.
Both molecules signal through the recruitment of TRAFs to their
cytoplasmic tail. In B lineage cells, CD40 has been shown to recruit
TRAFs 1, 2, 3, 4, 5, and 6 (42), but little is known about TRAFs
recruited by CD27 in primary B cells. Two putative TRAF binding
domains, PIQED and EEEG, have been identified in the cytoplasmic tail of CD27 (19, 20), and they have been shown, in
other cell lineages, to recruit TRAFs 2, 3, and 5. In this study, we
cloned and expressed FL CD27 and three deletion mutants that
either retained both PIQED and EEEG domains, lacked both
domains, or lacked only the PIQED domain, and used them to
transfect or virally transduce primary B cells in culture. We found
that ligation of the overexpressed FL molecule or the D-16 molecule, which lacks the PIQED domain, or the D-23 molecule,
which lacks both domains, abrogates plasma cell generation, as
read out by CD138 and BLIMP-1 staining. However, ligation of
the D-10 molecule, which contains the PIQED domain, continues
to inhibit plasma cell generation, albeit not completely (Fig. 2).
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FIGURE 7. IRF4 and BLIMP-1, but not Bach2, are modulated after
CD40 ligation. A, Induction of CD138 on, and of BLIMP-1 and IRF4 in,
cells treated with LPS alone or with LPS plus anti-CD40 in the presence or
absence of JNK-II inhibitor. B, Bar diagrammatic representation of similar
data from five experiments. C, Bach2 expression in untreated B cells
(dotted line) and in B cells treated with LPS in the absence (solid line) or
presence (dashed line) of anti-CD40. Isotype control: shaded histogram.
Representative of three experiments. *p = 0.0003; **p = 0.0006; ***p =
0.00001. NS, not significant.
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SIGNALING THROUGH JNK INHIBITS PLASMA CELL GENERATION
because JNK signaling is uniquely involved in this effect, our data
indicate that intervention strategies involving this molecule may
be possible for the rational design of vaccines.
Disclosures
The authors have no financial conflicts of interest.
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