Stages of Germinal Center Transit Are
Defined by B Cell Transcription Factor
Coexpression and Relative Abundance
This information is current as
of June 14, 2017.
Giorgio Cattoretti, Rita Shaknovich, Paula M. Smith,
Hans-Martin Jäck, Vundavalli V. Murty and Bachir Alobeid
J Immunol 2006; 177:6930-6939; ;
doi: 10.4049/jimmunol.177.10.6930
http://www.jimmunol.org/content/177/10/6930
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References
The Journal of Immunology
Stages of Germinal Center Transit Are Defined by B Cell
Transcription Factor Coexpression and Relative Abundance
Giorgio Cattoretti,1*† Rita Shaknovich,‡ Paula M. Smith,† Hans-Martin Jäck,§
Vundavalli V. Murty,*† and Bachir Alobeid*
T
he development and maturation of B cells from multipotential stem cells is dictated by multiple interacting transcription factors (TF),2 active at different defining steps
throughout the process (1–3). Pax5 is the single TF necessary and
sufficient for B cell development past the pro-B cell stage (4), a
negative regulator of the B cell to plasma cell transition (5), and is
expressed throughout all B cell stages, except in plasma cells (6).
PU.1, a member of the large ets family of TF (7), enforces the B
cell lineage commitment by limiting other differentiation choices
(7, 8), and is then expressed along the B cell, but not the plasma
cell lineage (9), together with other B cell-specific TF (9, 10). PU.1
heterodimerizes with members of the IFN regulatory factor family
(IRFs) (10 –12) in cell- and differentiation stage-specific combinations. IRF8 promotes genes crucial for germinal center (GC) development and function (B cell lymphoma (BCL) 6 and activationinduced cytidine deaminase (AID)) (13) and, with IRF4, is needed
for pre-B to B cell transition (14). IRF4 has been shown to be
expressed at high levels in centrocytes, believed to be at the preplasma cell stage (15). Lack of IRF4 prevents full mature B cell
transit and plasma cell differentiation but not memory B cell de*Department of Pathology, Columbia University Medical Center, New York, NY
10032; †Institute for Cancer Genetics, Columbia University, New York, NY 10032;
‡
Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10467;
and §Division of Molecular Immunology, Department of Internal Medicine, NikolausFiebiger-Center, University of Erlangen-Nürnberg, Erlangen, Germany
Received for publication June 22, 2006. Accepted for publication August 24, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Giorgio Cattoretti at the current
address: Department of Pathology, Azienda Ospedaliera San Gerardo, Via Pergolesi
33, 20052 Monza (MI), Italy. E-mail address: [email protected]
2
Abbreviations used in this paper: TF, transcription factor; IRF, IFN regulatory factor; GC, germinal center; AID, activation-induced cytidine deaminase; BCL, B cell
lymphoma; DLBCL, diffuse large BCL; IHC, immunohistochemistry; DAPI, 4⬘,6⬘diamidino-2-phenylindole; TMA, tissue microarray; FCM, flow cytometry; CLL,
chronic lymphocytic leukemia; PRDM1, positive regulatory domain 1.
Copyright © 2006 by The American Association of Immunologists, Inc.
velopment (16, 17). BCL6, which is highly expressed and necessary for GC B cell development (18, 19), has an inverse distribution with regards to IRF4 inside the GC (1, 15). The relative
expression of these TF correlates roughly with the different molecular subtypes of diffuse large B cell lymphomas (DLBCL), including a group of GC-derived neoplasms characterized by V gene
mutation and lack of terminal differentiation (20). Detailed molecular analysis has shown that IRF4 expression defines a group of
DLBCL characterized by the expression of activation-associated
genes (21, 22), low BCL6 expression (21), with frequent polysomy
or rearrangement of chromosome 3q27 (23–25) and deletion of
positive regulatory domain 1 (PRDM1) (26). Coexpression of
BCL6 and IRF4 can be seen in these DLBCLs (15), as opposed to
the normal GC, where the two are rigorously mutually exclusive
(15). Pre- (mantle cell) and post-GC (marginal zone) type lymphomas are usually negative for BCL6 (27, 28) and MUM1 (29,
30), similar to the normal counterpart B cells (mantle and marginal
zone B cells, respectively) (15), which are negative for both by
routine immunohistochemistry (IHC).
Comparison of mRNA transcription with posttranslational
expression of the final protein product of these TF shows discrepant results. In normal B cells, BCL6 shows posttranscriptional down-regulation outside the GC (31), thus accounting for
the general lack of BCL6 detection in a range of mature B cells,
including pre- and post-GC B cells, which are known to possess
mRNA (18, 32–34). Similarly, IRF4, which is necessary for
mature naive B cell and dendritic cell development (14, 35), is
usually negative in these cells by IHC staining using the MUM1
mAb (15).
In this study, we show the expression of several TF in mouse
and human lymphoid cells, with a special emphasis on BCL6
and IRF4. We show broad coexpression of all TF before, but not
within, the GC reaction. In addition, we demonstrate that IRF4
is expressed at the protein level in a much broader range of
0022-1767/06/$02.00
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The transit of T cell-activated B cells through the germinal center (GC) is controlled by sequential activation and repression of
key transcription factors, executing the pre- and post-GC B cell program. B cell lymphoma (BCL) 6 and IFN regulatory factor
(IRF) 8 are necessary for GC formation and for its molecular activity in Pax5ⴙPU.1ⴙ B cells. IRF4, which is highly expressed in
BCL6ⴚ GC B cells, is necessary for class switch recombination and the plasma cell differentiation at exit from the GC. In this
study, we show at the single-cell level broad coexpression of IRF4 with BCL6, Pax5, IRF8, and PU.1 in pre- and post-GC B cells
in human and mouse. IRF4 is down-regulated in BCL6ⴙ human GC founder cells (IgDⴙCD38ⴙ), is absent in GC centroblasts, and
is re-expressed in positive regulatory domain 1-positive centrocytes, which are negative for all the B cell transcription factors.
Activated (CD30ⴙ) and activation-induced cytidine deaminase-positive extrafollicular blasts coexpress Pax5 and IRF4. PU.1negative plasma cells and CD30ⴙ blasts uniquely display the conformational epitope of IRF4 recognized by the MUM1 Ab, an
epitope that is absent from any other IRF4ⴙPU.1ⴙ lymphoid and hemopoietic subsets. Low grade B cell lymphomas, representing
the malignant counterpart of pre- and post-GC B cells, accordingly express IRF4. However, a fraction of BCL6ⴙ diffuse large B
cell lymphomas express IRF4 bearing the MUM1 epitope, indicative of a posttranscriptional modification of IRF4 not seen in the
normal counterpart. The Journal of Immunology, 2006, 177: 6930 – 6939.
The Journal of Immunology
6931
Table I. Abs used
Clone or Serum
Immunogena
Reactive onb
Species
Source
Dilution
PU.1 (Spi1)
PU.1 (Spi1)
PU.1 (Spi1)
IRF4
IRF4
IRF4
IRF4
IRF8 (ICSBP)
BCL6
BCL6
BCL6
BCL6
BCL6
Pax-5
Pax-5
Pax-5
Oct-2
CD20
CD20
MCM7
Ki-67
Ki-67
PRDM1
PRDM1
PRDM1
AID
IRTA1
Neg. control
Neg. control
sc-352
sc-5949
G148-74
sc-6059
No. 4964
sc-28696
MUM1
sc-6058
PIF6
PGB6 276
PGB6p 594
sc-858
No. 4242
24
sc-1974
RB-9406
sc-233
RB-9013
L26
47DC141
SP6
MIB 1
Serum
3H2E8
6D3
EK2-5G9
mIRTA1
C-term mouse
N-term mouse
Full length
C-term mouse
Asp175
AA 128 –267h
AA 144 – 451h
C-term mouse
N-term human
N-term human
N-term human
N-term human
C-term human
AA 151–306h
C-term human
C-term human
C-term human
C-term human
Cytoplasm. CD20
hCDC47
C-term human
rKi-67
AA 1–350
AA 199 – 409
rBlimp-1
AA 185–198
AA 102–373
h, m
m
h, m
h, m
h
h, m
h
h, m
h, m
h, m
h
h, m
h, m
h, m
h, m
h, m
h, m
h
h
h, m
h, m
h
h, m
h, m
h, m
h
h
Rabbit
Goat
Mouse
Goat
Rabbit
Rabbit
Mouse
Goat
Mouse
Mouse
Mouse
Rabbit
Rabbit
Mouse
Goat
Rabbit
Rabbit
Rabbit
Mouse
Mouse
Rabbit
Mouse
Rabbit
Mouse
Rat
Rat
Mouse
Rabbit
Mouse
SCBT
SCBT
BD
SCBT
CST
SCBT
B. Falini
SCBT
Novocastra
B. Falini
B. Falini
SCBT
CST
BD
SCBT
LabVision
SCBT
LabVision
DakoCytomation
LabVision
LabVision
J. Gerdes
H.-M. Jäck
K. L. Calame
L. Corcoran
E. Kremmer
B. Falini
Sigma-Aldrich
Sigma-Aldrich
1 ␮g/ml
0.1 ␮g/ml
1 ␮g/ml
0.2 ␮g/ml
1/100
1 ␮g/ml
1/50
1 ␮g/ml
1/100
1/10
1/10
0.1 ␮g
1/100
1 ␮g/ml
1 ␮g/ml
1 ␮g/ml
0.2 ␮g/ml
1/200
1/200
0.5 ␮g/ml
1/100
1/50
1/5000
1/30
1 ␮g/ml
1/200
1/10
1 ␮g/ml
1 ␮g/ml
a
b
C-term, C-terminal; N-term, N-terminal; Cytoplasm., Cytoplasmic.
h, Human; m, murine.
mature B cell types, as detected by other Abs not limited to a
conformational epitope recognized by the MUM1 Ab.
Materials and Methods
Cells and tissues
IRF4 knockout (17), BCL6 knockout mice (19), and their wild-type littermates (C57BL6 and F1 from C57BL6 ⫻ 129Sv) were gifts from U. Klein
and R. Dalla-Favera (Columbia University, New York, NY). Anonymous
tonsil cell suspensions and tumor samples were provided and processed as
published (36). A tissue microarray containing B cell lymphomas and reference tissues was published previously (37).
Antibodies
Abs used in this study are listed in Table I. Multiple different anti-BCL6
Abs, which all reacted identically in various assays and combinations, will
be referred to collectively as “BCL6.” The term “MUM1” will be restricted
to anti-IRF4 Abs directed against aa 128 –267 or aa 144 – 451 of the human
protein.
Immunohistochemistry
Sections were essentially stained as previously published (36, 38), including double staining on 1 mM EDTA (pH 8) Ag-retrieved slides. Speciesspecific, alkaline phosphatase-conjugated secondary Abs, prescreened for
specificity and absence of cross-reactivity, were developed with nitroblue
tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (Roche). The
percentage and intensity of staining of CD20⫹ neoplastic B cells were
independently scored in the DLBCL tissue microarray (TMA), each using
a 10-tiered scale (0 –9). The product of both was used as a case score and
a value of 10 or greater was considered positive for ␹2 calculations.
Immunofluorescence
Double immunofluorescence was performed with primary Abs raised in
different species or of different isotypes, counterstained with FITC- or Cy3conjugated, species- or isotype-specific secondary Abs (Jackson ImmunoResearch Laboratories) or biotin-conjugated secondary Abs, followed by
conjugated avidin (Jackson ImmunoResearch Laboratories). Tyramide sig-
nal amplification for single or double labeling (39) was performed according to the manufacturer’s instructions (PerkinElmer): briefly, avidin-HRPcounterstained immunostains were followed by tyramide-FITC or -biotin
and Avidin Cy-3 (Jackson ImmunoResearch Laboratories) or AvidinAlexa350 (Molecular Probes). The slides were counterstained with 4⬘,6⬘diamidino-2-phenylindole (DAPI) (Molecular Probes) when indicated and
mounted.
Image acquisition and analysis
Gray scale or color images were taken on an E600-Nikon Microscope,
fitted with Planachromat ⫻4/0.10/30.0, ⫻10/0.25/10.5, ⫻20/0.40/1.3,
PlanApo ⫻40/0.095/0.12– 0.16 (light microscopy) or PlanFluor ⫻10/0.30/
16.0, ⫻40/0.75/0.72, ⫻60/0.80/0.3 (immunofluorescence) objectives, with
a SPOT-2 charge-coupled device camera and software (Diagnostic Instruments). A Zeiss LSM 510 NLO Multiphoton Confocal Microscope (Carl
Zeiss) equipped with ⫻10/0.3 and ⫻40/1.3 Fluor objectives, one 25-mW
argon laser exciting at 458, 488, and 514 nm and one 1 mW helium-neon
laser exciting at 543 nm, and proprietary image acquisition software was
used for confocal analysis.
All images were edited for optimal color contrast with Adobe Photoshop
7 and Adobe Illustrator 10 (Adobe Systems), on a G4 Apple computer.
Flow cytometry
Live human or murine cell suspensions were stained with appropriate combinations of fluorochrome- or biotin-conjugated primary Abs for 15 min on
ice in PBS-BSA-NaN3, washed twice with the same medium and further
processed for nuclear staining. For BCL6 staining, cells were fixed with
FACS lysing solution (BD Biosciences) for 10 min on ice, washed twice in
PBS-BSA-NaN3, incubated overnight with negative mouse IgG1 (1 ␮g/ml)
or PGB6 clone 276 (1/1000) ⫹ P1F6 (1/100) mouse IgG1 anti BCL6
mixture. After two washes in PBS-BSA-NaN3, the cells were counterstained with rat anti-mouse IgG1 PE-(1/200) or biotin-conjugated (1/300;
BD Pharmingen), this latter followed by fluorochrome-conjugated avidin
(1/500). IRF4, Pax5, and PU.1 Ab stainings requires a modified fixation,
which destroys prebound fluorochromes and some surface Ags, but not
biotin. Therefore, cells were first stained with biotin-conjugated Abs, fixed
as above, washed in PBS, fixed in absolute cold methanol for 10 min,
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Ab
6932
washed in PBS, transferred to PBS-BSA-NaN3, and processed for intracellular staining. Counterstaining of the nuclear Ag was followed by fluorochrome-avidin staining of the biotin-labeled Ab and staining for Ags
which survive the double fixation (e.g., IgD, CD38). IgD was stained with
a PE-conjugated rat anti-mouse IgD (Southern Biotechnology Associates)
or with a PE-conjugated mouse anti-human IgD (BD Biosciences), preceded by excessive cold mouse Ig blocking of residual anti-mouse IgG
moieties. BCL6 staining is not affected by the methanol postfixation.
Goat and rabbit primary Abs (IRF4, PU.1, negative control) were used
at 1 ␮g/ml, incubated overnight, and counterstained with either donkey
anti-rabbit Cy5 F(ab⬘)2 (1/500; Jackson ImmunoResearch Laboratories) or
donkey anti-goat-FITC (1/500; Jackson ImmunoResearch Laboratories).
For double IRF4, PU.1 and BCL6 stainings, human cells were surfacestained first with mouse anti CD3-biotin (BD Pharmingen), followed by
nuclear staining, exclusion by gating of the CD3⫹ cells, and analysis of the
non-T cells (⬎95% B cells).
More than 30,000 events were acquired on a FACSCalibur (BD Biosciences) and CellQuest acquisition software and analyzed with FlowJo 6.4
(Tree Star).
cDNA microarray analysis of human samples
Data were obtained from published databases (1), obtained by Affymetrix
U95A chip analysis of normal tonsil subsets.
LSI BCL6 dual-color, break-apart rearrangement, and Spectrum Greenlabeled CEP 8 probes were obtained from Abbott Molecular. Four 5-␮M
thick tissue sections of two TMAs were cut onto adhesive-coated slides.
Paraffin-sections were baked overnight at 60°C before hybridization. TMA
slides were subjected to protease treatment using paraffin pretreatment kit
(Abbott Molecular). Fluorescence in situ hybridization was performed by
standard methods and hybridization signals were scored on at least 200
interphase nuclei on DAPI-stained slides (26). The sensitivity of hybridization on paraffin-embedded tissues was determined by performing the
same analysis on analogous sections from normal tonsils and similarly
processed cell lines of known genomic asset. Cases were diagnosed as
rearranged if the fraction of cells showing the rearrangement in ⬎5% cells.
Results
Distribution of B cell TF by tissue IHC
The distribution of the transcription factors Pax-5, Oct-2, PU.1,
and IRF8 (Fig. 1) in mouse and human lymphoid tissues is broad
and overlaps among different B cell subsets (morphologically and
phenotypically defined follicular mantle, marginal, and GC cells)
as shown by in situ immunostaining with Abs validated for specificity (Fig. 2). BCL6 and IRF4 instead show a mutually exclusive
distribution; BCL6 is highly expressed in GC, IRF4 instead is positive in mantle and marginal zone B cells, and negative in most GC
B cells. The expression of IRF4 in IRF8⫹, PU.1⫹, Oct-2⫹, and
Pax5⫹ follicular mantle B cells could be seen only with Abs directed against the C-terminal and Asp175, and not with two other
Abs directed against aa 128 –267 of the IRF4 protein (hence named
MUM1 epitope). Abs against this region of IRF4 detect only
plasma cells and a fraction of GC centrocytes (15).
We then compared the levels and distribution of B cell TF proteins in human tonsil tissue with the RNA levels, analyzed by gene
expression profiling in purified B cell subsets (1). As shown in Fig.
1, the RNA levels of each TF in GC, mantle, and marginal zone B
cells correspond to the distribution of the respective protein Ags,
including IRF4 by C-terminal and Asp175 Ab staining.
Single-color staining and tissue RNA or protein extraction do
not allow a detailed analysis of the coexpression of two given Ags
on a cell-by-cell basis. Therefore, we analyzed by double immunofluorescence human (Fig. 3, D–N) and murine (data not shown)
samples. As predicted from the observed distribution by singlecolor staining, IRF4 was largely mutually exclusive with BCL6
(15), PU.1, IRF8, and Pax5 in GC, while coexpressed on PU.1⫹,
IRF8⫹, and Pax5⫹ mantle cells.
Staining with all IRF4 Abs was both nuclear and cytoplasmic,
the latter was least evident with Abs directed against aa 128 –267.
The GC contains morphologically and phenotypically different
and well-defined zones, comprised predominantly of B cells,
which correspond to different functional and/or maturational subsets. Extrafollicular B cells, however, are more dispersed and
therefore better defined by double staining (36, 40) rather than by
topographic location. We analyzed the coexpression of IRF4 and
other B cell TF in four B cell subsets: the CD30⫹ activated cells,
the extrafollicular AID⫹ blasts (36), the centrocytes in the GC
light zone, and the extrafollicular memory/marginal zone IRTA1⫹
cells (41).
CD30⫹ extrafollicular blasts are largely B cells showing evidence of BCR plus cofactor-mediated acute activation (36), and
IRF4 has been shown to be part of this signature (42). Accordingly, CD30⫹ cells were largely IRF4⫹ and Pax5⫹. The intensity
of Pax5 expression was inversely related to intensity of IRF4 expression (Fig. 3H).
B cells express AID upon in vitro activation (43) and IRF4 is
necessary for AID expression (17). Approximately half of the
AID⫹ extrafollicular blasts were IRF4⫹, MUM1⫺ (Fig. 3, A and
B), with the intensity of IRF4 often noticeably dim, compared with
that of CD30⫹ extrafollicular blasts. This was in sharp contrast to
the GC where AID and IRF4 were rigorously mutually exclusive
both in the dark and outer zone (Fig. 3A). Thus, appreciable levels
of IRF4 are detectable in extrafollicular AID⫹ cells, possibly before they enter the GC reaction, when IRF4 is shut off.
Commitment to the plasma cell lineage within the GC is marked
by PRDM1 expression, at first in Pax5⫹ centrocytes, then in
CD20⫺, Pax5⫺, and CD138⫺ preplasma cells (38). The bulk of
bright IRF4⫹ centrocytes coexpress PRDM1 and are Pax5 negative (Fig. 3, I, L, and M). In addition, a minority of Pax5-positive
or Pax5 weakly positive centrocytes express IRF4 and/or PRDM1
(Fig. 3I). The MUM1 epitope is not detected on these latter cells
(Fig. 3L).
The fourth B cell subset we focused on is defined by the FCRL4/
IRTA1 Ag (41, 44). IRTA1⫹ cells are monocytoid B cells with
evidence of Ag selection and variable Ig gene mutation (44), thus
memory B cells, located within the tonsil festooned epithelium and
scattered in the interfollicular areas. Although intraepithelial
IRTA1⫹ cells were largely IRF4⫺, interfollicular and intrafollicular IRTA1⫹ cells contained IRF4⫹ (Fig. 3C). This is consistent
with the reported expression of IRF4 by gene expression profiling
in purified memory B cells (1) (Fig. 1). IRTA1⫹ B cells were
previously reported negative for IRF4 with the MUM1 Ab.
In summary, IRF4 is coexpressed with the other B cell TF in
pre- and post-GC B cells, but not inside the GC, where it is specifically down-regulated during transit.
Distribution of IRF4 and BCL6 in lymphoid tissues by flow
cytometry
BCL6 staining in the follicle mantle is usually negative, however,
occasionally we are able to detect it by IHC only in selected mouse
samples (Fig. 2). BCL6 is among the most variably expressed
genes in the mouse (45) and our inconsistent results may be due to
either sample-to-sample variation and/or insufficient sensitivity.
To assess in a quantitative fashion BCL6 and IRF4, we developed
and validated (Fig. 2) a flow cytometry (FCM) assay, which has
the additional advantage of allowing multiparameter phenotypic
characterization of B cell subsets.
Mouse cells of defined BCL6 genomic dosage showed specific,
gene-dependent, near-ubiquitous biallelic BCL6 staining in most B
cell subsets (Fig. 2), thymus and myeloid cells (data not shown). In
particular, follicular B cells (B220⫹IgD⫹) showed detectable
BCL6, consistent with the occasional staining seen by IHC. IRF4
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Fluorescence in situ hybridization
IRF4, BCL6, AND B CELL TF COEXPRESSION
The Journal of Immunology
6933
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FIGURE 1. B cell TF staining in mouse spleen and human tonsil. A, Serial sections of immunized mouse spleen are stained for B cell TF and
proliferation-associated Ag (MCM7). Low-power (⫻10) images are on top, high power (⫻40) on bottom. oPALS, outer periarteriolar lymphoid sheet; MC,
mantle cells; MZ, marginal zone. Note the reciprocal staining pattern of IRF4 vs the other TF in the GC, and the coexpression in the MZ and MC. B, Serial
sections of human tonsil are stained for B cell TF and proliferation-associated Ag (MCM7). Low-power (⫻4) images are on top, high power (⫻40) in the
middle. Lower panel, The U95 Affymetrix cDNA chip raw values, depicted as bars, indicating cDNA expression for the corresponding transcription factors
in memory (Mem), naive, and GC-purified cells. IE, intraepithelial; SE, subepithelial; MC, mantle cell; GC, germinal center. Note the reciprocal staining
pattern of IRF4 vs the other TF in the GC, and the coexpression in the MC and IE.
6934
IRF4, BCL6, AND B CELL TF COEXPRESSION
tained a BCL6⫹,IRF4⫹ population, corresponding to mantle zone
B cells and a BCL6⫹⫹, IRF4 negative or weak population, corresponding to GC founder cells. MUM1 Ab, shown to be reactive
with IRF4 by FCM on lymphoma cell lines (data not shown), was
negative on all these B cell fractions.
We conclude that each B cell subset coexpressing BCL6, IRF4,
and the other B cell TF is characterized by discrete protein levels
of such TF, which are typical of each cell type.
Distribution of IRF4 and BCL6 in B cell non-Hodgkin
lymphoma
knockout splenic B cells were negative for IRF4 staining, compared with positive wild-type littermate B cells (Fig. 2).
As predicted by gene expression profiling of human tonsils,
BCL6 and IRF4 were expressed in mantle zone B cells (CD20⫹,
IgD⫹CD38⫺/dim) (Fig. 4). Plasmacytoid cells (CD20⫹,
IgD⫺, CD38⫹⫹) were BCL6⫺IRF4⫹⫹. On the contrary, GC cells
(CD20⫹, IgD⫺, CD38⫹) were BCL6⫹⫹,IRF4⫺. Memory B cells
(CD20⫹, IgD⫺, CD38⫺) showed IRF4 positivity and traces of
BCL6. Interestingly, GC founder cells (CD20⫹, IgD⫹, CD38⫹⫹)
(46) contained lower levels of IRF4 than mantle zone or memory
B cells, and two populations of BCL6low and BCL6high B cells
(Fig. 4).
Furthermore, tonsil cell suspensions were stained for BCL6,
IRF4, Pax5, CD3, and IgD, to define total (CD3⫺), GC (IgD⫺),
and mantle zone B cells (IgD⫹) (Fig. 5). Pax5⫹,IgD⫺ GC B cells
contained no IRF4 and high levels of BCL6. The Pax5⫹,IgD⫹
fraction (containing mantle zone and IgD⫹ GC founder cells) con-
Discussion
BCL6 is a potent transcriptional repressor expressed at high levels
in GC cells, and presumably prevents terminal B cell differentiation by repressing key genes, such as PRDM1/Blimp-1(48). BCL6
may also have other important functions, such as antiapoptotic
activity (49). Recently, Pax5 has been shown to have an essential
role in maintaining the B cell phenotype in GC cells (5). IRF8 and
IRF4 are also needed to complete essential activities within the
GC, such as somatic mutation and class switch recombination of Ig
genes through AID induction (13, 17). The emerging picture requires an understanding of the topographic distribution of all these
actors before, after, and through the GC reaction, such knowledge
has been lacking or largely incomplete. It is also important to know
not only the distribution but also the abundance of these factors.
PU.1 is an example of that, because levels PU.1 are critical for
normal physiology of multiple cell types (50). These data have
deep implications in interpretation of molecular interaction data in
the right normal physiologic context.
In the present study, we show that BCL6 and IRF4 are broadly
coexpressed, together with Pax5, PU.1, and Oct-2, in mature B
cells, before and after entering the GC reaction, a finding largely
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FIGURE 2. Validation of the BCL6 and IRF4 staining. A, A murine
wild-type (wt) lymph node (center left) is stained by BCL6 Ab in a GC
(strongly) and in the follicles (weakly). The negative control Ab and the
BCL6 Ab on a BCL6 knockout (KO) lymph node are negative. A wild-type
and knockout mouse spleen are stained by IRF4; the knockout spleen is
negative. B, Four mice, each bearing none, one, two BCL6 alleles, and one
with one C-terminally truncated allele, are analyzed in the thymus and
spleen for BCL6 expression by flow cytometry. The gray histogram is an
isotype-matched negative control. Note the increased intensity (rightward
shift of the peak fluorescence) of BCL6 staining with increased genetic
dosage. The gating for the two B cell subsets are indicated at right. The
truncated allele produces increased amounts of protein, because of the lack
of autoregulation (Wang et al. (52)). C, Spleen cells from an IRF4 knockout mouse (KO) and a wild-type littermate (wt) were stained for B220,
CD11b, negative control (gray histogram), or IRF4 (black line). Note absence of specific staining in the IRF4 KO mouse.
We then evaluated by IHC the mutual distribution of IRF4 and
BCL6 in a series of 40 DLBCL. We found 45% (18 of 40) coexpressed BCL6 and IRF4, 45% were BCL6⫹ only, 7.5% (3 of 40)
were IRF4⫹ only, and 2 were negative for both (Fig. 6). A semiquantitative evaluation of the staining showed two distinctly distributed groups of predominantly BCL6- or IRF4-positive cases
and a heterogeneous cluster of coexpressing cases (Fig. 6A). A
similar inversely related distribution has been obtained for the
RNA expression levels by gene expression profiling (Ref. 47 and
data not shown). Cases with known BCL6 translocation were low
in BCL6 and (3 of 4) coexpressed IRF4.
IRF4 and MUM1 staining were highly correlated (Fig. 6B), with
only two IRF4⫹ cases not expressing the MUM1 epitope (Fig. 6B).
To understand the mutual relationship of BCL6 and IRF4 at the
single-cell level, we costained for both selected double-positive
DLBCL cases. A range of coexpression patterns was obtained,
from mutual exclusion to substantial costaining (Fig. 7).
A small group of low-grade lymphomas representative of the
neoplastic counterpart of pre- and post-GC cells was also evaluated. Six cases of chronic lymphocytic leukemia (CLL), two mantle cell lymphomas, and two marginal zone lymphomas, showed
more extensive but not consistent IRF4 staining, compared with
MUM1 (Fig. 8). One CLL case was BCL6⫹ by IHC (data not
shown). BCL6 and IRF4 expression was confirmed by FCM in two
representative CLL cases, one of which showed coexpression (data
not shown).
Thus, B cell lymphomas, as their normal B cell counterparts,
show coexpression of BCL6 and IRF4. However, in tumors of
putative GC origin, such as a group of DLBCL, IRF4 bears the
MUM1 conformational epitope in BCL6⫹ cells.
The Journal of Immunology
6935
expected according to previously published molecular and immunohistochemical studies (1, 31, 51). The mutual relationship of
these TF outside the GC is fine tuned by the amount of each TF in
a cell type-specific fashion, as demonstrated by quantitative flow
cytometry.
The role of BCL6 in pre- and post-GC B cells is unknown and
probably redundant for B cell maturation and survival, because
mantle zone B cells in BCL6 knockout mice are unaffected (19). In
addition, the protein levels of BCL6 in pre-GC cells are maintained
low by posttranscriptional down-regulation (31) and transcriptionalnegative autoregulation (52). However, unimmunized BCL6-transgenic mice with only moderately raised baseline BCL6 levels in
pre-GC cells experience GC formation to a level comparable to
that of a potent polyclonal immunization (53), suggesting a “tonic”
role for such low protein levels. What is probably crucial is the
ability to rapidly increase BCL6 levels upon activation. We suggest this increase to happen in GC founder cells.
Low levels of BCL6 are also physiologically relevant in myeloid-derived cells, in which BCL6, undetectable by routine IHC,
but detected by FCM, is necessary to repress an otherwise lethal
immune dysregulation (33) and to control IL-6 signaling (32).
Thus, BCL6 is continuously expressed at various levels before and
during the GC reaction, and is eventually down-regulated in terminally differentiated plasma cells.
The presence of IRF4 in pre-GC B cells is expected because it
is required for IgD⫹IgM⫹ B cell phenotypic maturation (16). The
levels of IRF4 in human and murine mantle and marginal zone B
cells are quantitatively lower than the levels found in plasma cells
and, notably, qualitatively different. The large majority of IRF4⫹
cells in mouse and human tissue cannot be detected by MUM1-like
Abs directed against aa 128 –267 of IRF4. The fact that these Abs
recognize denatured IRF4 in a Western blot indicates that the molecule is in a peculiar conformational configuration which masks
the MUM1-like epitope in most B cells and myelomonocytic cells.
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FIGURE 3. Coexpression pattern of IRF4 with other B cell TF in human tonsil. A, AID (brown) decorates IRF4-GC cells (✮) and extrafollicular blast
with variable amounts of IRF4 (purple). Light zone IRF4⫹ centrocytes are AID⫺. Original magnification, ⫻10; scale bar, 15 ␮m. B, Enlargement of portion
of extrafollicular area detailing AID⫹ (brown) cells containing variable amounts of IRF4 (purple). The edge of a GC is shown (arrow). Original magnification, ⫻40; scale bar, 15 ␮m. C, IRTA1⫹ (brown) lymphocytes express IRF4 (purple) in the mantle/marginal zone (yellow arrows, enlarged in the inset),
but not in the intraepithelial location (far right). ✮, GC. Original magnification, ⫻40; scale bar, 15 ␮m. D–G, Low-power (left) and high-power LZ detail
(right) of coexpression or IRF4 (green) with PU.1, IRF8, and BCL6 (all red) and of PU.1 (green) and BCL6 (red). Note in D coexpression of IRF4 and
PU.1 in mantle zone B cells (✮) but not in centrocytes. where IRF4, BCL6, and IRF8 are mutually exclusive. PU.1 and BCL6 show coexpression in part
of the GC cells. Left, Original magnification, ⫻4; scale bar, 100 ␮m. Right, Original magnification, ⫻40; scale bar, 5 ␮m. H, CD30⫹ (blue) interfollicular
blasts coexpress IRF4 (green) and Pax5 (red). The boxed area is magnified and the color split on the right. Note two IRF4⫹Pax5⫹CD30⫺ blasts (arrows)
at the edge of the GC (✮). Original magnification ⫻40, scale bar 5 ␮m. I, Detail of GC light zone, containing centrocytes variably expressing IRF4 (green)
and PRDM1 (red). Two cells also express Pax5 (blue, arrows). One Pax5⫹PRDM1⫹ blast is shown (arrowhead). K, Plasma cells outside the GC coexpress
IRF4 (green) and PRDM1 (red) but not Pax5 (blue). Note the heterogeneity in Ag expression. A plasmacytoid cell is PRDM1⫹IRF4⫺. L, Light zone
centrocytes are IRF4⫹ (green), PRDM1⫹ (red), and a minority express MUM1 (blue). I, K, and L, magnification, ⫻40; scale bar, 15 ␮m. M, Confocal
analysis of IRF4 (green), MUM1 (red), and DAPI (blue) staining in GC light zone, showing two IRF4-only centrocytes (arrows) and, on the right,
nonidentical distribution of IRF4 and MUM1 in the nuclei. Original magnification, ⫻40; scale bar, 5 ␮m. N, Light zone centrocytes show relocation of
cREL (red, arrows) in IRF4⫺ nuclei (green). Nuclei are stained with DAPI (blue). Original magnification, ⫻40; scale bar, 15 ␮m.
6936
IRF4, BCL6, AND B CELL TF COEXPRESSION
FIGURE 4. BCL6 expression in human tonsil B cells subsets. Five human B cells subsets, defined by CD38 and IgD staining on CD3-negative,
CD20⫹ B cells, are analyzed for BCL6, IRF4, and a negative control (gray histograms). Note expression of both BCL6 and IRF4 in mantle zone
B cells (IgD⫹CD38⫺), reduced IRF4 and emergence of a strong BCL6⫹ peak in GC founder cells (IgD⫹CD38⫹), disappearance of IRF4 in GC cells
(IgD⫺CD38⫹), expression of IRF4 but minimal BCL6 in memory B cells (IgD⫺CD38⫺) and high IRF4 expression in BCL6- plasmacytoid cells
(IgD⫺CD38⫹⫹).
FIGURE 5. Coexpression of BCL6, IRF4, and Pax5 in IgD⫹ and IgD⫺
tonsil B cells. Two fractions of tonsil CD3⫺ B cells (IgDneg and IgDpos)
are costained for IRF4, negative control, BCL6, and Pax5. The shaded gray
area indicate IRF4 vs negative control. The contour image superimposed
shows IRF4 vs a second marker distribution. At each side, the single distribution of the negative isotype control (gray histogram) and the respective
positive Ab (black line) are represented. Note two populations, one BCL6⫹
IRF4 weak/negative and one BCL6 weak/IRF4 weak in the IgD⫹ fraction.
At the entry of the GC, IRF4 is quickly down-regulated, while
BCL6 is up-regulated, as demonstrated by the absence of double
IRF4-BCL6-positive cells by FCM and IHC inside the GC. The
situation is reversed in the light zone centrocytes committed to the
plasma cell lineage. There, down-regulation of BCL6 is followed
by PRDM1 expression in cells which still express Pax5. Then,
Pax5, IRF8, and PU.1 are gradually down-regulated and IRF4 is
substantially up-regulated in cells which have already lost the B
cell program (38, 48) (Fig. 9). BCL6 is eventually totally lost on
these cells. The factors initiating the exit process are unknown at
the present time. We detected nuclear relocation of cREL, an
NF-␬B member, in BCL6⫹ and negative centrocytes in the light
zone (Ref. 57 and data not shown), largely in IRF4-negative but
also in some IRF4⫹ and PRDM1⫹ cells (38). Unfortunately, we
could not investigate nuclear relocation of other NF-␬B members,
therefore, the role of this group of TF in down-regulating BCL6
remains elusive. Recent studies have suggested that Pax5, not
BCL6, may be the key regulator to be switched off before plasma
cell differentiation (5), and PRDM1 would be the TF that seals the
plasma cell fate (58).
The light zone contains Pax5⫹ centrocytes which lack both high
levels of BCL6, as well as PRDM1 and IRF4. Some of these may
be memory B cell precursors, which did not acquire yet memory B
cell Ags, such as IRTA1 (41), usually not detectable in the GC.
However, the lack of IRF4 would be in contrast with the gene
expression and flow cytometry data that we have generated, indicating that memory B cells are IRF4⫹. One possibility is that
FIGURE 6. Distribution and coexpression of BCL6 and IRF4 (MUM1
and polyclonal Ab) in DLBCL. A, Bivariate dot plot showing the distribution of BCL6 and IRF4 in 40 DLBCL cases, according to the score of
positive cells. u, The 10% lower limit for positivity for score (see Materials and Methods). The number of cases in each quadrant is shown in
italics. Gray symbols represent cases polysomic for 3q27; asterisks represent cases with 3q27 breaks (BCL6 rearrangement). Other cases are either
normal or not tested (n ⫽ 11). B, The correlation of IRF4 and MUM1
staining in 40 DLBCL cases is shown. u, The 10% lower limit for positivity for score.
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IRF4 is structurally modified by an immunophilin (FKBP52 or
FKBP4) with peptidyl-prolyl isomerase and chaperone-like activity (54), broadly expressed in all B cell subsets at the mRNA level
(data obtained from Ref. 1) which binds to IRF4 in a segment
centered on aa 150 –237. FKBP4 binding induces a structural conformation which reduces the interaction of IRF4 with PU.1 and the
transcriptional activity on PU.1-binding target genes (54). FKBP4
binding is inhibited when the C-terminal autoinhibitory domain
folds back on the N-terminal DNA-binding domain (54).
Interestingly enough, the aa 128 –267/MUM1 epitope is found
predominantly on PU.1-negative cells (T cells, plasma cells, and
melanocytes) (15, 55) and never present in myelomonocytic cells
(G. Cattoretti, manuscript in preparation) or mantle zone B cells,
where IRF4 has been shown to be functionally active and required,
in cooperation with PU.1 (16, 56). This suggests that the acquisition of the MUM1⫹ conformation by IRF4 is associated with a
change in interacting partners or with reduced interaction with
PU.1. Future studies with biochemical assays are needed to identify the interacting partners and the target DNA sequences.
The Journal of Immunology
6937
FIGURE 7. Coexpression of BCL6 and IRF4 in DLBCL. Four representative cases of DLBCL (A–D), double stained for BCL6 and IRF4
(MUM1), are shown, the two stains are split and shown in black and white.
Arrowheads indicate nuclei with mutually exclusive staining, arrows coexpression. Note the variety of ratios of the two Ags.
FIGURE 9. Scheme of B cell TF distribution across the human GC
transit. A, B cell Ags and TF distribution is shown from naive mantle cell
(MC) B cells, through the outer and dark zone (OZ, DZ) of the GC, the GC
light zone (LZ), and post GC. Only the plasma cell exit is shown. T, T cell;
FDC, follicular dendritic cell; NcREL, nuclear cRel. B, B cell Ags and TF
distribution in the GC light zone (LZ) and in the memory cell (left) and
plasma cell (right) arms of post-GC differentiation. Extrafollicular CD30⫹
cells are shown on the left as precursors of both the GC and the extrafollicular memory B cell pathway. On the right, a CD30⫹ intermediate is
hypothesized (dashed) although no evidence of plasma cell commitment
has been identified so far. Vertical gray bars are drawn to identify boundaries of Ag expression per cell type or stage of differentiation. Extent of
coexpression is approximate and not quantitative.
FIGURE 8. Comparison of MUM1 and IRF4 polyclonal Ab staining on
a sample of low grade non-Hodgkin lymphoma. Selected cases of low
grade B cell lymphomas (CLL; MC, mantle cell lymphoma, MZ, marginal
zone lymphoma) and tonsil are stained respectively with the MUM1 and
the polyclonal IRF4 Ab. Identical fields on serial sections are shown. A
low-power field (⫻4) is shown at the sides. The polygon marks the tonsil
GC, the star the mantle. Note the more frequent positivity of the polyclonal
IRF4 Ab.
IRF4⫹ and/or PRDM1⫹ cells may still be able to enter the memory
B cell lineage and would then quickly exit the GC. Promiscuity in
lineage commitment is not a new concept in B and myeloid TF
literature (59). The hypothesis that PRDM1 is expressed in a common memory and plasma cell precursor has been published in the
past (60). Both hypotheses would require a “common centrocyte”
(Fig. 9) which would then give rise to either memory or plasma
cells or both.
This putative “common centrocyte” is an AID-negative cell. We
have previously shown that AID⫹ cells in the dark and outer zone
of the GC are MUM1 negative and now we confirm these data with
a broader anti-IRF4 reagent. Because IRF4 is needed for class
switch recombination (17), its absence in AID⫹ cells through the
GC is in contrast with the common knowledge that the light zone
is the site where such activity occurs. It is possible that IRF4 function in the GC is replaced by another TF of the same class; IRF8
and IRF4 have been previously shown to be partially redundant
(56), thus IRF8 (which promotes AID expression (13)) may be a
candidate. Yet, the absence of AID in centrocytes suggests that the
molecular lesions initiating class switch recombination occur in a
cell upstream, which would be AID⫹ and may express IRF4 at a
certain point. We have identified putative cells fulfilling such criteria. IRF4 is expressed in rare AID⫹ blasts at the edge of the GC
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Pax5⫹ memory B cell precursors are rapidly exported from the GC
and they acquire memory B cell Ags and IRF4 outside the GC
boundaries. The detection of IRF4 in IRTA1⫹ cells at the edge of
the GC may be evidence of such maturation. Another nonexclusive
possibility is that centrocytes may be quite promiscuous in terms
of lineage commitment, once they reach the BCL6-negative stage.
6938
Acknowledgments
We thank Ulf Klein, Masumichi Saito, Riccardo Dalla-Favera, and Michael
L. Shelanski for discussion, scientific support, continuous encouragement,
and advice. Attilio Orazi (Indiana University, Indianapolis, IN) contributed
cases to the TMA. Maryellen Benito provided outstanding technical help.
Lin Yang, the Molecular Pathology Facility and the Optical Microscopy
Facility, Herbert Irving Comprehensive Cancer Center, Columbia University, provided superb histology service and excellent confocal analysis.
Brunangelo Falini (Perugia University, Perugia, Italy), Lynn Corcoran
(Walter and Eliza Hall Institute, Melbourne, Australia), Elisabeth Kremmer
(GSF-National Research Center for Environment and Health, Münich, Germany), and Johannes Gerdes (Molecular Immunology, Borstel, Germany)
generously provided Abs.
Disclosures
The authors have no financial conflict of interest.
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shown that these cells are losing the phenotype of the acute BCR
stimulation typical of CD30⫹ blasts (Myc, JunB, CCL22) and acquire characteristics that are similar to GC cells (36). Once these
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