Stages of Germinal Center Transit Are Defined by Coexpression and Relative Abundance

This information is current as Giorgio Cattoretti, Rita Shaknovich, Paula M. Smith, of October 4, 2021. 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 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 Ja¨ck,§ Vundavalli V. Murty,*† and Bachir Alobeid*

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 Pax5؉PU.1؉ B cells. IRF4, which is highly expressed in) BCL6؊ GC B cells, is necessary for class switch recombination and the 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 BCL6؉ human GC founder cells (IgD؉CD38؉), 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. Downloaded from Activated (CD30؉) and activation-induced cytidine deaminase-positive extrafollicular blasts coexpress Pax5 and IRF4. PU.1- negative plasma cells and CD30؉ blasts uniquely display the conformational epitope of IRF4 recognized by the MUM1 Ab, an epitope that is absent from any other IRF4؉PU.1؉ 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 BCL6؉ 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. http://www.jimmunol.org/

he development and maturation of B cells from multipo- velopment (16, 17). BCL6, which is highly expressed and neces- tential stem cells is dictated by multiple interacting tran- sary for GC B cell development (18, 19), has an inverse distribu- T scription factors (TF),2 active at different defining steps tion with regards to IRF4 inside the GC (1, 15). The relative throughout the process (1–3). Pax5 is the single TF necessary and expression of these TF correlates roughly with the different mo- sufficient for B cell development past the pro-B cell stage (4), a lecular subtypes of diffuse large B cell lymphomas (DLBCL), in- negative regulator of the B cell to plasma cell transition (5), and is cluding a group of GC-derived neoplasms characterized by V expressed throughout all B cell stages, except in plasma cells (6). mutation and lack of terminal differentiation (20). Detailed molec- PU.1, a member of the large ets family of TF (7), enforces the B ular analysis has shown that IRF4 expression defines a group of by guest on October 4, 2021 cell lineage commitment by limiting other differentiation choices DLBCL characterized by the expression of activation-associated (7, 8), and is then expressed along the B cell, but not the plasma (21, 22), low BCL6 expression (21), with frequent polysomy cell lineage (9), together with other B cell-specific TF (9, 10). PU.1 or rearrangement of 3q27 (23–25) and deletion of heterodimerizes with members of the IFN regulatory factor family positive regulatory domain 1 (PRDM1) (26). Coexpression of (IRFs) (10–12) in cell- and differentiation stage-specific combina- BCL6 and IRF4 can be seen in these DLBCLs (15), as opposed to tions. IRF8 promotes genes crucial for germinal center (GC) de- velopment and function (B cell lymphoma (BCL) 6 and activation- the normal GC, where the two are rigorously mutually exclusive induced cytidine deaminase (AID)) (13) and, with IRF4, is needed (15). Pre- (mantle cell) and post-GC (marginal zone) type lym- for pre-B to B cell transition (14). IRF4 has been shown to be phomas are usually negative for BCL6 (27, 28) and MUM1 (29, expressed at high levels in centrocytes, believed to be at the pre- 30), similar to the normal counterpart B cells (mantle and marginal plasma cell stage (15). Lack of IRF4 prevents full mature B cell zone B cells, respectively) (15), which are negative for both by transit and plasma cell differentiation but not memory B cell de- routine immunohistochemistry (IHC). Comparison of mRNA transcription with posttranslational expression of the final product of these TF shows dis- *Department of Pathology, Columbia University Medical Center, New York, NY 10032; †Institute for Cancer Genetics, Columbia University, New York, NY 10032; crepant results. In normal B cells, BCL6 shows posttranscrip- ‡Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10467; tional down-regulation outside the GC (31), thus accounting for and §Division of Molecular Immunology, Department of Internal Medicine, Nikolaus- Fiebiger-Center, University of Erlangen-Nu¨rnberg, Erlangen, Germany the general lack of BCL6 detection in a range of mature B cells, Received for publication June 22, 2006. Accepted for publication August 24, 2006. including pre- and post-GC B cells, which are known to possess mRNA (18, 32–34). Similarly, IRF4, which is necessary for 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 mature naive B cell and dendritic cell development (14, 35), is with 18 U.S.C. Section 1734 solely to indicate this fact. usually negative in these cells by IHC staining using the MUM1 1 Address correspondence and reprint requests to Dr. Giorgio Cattoretti at the current mAb (15). address: Department of Pathology, Azienda Ospedaliera San Gerardo, Via Pergolesi 33, 20052 Monza (MI), Italy. E-mail address: [email protected] In this study, we show the expression of several TF in mouse 2 Abbreviations used in this paper: TF, transcription factor; IRF, IFN regulatory fac- and human lymphoid cells, with a special emphasis on BCL6 tor; GC, germinal center; AID, activation-induced cytidine deaminase; BCL, B cell and IRF4. We show broad coexpression of all TF before, but not lymphoma; DLBCL, diffuse large BCL; IHC, immunohistochemistry; DAPI, 4Ј,6Ј- diamidino-2-phenylindole; TMA, tissue microarray; FCM, flow cytometry; CLL, within, the GC reaction. In addition, we demonstrate that IRF4 chronic lymphocytic leukemia; PRDM1, positive regulatory domain 1. is expressed at the protein level in a much broader range of

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 6931

Table I. Abs used

Ab Clone or Serum Immunogena Reactive onb Species Source Dilution

PU.1 (Spi1) sc-352 C-term mouse h, m Rabbit SCBT 1 ␮g/ml PU.1 (Spi1) sc-5949 N-term mouse m Goat SCBT 0.1 ␮g/ml PU.1 (Spi1) G148-74 Full length h, m Mouse BD 1 ␮g/ml IRF4 sc-6059 C-term mouse h, m Goat SCBT 0.2 ␮g/ml IRF4 No. 4964 Asp175 h Rabbit CST 1/100 IRF4 sc-28696 AA 128–267h h, m Rabbit SCBT 1 ␮g/ml IRF4 MUM1 AA 144–451h h Mouse B. Falini 1/50 IRF8 (ICSBP) sc-6058 C-term mouse h, m Goat SCBT 1 ␮g/ml BCL6 PIF6 N-term human h, m Mouse Novocastra 1/100 BCL6 PGB6 276 N-term human h, m Mouse B. Falini 1/10 BCL6 PGB6p 594 N-term human h Mouse B. Falini 1/10 BCL6 sc-858 N-term human h, m Rabbit SCBT 0.1 ␮g BCL6 No. 4242 C-term human h, m Rabbit CST 1/100 Pax-5 24 AA 151–306h h, m Mouse BD 1 ␮g/ml Pax-5 sc-1974 C-term human h, m Goat SCBT 1 ␮g/ml Pax-5 RB-9406 C-term human h, m Rabbit LabVision 1 ␮g/ml Oct-2 sc-233 C-term human h, m Rabbit SCBT 0.2 ␮g/ml CD20 RB-9013 C-term human h Rabbit LabVision 1/200 CD20 L26 Cytoplasm. CD20 h Mouse DakoCytomation 1/200 Downloaded from MCM7 47DC141 hCDC47 h, m Mouse LabVision 0.5 ␮g/ml Ki-67 SP6 C-term human h, m Rabbit LabVision 1/100 Ki-67 MIB 1 rKi-67 h Mouse J. Gerdes 1/50 PRDM1 Serum AA 1–350 h, m Rabbit H.-M. Ja¨ck 1/5000 PRDM1 3H2E8 AA 199–409 h, m Mouse K. L. Calame 1/30 PRDM1 6D3 rBlimp-1 h, m Rat L. Corcoran 1 ␮g/ml AID EK2-5G9 AA 185–198 h Rat E. Kremmer 1/200

IRTA1 mIRTA1 AA 102–373 h Mouse B. Falini 1/10 http://www.jimmunol.org/ Neg. control Rabbit Sigma-Aldrich 1 ␮g/ml Neg. control Mouse Sigma-Aldrich 1 ␮g/ml

a C-term, C-terminal; N-term, N-terminal; Cytoplasm., Cytoplasmic. b h, Human; m, murine.

mature B cell types, as detected by other Abs not limited to a nal amplification for single or double labeling (39) was performed accord- conformational epitope recognized by the MUM1 Ab. ing to the manufacturer’s instructions (PerkinElmer): briefly, avidin-HRP-

counterstained immunostains were followed by tyramide-FITC or -biotin by guest on October 4, 2021 and Avidin Cy-3 (Jackson ImmunoResearch Laboratories) or Avidin- Materials and Methods Alexa350 (Molecular Probes). The slides were counterstained with 4Ј,6Ј- Cells and tissues diamidino-2-phenylindole (DAPI) (Molecular Probes) when indicated and mounted. IRF4 knockout (17), BCL6 knockout mice (19), and their wild-type litter- ϫ mates (C57BL6 and F1 from C57BL6 129Sv) were gifts from U. Klein and R. Dalla-Favera (Columbia University, New York, NY). Anonymous Image acquisition and analysis tonsil cell suspensions and tumor samples were provided and processed as Gray scale or color images were taken on an E600-Nikon Microscope, published (36). A tissue microarray containing B cell lymphomas and ref- fitted with Planachromat ϫ4/0.10/30.0, ϫ10/0.25/10.5, ϫ20/0.40/1.3, erence tissues was published previously (37). PlanApo ϫ40/0.095/0.12–0.16 (light microscopy) or PlanFluor ϫ10/0.30/ ϫ ϫ Antibodies 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 Instru- Abs used in this study are listed in Table I. Multiple different anti-BCL6 ments). A Zeiss LSM 510 NLO Multiphoton Confocal Microscope (Carl Abs, which all reacted identically in various assays and combinations, will Zeiss) equipped with ϫ10/0.3 and ϫ40/1.3 Fluor objectives, one 25-mW be referred to collectively as “BCL6.” The term “MUM1” will be restricted argon laser exciting at 458, 488, and 514 nm and one 1 mW helium-neon to anti-IRF4 Abs directed against aa 128–267 or aa 144–451 of the human laser exciting at 543 nm, and proprietary image acquisition software was protein. used for confocal analysis. All images were edited for optimal color contrast with Adobe Photoshop Immunohistochemistry 7 and Adobe Illustrator 10 (Adobe Systems), on a G4 Apple computer. Sections were essentially stained as previously published (36, 38), includ- ing double staining on 1 mM EDTA (pH 8) Ag-retrieved slides. Species- Flow cytometry specific, alkaline phosphatase-conjugated secondary Abs, prescreened for specificity and absence of cross-reactivity, were developed with nitroblue Live human or murine cell suspensions were stained with appropriate com- tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (Roche). The binations of fluorochrome- or biotin-conjugated primary Abs for 15 min on ϩ ice in PBS-BSA-NaN , washed twice with the same medium and further percentage and intensity of staining of CD20 neoplastic B cells were 3 independently scored in the DLBCL tissue microarray (TMA), each using processed for nuclear staining. For BCL6 staining, cells were fixed with a 10-tiered scale (0–9). The product of both was used as a case score and FACS lysing solution (BD Biosciences) for 10 min on ice, washed twice in PBS-BSA-NaN , incubated overnight with negative mouse IgG1 (1 ␮g/ml) a value of 10 or greater was considered positive for ␹2 calculations. 3 or PGB6 clone 276 (1/1000) ϩ P1F6 (1/100) mouse IgG1 anti BCL6 Immunofluorescence mixture. After two washes in PBS-BSA-NaN3, the cells were counter- stained with rat anti-mouse IgG1 PE-(1/200) or biotin-conjugated (1/300; Double immunofluorescence was performed with primary Abs raised in BD Pharmingen), this latter followed by fluorochrome-conjugated avidin different species or of different isotypes, counterstained with FITC- or Cy3- (1/500). IRF4, Pax5, and PU.1 Ab stainings requires a modified fixation, conjugated, species- or isotype-specific secondary Abs (Jackson Immu- which destroys prebound fluorochromes and some surface Ags, but not noResearch Laboratories) or biotin-conjugated secondary Abs, followed by biotin. Therefore, cells were first stained with biotin-conjugated Abs, fixed conjugated avidin (Jackson ImmunoResearch Laboratories). Tyramide sig- as above, washed in PBS, fixed in absolute cold methanol for 10 min, 6932 IRF4, BCL6, AND B CELL TF COEXPRESSION

washed in PBS, transferred to PBS-BSA-NaN3, and processed for intra- The GC contains morphologically and phenotypically different cellular staining. Counterstaining of the nuclear Ag was followed by flu- and well-defined zones, comprised predominantly of B cells, orochrome-avidin staining of the biotin-labeled Ab and staining for Ags which correspond to different functional and/or maturational sub- which survive the double fixation (e.g., IgD, CD38). IgD was stained with a PE-conjugated rat anti-mouse IgD (Southern Biotechnology Associates) sets. Extrafollicular B cells, however, are more dispersed and or with a PE-conjugated mouse anti-human IgD (BD Biosciences), pre- therefore better defined by double staining (36, 40) rather than by ceded by excessive cold mouse Ig blocking of residual anti-mouse IgG topographic location. We analyzed the coexpression of IRF4 and moieties. BCL6 staining is not affected by the methanol postfixation. other B cell TF in four B cell subsets: the CD30ϩ activated cells, Goat and rabbit primary Abs (IRF4, PU.1, negative control) were used ϩ ␮ the extrafollicular AID blasts (36), the centrocytes in the GC at 1 g/ml, incubated overnight, and counterstained with either donkey ϩ Ј light zone, and the extrafollicular memory/marginal zone IRTA1 anti-rabbit Cy5 F(ab )2 (1/500; Jackson ImmunoResearch Laboratories) or donkey anti-goat-FITC (1/500; Jackson ImmunoResearch Laboratories). cells (41). For double IRF4, PU.1 and BCL6 stainings, human cells were surface- CD30ϩ extrafollicular blasts are largely B cells showing evi- stained first with mouse anti CD3-biotin (BD Pharmingen), followed by ϩ dence of BCR plus cofactor-mediated acute activation (36), and nuclear staining, exclusion by gating of the CD3 cells, and analysis of the Ͼ IRF4 has been shown to be part of this signature (42). Accord- non-T cells ( 95% B cells). ϩ ϩ ϩ More than 30,000 events were acquired on a FACSCalibur (BD Bio- ingly, CD30 cells were largely IRF4 and Pax5 . The intensity sciences) and CellQuest acquisition software and analyzed with FlowJo 6.4 of Pax5 expression was inversely related to intensity of IRF4 ex- (Tree Star). pression (Fig. 3H). cDNA microarray analysis of human samples B cells express AID upon in vitro activation (43) and IRF4 is necessary for AID expression (17). Approximately half of the Data were obtained from published databases (1), obtained by Affymetrix AIDϩ extrafollicular blasts were IRF4ϩ, MUM1Ϫ (Fig. 3, A and

U95A chip analysis of normal tonsil subsets. Downloaded from B), with the intensity of IRF4 often noticeably dim, compared with Fluorescence in situ hybridization that of CD30ϩ extrafollicular blasts. This was in sharp contrast to LSI BCL6 dual-color, break-apart rearrangement, and Spectrum Green- the GC where AID and IRF4 were rigorously mutually exclusive labeled CEP 8 probes were obtained from Abbott Molecular. Four 5-␮M both in the dark and outer zone (Fig. 3A). Thus, appreciable levels thick tissue sections of two TMAs were cut onto adhesive-coated slides. of IRF4 are detectable in extrafollicular AIDϩ cells, possibly be- Paraffin-sections were baked overnight at 60°C before hybridization. TMA slides were subjected to protease treatment using paraffin pretreatment kit fore they enter the GC reaction, when IRF4 is shut off. (Abbott Molecular). Fluorescence in situ hybridization was performed by Commitment to the plasma cell lineage within the GC is marked http://www.jimmunol.org/ standard methods and hybridization signals were scored on at least 200 by PRDM1 expression, at first in Pax5ϩ centrocytes, then in interphase nuclei on DAPI-stained slides (26). The sensitivity of hybrid- CD20Ϫ, Pax5Ϫ, and CD138Ϫ preplasma cells (38). The bulk of ization on paraffin-embedded tissues was determined by performing the ϩ same analysis on analogous sections from normal tonsils and similarly bright IRF4 centrocytes coexpress PRDM1 and are Pax5 nega- processed cell lines of known genomic asset. Cases were diagnosed as tive (Fig. 3, I, L, and M). In addition, a minority of Pax5-positive rearranged if the fraction of cells showing the rearrangement in Ͼ5% cells. or Pax5 weakly positive centrocytes express IRF4 and/or PRDM1 (Fig. 3I). The MUM1 epitope is not detected on these latter cells Results (Fig. 3L). Distribution of B cell TF by tissue IHC The fourth B cell subset we focused on is defined by the FCRL4/ by guest on October 4, 2021 The distribution of the transcription factors Pax-5, Oct-2, PU.1, IRTA1 Ag (41, 44). IRTA1ϩ cells are monocytoid B cells with and IRF8 (Fig. 1) in mouse and human lymphoid tissues is broad evidence of Ag selection and variable Ig gene mutation (44), thus and overlaps among different B cell subsets (morphologically and memory B cells, located within the tonsil festooned epithelium and phenotypically defined follicular mantle, marginal, and GC cells) scattered in the interfollicular areas. Although intraepithelial as shown by in situ immunostaining with Abs validated for spec- IRTA1ϩ cells were largely IRF4Ϫ, interfollicular and intrafollicu- ificity (Fig. 2). BCL6 and IRF4 instead show a mutually exclusive lar IRTA1ϩ cells contained IRF4ϩ (Fig. 3C). This is consistent distribution; BCL6 is highly expressed in GC, IRF4 instead is pos- with the reported expression of IRF4 by profiling itive in mantle and marginal zone B cells, and negative in most GC in purified memory B cells (1) (Fig. 1). IRTA1ϩ B cells were B cells. The expression of IRF4 in IRF8ϩ, PU.1ϩ, Oct-2ϩ, and previously reported negative for IRF4 with the MUM1 Ab. Pax5ϩ follicular mantle B cells could be seen only with Abs di- In summary, IRF4 is coexpressed with the other B cell TF in rected against the C-terminal and Asp175, and not with two other pre- and post-GC B cells, but not inside the GC, where it is spe- Abs directed against aa 128–267 of the IRF4 protein (hence named cifically down-regulated during transit. 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 pro- Distribution of IRF4 and BCL6 in lymphoid tissues by flow teins in human tonsil tissue with the RNA levels, analyzed by gene cytometry expression profiling in purified B cell subsets (1). As shown in Fig. BCL6 staining in the follicle mantle is usually negative, however, 1, the RNA levels of each TF in GC, mantle, and marginal zone B occasionally we are able to detect it by IHC only in selected mouse cells correspond to the distribution of the respective protein Ags, samples (Fig. 2). BCL6 is among the most variably expressed including IRF4 by C-terminal and Asp175 Ab staining. genes in the mouse (45) and our inconsistent results may be due to Single-color staining and tissue RNA or protein extraction do either sample-to-sample variation and/or insufficient sensitivity. not allow a detailed analysis of the coexpression of two given Ags To assess in a quantitative fashion BCL6 and IRF4, we developed on a cell-by-cell basis. Therefore, we analyzed by double immu- and validated (Fig. 2) a flow cytometry (FCM) assay, which has nofluorescence human (Fig. 3, D–N) and murine (data not shown) the additional advantage of allowing multiparameter phenotypic samples. As predicted from the observed distribution by single- characterization of B cell subsets. color staining, IRF4 was largely mutually exclusive with BCL6 Mouse cells of defined BCL6 genomic dosage showed specific, (15), PU.1, IRF8, and Pax5 in GC, while coexpressed on PU.1ϩ, gene-dependent, near-ubiquitous biallelic BCL6 staining in most B IRF8ϩ, and Pax5ϩ mantle cells. cell subsets (Fig. 2), thymus and myeloid cells (data not shown). In Staining with all IRF4 Abs was both nuclear and cytoplasmic, particular, follicular B cells (B220ϩIgDϩ) showed detectable the latter was least evident with Abs directed against aa 128–267. BCL6, consistent with the occasional staining seen by IHC. IRF4 The Journal of Immunology 6933 Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

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 (ϫ10) images are on top, high power (ϫ40) 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 (ϫ4) images are on top, high power (ϫ40) 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 BCL6ϩ,IRF4ϩ population, corresponding to mantle zone B cells and a BCL6ϩϩ, IRF4 negative or weak population, corre- sponding 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- We then evaluated by IHC the mutual distribution of IRF4 and BCL6 in a series of 40 DLBCL. We found 45% (18 of 40) coex- pressed BCL6 and IRF4, 45% were BCL6ϩ only, 7.5% (3 of 40) were IRF4ϩ only, and 2 were negative for both (Fig. 6). A semi- quantitative evaluation of the staining showed two distinctly dis- tributed 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 Downloaded from 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 IRF4ϩ cases not expressing the MUM1 epitope (Fig. 6B).

To understand the mutual relationship of BCL6 and IRF4 at the http://www.jimmunol.org/ 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 evalu- 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 ated. Six cases of chronic lymphocytic leukemia (CLL), two man- (strongly) and in the follicles (weakly). The negative control Ab and the tle cell lymphomas, and two marginal zone lymphomas, showed BCL6 Ab on a BCL6 knockout (KO) lymph node are negative. A wild-type more extensive but not consistent IRF4 staining, compared with ϩ and knockout mouse spleen are stained by IRF4; the knockout spleen is MUM1 (Fig. 8). One CLL case was BCL6 by IHC (data not by guest on October 4, 2021 negative. B, Four mice, each bearing none, one, two BCL6 alleles, and one shown). BCL6 and IRF4 expression was confirmed by FCM in two with one C-terminally truncated allele, are analyzed in the thymus and representative CLL cases, one of which showed coexpression (data spleen for BCL6 expression by flow cytometry. The gray histogram is an not shown). isotype-matched negative control. Note the increased intensity (rightward Thus, B cell lymphomas, as their normal B cell counterparts, shift of the peak fluorescence) of BCL6 staining with increased genetic show coexpression of BCL6 and IRF4. However, in tumors of dosage. The gating for the two B cell subsets are indicated at right. The putative GC origin, such as a group of DLBCL, IRF4 bears the truncated allele produces increased amounts of protein, because of the lack ϩ of autoregulation (Wang et al. (52)). C, Spleen cells from an IRF4 knock- MUM1 conformational epitope in BCL6 cells. out mouse (KO) and a wild-type littermate (wt) were stained for B220, CD11b, negative control (gray histogram), or IRF4 (black line). Note ab- Discussion sence of specific staining in the IRF4 KO mouse. BCL6 is a potent transcriptional repressor expressed at high levels in GC cells, and presumably prevents terminal B cell differentia- tion by repressing key genes, such as PRDM1/Blimp-1(48). BCL6 knockout splenic B cells were negative for IRF4 staining, com- may also have other important functions, such as antiapoptotic pared with positive wild-type littermate B cells (Fig. 2). activity (49). Recently, Pax5 has been shown to have an essential As predicted by gene expression profiling of human tonsils, role in maintaining the B cell phenotype in GC cells (5). IRF8 and BCL6 and IRF4 were expressed in mantle zone B cells (CD20ϩ, IRF4 are also needed to complete essential activities within the IgDϩCD38Ϫ/dim) (Fig. 4). Plasmacytoid cells (CD20ϩ, GC, such as somatic mutation and class switch recombination of Ig IgDϪ, CD38ϩϩ) were BCL6ϪIRF4ϩϩ. On the contrary, GC cells genes through AID induction (13, 17). The emerging picture re- (CD20ϩ, IgDϪ, CD38ϩ) were BCL6ϩϩ,IRF4Ϫ. Memory B cells quires an understanding of the topographic distribution of all these (CD20ϩ, IgDϪ, CD38Ϫ) showed IRF4 positivity and traces of actors before, after, and through the GC reaction, such knowledge BCL6. Interestingly, GC founder cells (CD20ϩ, IgDϩ, CD38ϩϩ) has been lacking or largely incomplete. It is also important to know (46) contained lower levels of IRF4 than mantle zone or memory not only the distribution but also the abundance of these factors. B cells, and two populations of BCL6low and BCL6high B cells PU.1 is an example of that, because levels PU.1 are critical for (Fig. 4). normal physiology of multiple cell types (50). These data have Furthermore, tonsil cell suspensions were stained for BCL6, deep implications in interpretation of molecular interaction data in IRF4, Pax5, CD3, and IgD, to define total (CD3Ϫ), GC (IgDϪ), the right normal physiologic context. and mantle zone B cells (IgDϩ) (Fig. 5). Pax5ϩ,IgDϪ GC B cells In the present study, we show that BCL6 and IRF4 are broadly contained no IRF4 and high levels of BCL6. The Pax5ϩ,IgDϩ coexpressed, together with Pax5, PU.1, and Oct-2, in mature B fraction (containing mantle zone and IgDϩ GC founder cells) con- cells, before and after entering the GC reaction, a finding largely The Journal of Immunology 6935 Downloaded from http://www.jimmunol.org/

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 IRF4ϩ centrocytes are AIDϪ. Original magnification, ϫ10; scale bar, 15 ␮m. B, Enlargement of portion of extrafollicular area detailing AIDϩ (brown) cells containing variable amounts of IRF4 (purple). The edge of a GC is shown (arrow). Original magni- fication, ϫ40; scale bar, 15 ␮m. C, IRTA1ϩ (brown) lymphocytes express IRF4 (purple) in the mantle/marginal zone (yellow arrows, enlarged in the inset), by guest on October 4, 2021 but not in the intraepithelial location (far right). ✮, GC. Original magnification, ϫ40; 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, ϫ4; scale bar, 100 ␮m. Right, Original magnification, ϫ40; scale bar, 5 ␮m. H, CD30ϩ (blue) interfollicular blasts coexpress IRF4 (green) and Pax5 (red). The boxed area is magnified and the color split on the right. Note two IRF4ϩPax5ϩCD30Ϫ blasts (arrows) at the edge of the GC (✮). Original magnification ϫ40, 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 Pax5ϩPRDM1ϩ 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 PRDM1ϩIRF4Ϫ. L, Light zone centrocytes are IRF4ϩ (green), PRDM1ϩ (red), and a minority express MUM1 (blue). I, K, and L, magnification, ϫ40; 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, ϫ40; scale bar, 5 ␮m. N, Light zone centrocytes show relocation of cREL (red, arrows) in IRF4Ϫ nuclei (green). Nuclei are stained with DAPI (blue). Original magnification, ϫ40; scale bar, 15 ␮m. expected according to previously published molecular and immu- Low levels of BCL6 are also physiologically relevant in my- nohistochemical studies (1, 31, 51). The mutual relationship of eloid-derived cells, in which BCL6, undetectable by routine IHC, these TF outside the GC is fine tuned by the amount of each TF in but detected by FCM, is necessary to repress an otherwise lethal a cell type-specific fashion, as demonstrated by quantitative flow immune dysregulation (33) and to control IL-6 signaling (32). cytometry. Thus, BCL6 is continuously expressed at various levels before and The role of BCL6 in pre- and post-GC B cells is unknown and during the GC reaction, and is eventually down-regulated in ter- probably redundant for B cell maturation and survival, because minally differentiated plasma cells. mantle zone B cells in BCL6 knockout mice are unaffected (19). In The presence of IRF4 in pre-GC B cells is expected because it addition, the protein levels of BCL6 in pre-GC cells are maintained is required for IgDϩIgMϩ B cell phenotypic maturation (16). The low by posttranscriptional down-regulation (31) and transcriptional- levels of IRF4 in human and murine mantle and marginal zone B negative autoregulation (52). However, unimmunized BCL6-trans- cells are quantitatively lower than the levels found in plasma cells genic mice with only moderately raised baseline BCL6 levels in and, notably, qualitatively different. The large majority of IRF4ϩ pre-GC cells experience GC formation to a level comparable to cells in mouse and human tissue cannot be detected by MUM1-like that of a potent polyclonal immunization (53), suggesting a “tonic” Abs directed against aa 128–267 of IRF4. The fact that these Abs role for such low protein levels. What is probably crucial is the recognize denatured IRF4 in a Western blot indicates that the mol- ability to rapidly increase BCL6 levels upon activation. We sug- ecule is in a peculiar conformational configuration which masks gest this increase to happen in GC founder cells. the MUM1-like epitope in most B cells and myelomonocytic cells. 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, CD20ϩ 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 (IgDϩCD38Ϫ), reduced IRF4 and emergence of a strong BCL6ϩ peak in GC founder cells (IgDϩCD38ϩ), disappearance of IRF4 in GC cells (IgDϪCD38ϩ), expression of IRF4 but minimal BCL6 in memory B cells (IgDϪCD38Ϫ) and high IRF4 expression in BCL6- plasmacytoid cells (IgDϪCD38ϩϩ).

IRF4 is structurally modified by an immunophilin (FKBP52 or At the entry of the GC, IRF4 is quickly down-regulated, while FKBP4) with peptidyl-prolyl isomerase and chaperone-like activ- BCL6 is up-regulated, as demonstrated by the absence of double ity (54), broadly expressed in all B cell subsets at the mRNA level IRF4-BCL6-positive cells by FCM and IHC inside the GC. The

(data obtained from Ref. 1) which binds to IRF4 in a segment situation is reversed in the light zone centrocytes committed to the Downloaded from centered on aa 150–237. FKBP4 binding induces a structural con- plasma cell lineage. There, down-regulation of BCL6 is followed formation which reduces the interaction of IRF4 with PU.1 and the by PRDM1 expression in cells which still express Pax5. Then, transcriptional activity on PU.1-binding target genes (54). FKBP4 Pax5, IRF8, and PU.1 are gradually down-regulated and IRF4 is binding is inhibited when the C-terminal autoinhibitory domain substantially up-regulated in cells which have already lost the B folds back on the N-terminal DNA-binding domain (54). cell program (38, 48) (Fig. 9). BCL6 is eventually totally lost on

Interestingly enough, the aa 128–267/MUM1 epitope is found these cells. The factors initiating the exit process are unknown at http://www.jimmunol.org/ predominantly on PU.1-negative cells (T cells, plasma cells, and the present time. We detected nuclear relocation of cREL, an melanocytes) (15, 55) and never present in myelomonocytic cells NF-␬B member, in BCL6ϩ and negative centrocytes in the light (G. Cattoretti, manuscript in preparation) or mantle zone B cells, zone (Ref. 57 and data not shown), largely in IRF4-negative but where IRF4 has been shown to be functionally active and required, also in some IRF4ϩ and PRDM1ϩ cells (38). Unfortunately, we in cooperation with PU.1 (16, 56). This suggests that the acquisi- could not investigate nuclear relocation of other NF-␬B members, tion of the MUM1ϩ conformation by IRF4 is associated with a therefore, the role of this group of TF in down-regulating BCL6 change in interacting partners or with reduced interaction with remains elusive. Recent studies have suggested that Pax5, not PU.1. Future studies with biochemical assays are needed to iden- BCL6, may be the key regulator to be switched off before plasma tify the interacting partners and the target DNA sequences. cell differentiation (5), and PRDM1 would be the TF that seals the by guest on October 4, 2021 plasma cell fate (58). The light zone contains Pax5ϩ 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, indi- cating that memory B cells are IRF4ϩ. 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 distri- FIGURE 5. Coexpression of BCL6, IRF4, and Pax5 in IgDϩ and IgDϪ bution of BCL6 and IRF4 in 40 DLBCL cases, according to the score of tonsil B cells. Two fractions of tonsil CD3Ϫ B cells (IgDneg and IgDpos) positive cells. u, The 10% lower limit for positivity for score (see Mate- are costained for IRF4, negative control, BCL6, and Pax5. The shaded gray rials and Methods). The number of cases in each quadrant is shown in area indicate IRF4 vs negative control. The contour image superimposed italics. Gray symbols represent cases polysomic for 3q27; asterisks repre- shows IRF4 vs a second marker distribution. At each side, the single dis- sent cases with 3q27 breaks (BCL6 rearrangement). Other cases are either tribution of the negative isotype control (gray histogram) and the respective normal or not tested (n ϭ 11). B, The correlation of IRF4 and MUM1 positive Ab (black line) are represented. Note two populations, one BCL6ϩ staining in 40 DLBCL cases is shown. u, The 10% lower limit for posi- IRF4 weak/negative and one BCL6 weak/IRF4 weak in the IgDϩ fraction. tivity for score. The Journal of Immunology 6937

FIGURE 7. Coexpression of BCL6 and IRF4 in DLBCL. Four repre- sentative 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 co- expression. Note the variety of ratios of the two Ags. Downloaded from Pax5ϩ 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 IRTA1ϩ 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. http://www.jimmunol.org/

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 CD30ϩ by guest on October 4, 2021 cells are shown on the left as precursors of both the GC and the extrafol- licular memory B cell pathway. On the right, a CD30ϩ intermediate is hypothesized (dashed) although no evidence of plasma cell commitment has been identified so far. Vertical gray bars are drawn to identify bound- aries of Ag expression per cell type or stage of differentiation. Extent of coexpression is approximate and not quantitative.

IRF4ϩ and/or PRDM1ϩ 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 com- mon 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 AIDϩ 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 AIDϩ 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 func- tion in the GC is replaced by another TF of the same class; IRF8 FIGURE 8. Comparison of MUM1 and IRF4 polyclonal Ab staining on and IRF4 have been previously shown to be partially redundant a sample of low grade non-Hodgkin lymphoma. Selected cases of low (56), thus IRF8 (which promotes AID expression (13)) may be a grade B cell lymphomas (CLL; MC, mantle cell lymphoma, MZ, marginal zone lymphoma) and tonsil are stained respectively with the MUM1 and candidate. Yet, the absence of AID in centrocytes suggests that the molecular lesions initiating class switch recombination occur in a the polyclonal IRF4 Ab. Identical fields on serial sections are shown. A ϩ low-power field (ϫ4) is shown at the sides. The polygon marks the tonsil cell upstream, which would be AID and may express IRF4 at a GC, the star the mantle. Note the more frequent positivity of the polyclonal certain point. We have identified putative cells fulfilling such cri- ϩ IRF4 Ab. teria. IRF4 is expressed in rare AID blasts at the edge of the GC 6938 IRF4, BCL6, AND B CELL TF COEXPRESSION and, more conspicuously, in extrafollicular AIDϩ cells. We have References shown that these cells are losing the phenotype of the acute BCR 1. Klein, U., Y. Tu, G. A. Stolovitzky, J. L. Keller, J. Haddad, Jr., V. Miljkovic, stimulation typical of CD30ϩ blasts (, JunB, CCL22) and ac- G. Cattoretti, A. Califano, and R. Dalla-Favera. 2003. Transcriptional analysis of Proc. Natl. Acad. Sci. USA quire characteristics that are similar to GC cells (36). Once these the B cell germinal center reaction. 100: 2639–2644. 2. Matthias, P., and A. G. Rolink. 2005. Transcriptional networks in developing and cells enter the GC reaction, if they do, then they may start the mature B cells. Nat. Rev. Immunol. 5: 497–508. molecular processes, completed later in the light zone. The re- 3. Shaffer, A. L., A. Rosenwald, E. M. Hurt, J. M. Giltnane, L. T. 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Gambacorta, blasts and some AIDϩ cells); IRF4 is also needed for plasma cell R. Pacini, C. Alunni, L. Natali-Tanci, B. Ugolini, et al. 2000. A monoclonal antibody (MUM1p) detects expression of the MUM1/IRF4 protein in a subset of differentiation (17), but is unclear whether the required form in this germinal center B cells, plasma cells, and activated T cells. Blood 95: Ϫ case is the modified type found in PU.1 cells. To add to the 2084–2092. by guest on October 4, 2021 complexity, DLBCL containing a modified IRF4 may at the same 16. Mittrucker, H. W., T. Matsuyama, A. Grossman, T. M. Kundig, J. Potter, A. Shahinian, A. Wakeham, B. Patterson, P. S. Ohashi, and T. W. Mak. 1997. time contain deregulated BCL6 or lack PRDM1 expression, the Requirement for the transcription factor LSIRF/IRF4 for mature B and T lym- other necessary factor to drive plasma cell differentiation (26, 63, phocyte function. Science 275: 540–543. 64). Finally, the mutual interactions between all these TF is only 17. Klein, U., S. Casola, G. Cattoretti, Q. Shen, M. Lia, T. Mo, T. Ludwig, K. Rajewsky, and R. Dalla-Favera. 2006. Transcription factor IRF4 controls partially elucidated and posttranscriptional regulation will add yet plasma cell differentiation and class-switch recombination. Nat. Immunol. 7: another layer of complexity to this multiplayer system. 773–82. Low-grade, pre-, or post-GC human B cell lymphomas (in con- 18. Cattoretti, G., C. C. Chang, K. Cechova, J. Zhang, B. H. Ye, B. Falini, D. C. Louie, K. Offit, R. S. Chaganti, and R. Dalla-Favera. 1995. BCL-6 protein trast to DLBCL) resemble their normal counterpart by expressing is expressed in germinal-center B cells. Blood 86: 45–53. low levels of IRF4 and BCL6. However, there is significant vari- 19. Ye, B. H., G. Cattoretti, Q. Shen, J. Zhang, N. Hawe, R. de Waard, C. Leung, ability among different cases, possibly reflecting the state of acti- M. Nouri-Shirazi, A. Orazi, R. S. Chaganti, et al. 1997. The BCL-6 proto-onco- gene controls germinal-centre formation and Th2-type inflammation. Nat. Genet. vation or the fluctuation of baseline BCL6 expression (45). From 16: 161–170. a practical point of view, use of MUM1 Ab for prognostication in 20. Jaffe, E. S., N. L. Harris, H. Stein, and W. V. Vardiman. 2001. Pathology and DLBCL should remain valid (61), but should be reconsidered for Genetics of Tumors of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon, ϩ France. any non-GC, PU.1 cell type. 21. Alizadeh, A. A., M. B. Eisen, R. E. Davis, C. Ma, I. S. Lossos, A. Rosenwald, J. C. Boldrick, H. Sabet, T. Tran, X. Yu, et al. 2000. Distinct types of diffuse large Acknowledgments B-cell lymphoma identified by gene expression profiling. Nature 403: 503–511. 22. Shaffer, A. L., A. Rosenwald, and L. M. Staudt. 2002. Lymphoid malignancies: We thank Ulf Klein, Masumichi Saito, Riccardo Dalla-Favera, and Michael the dark side of B-cell differentiation. 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