Stages of Germinal Center Transit Are Defined by B Cell Transcription Factor Coexpression and Relative Abundance This information is current as Giorgio Cattoretti, Rita Shaknovich, Paula M. Smith, of September 27, 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 References This article cites 63 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/177/10/6930.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 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 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 http://www.jimmunol.org/ normal counterpart. The Journal of Immunology, 2006, 177: 6930–6939. 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 gene expressed throughout all B cell stages, except in plasma cells (6). mutation and lack of terminal differentiation (20). Detailed molec- by guest on September 27, 2021 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 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 genes (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 chromosome 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 protein 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.