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Up-Regulation of BOB.1/OBF.1 Expression in Normal Germinal Center B Cells and Germinal Center-Derived Lymphomas

2000; Elsevier BV; Volume: 156; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)64754-2

ISSN

1525-2191

Autores

Andreas Greiner, Kerstin Müller, Jochen Heß, Klaus Pfeffer, H. K. Müller-Hermelink, Thomas Wirth,

Tópico(s)

Cancer-related molecular mechanisms research

Resumo

The BOB.1/OBF.1/OCAB.1 protein is a lymphocyte-specific transcriptional coactivator. It interacts with the Oct1 and Oct2 transcription factors and contributes to the transcriptional activity of octamer motifs. The analysis of established B cell lines had suggested that BOB.1/OBF.1 is constitutively expressed at all stages of B cell development. Here we show that expression of BOB.1/OBF.1 is regulated within the B cell lineage. Specifically, germinal center B cells show highly increased BOB.1/OBF.1 levels. We can induce the up-regulation by stimulating primary splenic B cells, eg, by triggering CD40 signaling in the presence of interleukin-4. Expression of BOB.1/OBF.1 is detectable but reduced in spleens from mice unable to undergo the germinal center reaction due to mutations in the TNF receptor p55 or lymphotoxin β (LTβ) receptor genes. Furthermore, we demonstrate that BOB.1/OBF.1 expression is highly regulated in human B cell lymphomas. Whereas lymphomas representing pre- and postfollicular B cell developmental stages are negative for BOB.1/OBF.1, high-level expression of BOB.1/OBF.1 is characteristic of germinal center-derived tumors. In these tumors BOB.1/OBF.1 is typically coexpressed with high levels of Bcl6. These results imply that overexpression of BOB.1/OBF.1, like overexpression of Bcl6, might play a role in the pathogenesis of germinal center-derived B cell lymphomas. Furthermore, overexpression of BOB.1/OBF.1 represents a characteristic feature of these tumors that is useful in their identification. The BOB.1/OBF.1/OCAB.1 protein is a lymphocyte-specific transcriptional coactivator. It interacts with the Oct1 and Oct2 transcription factors and contributes to the transcriptional activity of octamer motifs. The analysis of established B cell lines had suggested that BOB.1/OBF.1 is constitutively expressed at all stages of B cell development. Here we show that expression of BOB.1/OBF.1 is regulated within the B cell lineage. Specifically, germinal center B cells show highly increased BOB.1/OBF.1 levels. We can induce the up-regulation by stimulating primary splenic B cells, eg, by triggering CD40 signaling in the presence of interleukin-4. Expression of BOB.1/OBF.1 is detectable but reduced in spleens from mice unable to undergo the germinal center reaction due to mutations in the TNF receptor p55 or lymphotoxin β (LTβ) receptor genes. Furthermore, we demonstrate that BOB.1/OBF.1 expression is highly regulated in human B cell lymphomas. Whereas lymphomas representing pre- and postfollicular B cell developmental stages are negative for BOB.1/OBF.1, high-level expression of BOB.1/OBF.1 is characteristic of germinal center-derived tumors. In these tumors BOB.1/OBF.1 is typically coexpressed with high levels of Bcl6. These results imply that overexpression of BOB.1/OBF.1, like overexpression of Bcl6, might play a role in the pathogenesis of germinal center-derived B cell lymphomas. Furthermore, overexpression of BOB.1/OBF.1 represents a characteristic feature of these tumors that is useful in their identification. The octamer motif is an important regulatory element for B-cell-specific transcription.1Wirth T Staudt L Baltimore D An octamer oligonucleotide upstream of a TATA motif is sufficient for lymphoid-specific promoter activity.Nature. 1987; 329: 174-178Crossref PubMed Scopus (299) Google Scholar It is conserved in virtually all immunoglobulin heavy and light chain gene promoters as well as in several immunoglobulin enhancer elements. It is essential for the B-cell-specific promoter function and contributes to immunoglobulin enhancer activity in B cells.2Staudt LM Lenardo MJ Immunoglobulin gene transcription.Annu Rev Immunol. 1991; 9: 373-398Crossref PubMed Scopus (251) Google Scholar B-cell-specific transcription of octamer-dependent promoters has been shown to require additional coactivators, which functionally interact with the Oct1 and/or Oct2 transcription factors.3Pierani A Heguy A Fujii H Roeder RG Activation of octamer-containing promoters by either octamer-binding transcription factor 1 (OTF-1) or OTF-2 and requirement of an additional B-cell-specific component for optimal transcription of immunoglobulin promoters.Mol Cell Biol. 1990; 10: 6204-6215Crossref PubMed Scopus (84) Google Scholar, 4Luo Y Fujii H Gerster T Roeder RG A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors.Cell. 1992; 71: 231-241Abstract Full Text PDF PubMed Scopus (251) Google Scholar, 5Annweiler A Müller-Immerglück M Wirth T Oct2 transactivation from a remote enhancer position requires a B-cell-restricted activity.Mol Cell Biol. 1992; 12: 3107-3116Crossref PubMed Scopus (59) Google Scholar, 6Pfisterer P Annweiler A Ullmer C Corcoran L Wirth T Differential transactivation potential of Oct1 and Oct2 is determined by additional B cell-specific activities.EMBO J. 1994; 13: 1654-1663Crossref PubMed Scopus (65) Google Scholar The BOB.1/OBF.1 coactivator was identified a few years ago and was shown to be a critical determinant of octamer-dependent gene transcription in B lymphocytes.7Gstaiger M Knoepfel L Georgiev O Schaffner W Hovens CM A B-cell coactivator of octamer-binding transcription factors.Nature. 1995; 373: 360-362Crossref PubMed Scopus (290) Google Scholar, 8Strubin M Newell JW Matthias P OBF-1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer proteins.Cell. 1995; 80: 497-506Abstract Full Text PDF PubMed Scopus (354) Google Scholar, 9Luo Y Roeder RG Cloning, functional characterization, and mechanism of action of the B-cell-specific transcriptional coactivator OCA-B.Mol Cell Biol. 1995; 15: 4115-4124Crossref PubMed Scopus (250) Google Scholar, 10Pfisterer P Zwilling S Hess J Wirth T Functional characterization of the murine homolog of the B-cell-specific coactivator BOB.1/OBF.1.J Biol Chem. 1995; 270: 29870-29880Crossref PubMed Scopus (61) Google Scholar This coactivator does not recognize the octamer motif with high affinity,11Cepek KL Chasman DI Sharp PA Sequence-specific DNA binding of the B-cell-specific coactivator OCA-B.Genes Dev. 1996; 10: 2079-2088Crossref PubMed Scopus (86) Google Scholar but rather is recruited into the transcription complexes via protein-protein interactions with the POU domains of the Oct proteins.12Gstaiger M Georgiev O van Leeuwen H van der Vliet P Schaffner W The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with the Oct-1/Oct-2 factors, leading to differential promoter activation.EMBO J. 1996; 15: 2781-2790Crossref PubMed Scopus (125) Google Scholar Expression of BOB.1/OBF.1 is restricted largely to B lymphocytes. Analyses of BOB.1/OBF.1 expression in a variety of established B cell lines representing different stages of B cell development had suggested a constitutive, B-cell-specific expression pattern.13Schubart DB Sauter P Massa S Friedl EM Schwarzenbach H Matthias P Gene structure and characterization of the murine homologue of the B cell-specific transcriptional coactivator OBF-1.Nucleic Acids Res. 1996; 24: 1913-1920Crossref PubMed Scopus (44) Google Scholar Furthermore, cell fusion experiments between B cells and fibroblasts, where the resulting somatic cell hybrids show a dominance of the fibroblast phenotype, revealed that BOB.1/OBF.1 expression is extinguished in these hybrids.14Reich L Sharir H Ber R Wirth T Bergman Y Laskov R Coordinate suppression of myeloma-specific genes and expression of fibroblast-specific genes in myeloma × fibroblast somatic hybrids.Somatic Cell Mol Genet. 1996; 22: 1-20Crossref PubMed Scopus (7) Google Scholar Interestingly, expression of BOB.1/OBF.1 can be induced in T lymphocytes by costimulation with phorbol ester (PMA) and ionomycin.15Zwilling S Dieckmann A Pfisterer P Angel P Wirth T Inducible expression and phosphorylation of coactivator BOB.1/OBF.1 in T cells.Science. 1997; 277: 221-225Crossref PubMed Scopus (78) Google Scholar Furthermore, it was demonstrated that BOB.1/OBF.1 transactivation function in T cells is regulated by costimulation-induced phosphorylation of the transactivation domain.15Zwilling S Dieckmann A Pfisterer P Angel P Wirth T Inducible expression and phosphorylation of coactivator BOB.1/OBF.1 in T cells.Science. 1997; 277: 221-225Crossref PubMed Scopus (78) Google Scholar Recently, a specific up-regulation of BOB.1/OBF.1 expression in murine germinal center B cells has been demonstrated.16Qin XF Reichlin A Luo Y Roeder RG Nussenzweig MC OCA-B integrates B cell antigen receptor-, CD40L-, and IL 4-mediated signals for the germinal center pathway of B cell development.EMBO J. 1998; 17: 5066-5075Crossref PubMed Scopus (96) Google Scholar Mice deficient for the BOB.1/OBF.1 coactivator showed specific defects, which were largely restricted to late stages of B cell development.17Schubart DB Rolink A Kosco-Vilbois MH Botteri F Matthias P B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune response, and germinal centre formation.Nature. 1996; 383: 538-542Crossref PubMed Scopus (252) Google Scholar, 18Kim U Qin FF Gong S Stevens S Luo Y Nussenzweig M Roeder RG The B-cell-specific transcription coactivator OCA-B/OBF-1/Bob-1 is essential for normal production of immunoglobulin isotypes.Nature. 1996; 383: 542-547Crossref PubMed Scopus (219) Google Scholar, 19Nielsen PJ Georgiev O Lorenz B Schaffner WB lymphocytes are impaired in mice lacking the transcriptional co-activator Bob1/OCA-B/OBF1.Eur J Immunol. 1996; 26: 3214-3218Crossref PubMed Scopus (124) Google Scholar The most prominent phenotype was the complete absence of germinal centers in the secondary lymphoid organs of these mice. No gross alterations of antigen-independent B cell development was observed, although the actual number of mature B cells in the spleen was reduced two- to fourfold in these mice. Neither rearrangement of immunoglobulin genes nor expression of the μ-heavy chain were measurably affected in these mice. However, consistent with the failure of germinal center development, BOB.1/OBF.1-deficient mice showed a strong reduction in humoral responses to both T-cell-dependent and T-cell-independent antigens. The levels of secondary immunoglobulin isotypes other than IgM were dramatically reduced. Here we have analyzed the expression of BOB.1/OBF.1 in mouse and human primary B cells representing distinct stages of B cell development. Given the presumed role of BOB.1/OBF.1 in germinal center development, we were very interested in determining its expression levels in germinal center B cells compared to different B cell differentiation stages. In addition, we have investigated BOB.1/OBF.1 expression in a variety of human B cell lymphomas, which represented both germinal center-derived and non-germinal center-derived tumors. Immunohistochemical stainings were performed on 4-μm cryostat sections of fresh frozen surgical specimens. Biopsy tissues were kept at −70°C as snap-frozen blocks until sections were prepared at the time of the experiments. Tumor samples were selected randomly from the German lymph node registry of the Institute of Pathology in Würzburg and classified according to REAL classification.20Harris NL Jaffe ES Stein H Banks PM Chan JK Cleary ML Delsol G De Wolf-Peeters C Falini B Gatter KC A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar The immunoperoxidase method was applied using a three-step incubation procedure with diluted affinity-purified rabbit anti-BOB.1/OBF.1 antibodies and the preimmune serum as control, as described in detail elsewhere.21Greiner A Marx A Müller-Hermelink HK Schmausser B Petzold K Kruger H Vascular autoantibodies in amyotrophic lateral sclerosis.Lancet. 1992; 340: 378-379Abstract PubMed Scopus (12) Google Scholar For double stainings on cryostat sections the immunoperoxidase-alkaline phosphatase double staining technique was used according to Krenn et al.22Krenn V Schalhorn N Greiner A Molitoris R Konig A Gohlke F Müller-Hermelink HK Immunohistochemical analysis of proliferating and antigen-presenting cells in rheumatoid synovial tissue.Rheumatol Int. 1996; 15: 239-247Crossref PubMed Scopus (43) Google Scholar In brief, BOB.1/OBF.1 was first detected using indirect immunoperoxidase staining. Thereafter, the alkaline anti-alkaline phosphatase (APAAP. method was applied for Bcl6 detection (mAb D-8; Santa Cruz Biotechnology, Santa Cruz, CA). The BOB.1/OBF.1-specific antibody used in these studies has been described earlier.10Pfisterer P Zwilling S Hess J Wirth T Functional characterization of the murine homolog of the B-cell-specific coactivator BOB.1/OBF.1.J Biol Chem. 1995; 270: 29870-29880Crossref PubMed Scopus (61) Google Scholar In brief, the antibody was raised in rabbits against the amino terminal domain of murine BOB.1/OBF.1, which is highly homologous to the human protein. The rabbit serum was affinity-purified using the recombinant BOB.1/OBF.1 protein. Control experiments showed that the antibody reacts equally well with murine and human BOB.1/OBF.1 in all experimental procedures. Viable single-cell suspensions of lymphocytes were isolated from reactive human tonsils by density-gradient centrifugation and depleted of T cells and macrophages using magnetic beads coupled either with anti-CD2 or anti-CD14 (Dynal, Hamburg, Germany). Thereafter, lymphocytes were >95% CD19+ as determined by fluorescence-activated cell sorting (FACS). They were further purified by positive selection with anti-CD38 (clone ACT 13.5, Serotec, Heidelberg, Germany)-coupled magnetic beads to isolate human germinal center B cells and anti-IgD (clone HJ9, Sigma, Heidelberg)-coupled beads to isolate mantle zone B cells. The remaining memory B lymphocytes stained CD19+/IgD−/CD38−/sIg+ as described elsewhere.23Arpin C Banchereau J Liu YJ Memory B cells are biased towards terminal differentiation: a strategy that may prevent repertoire freezing.J Exp Med. 1997; 186: 931-940Crossref PubMed Scopus (133) Google Scholar, 24Greiner A Knörr C Qin Y Sebald W Schimpl A Banchereau J Müller-Hermelink HK Low-grade B cell lymphomas of mucosa-associated lymphoid tissue (MALT-type) require CD40-mediated signaling, and Th2-type cytokines for in vitro growth and differentiation.Am J Pathol. 1997; 150: 1583-1593PubMed Google Scholar If purity of cell isolates was 95% or better, cell suspensions were subjected to further analyses. Each fraction was checked by morphology using Pappenheim-stained cytosmears. Furthermore, three color flow cytometric analyses were performed with a FACScan (Becton Dickinson, Heidelberg, Germany) using an Argon ion laser tuned at 488 nm, with LYSIS II (FACS analysis software, Becton Dickinson) for data acquisition and analysis using directly conjugated mAb (CD19 HD 37, Sigma; CD3 UCHT-1, Sigma; IgD, HJ9, Sigma; CD14, Leu-M3, Becton Dickinson; CD38, ACT-13.5, Serotec; κ, KP-53, Sigma; λ, HP6054, Sigma). For Percoll gradient separation, single-cell suspensions of mouse spleens were depleted for T cells using a cocktail of Thy1-, CD4-, and CD8-specific antibodies, followed by treatment with low toxicity guinea pig complement prepared as described. Preparation of discontinuous Percoll gradients has been described previously.25Chen-Bettecken U Wecker E Schimpl A IgM RNA switch from membrane to secretory form is prevented by adding antireceptor antibody to bacterial lipopolysaccharide-stimulated murine primary B-cell cultures.Proc Natl Acad Sci USA. 1985; 82: 7384-7388Crossref PubMed Scopus (62) Google Scholar Preparation of whole cell and whole organ protein extracts, nuclear and cytoplasmic cell extracts, and isolation of total RNA have been described.15Zwilling S Dieckmann A Pfisterer P Angel P Wirth T Inducible expression and phosphorylation of coactivator BOB.1/OBF.1 in T cells.Science. 1997; 277: 221-225Crossref PubMed Scopus (78) Google Scholar, 26Lernbecher T Müller U Wirth T Distinct NF-κB/Rel transcription factors are responsible for tissue-specific, and inducible gene activation.Nature. 1993; 365: 767-770Crossref PubMed Scopus (224) Google Scholar, 27Lernbecher T Kistler B Wirth T Two distinct mechanisms contribute to the constitutive activation of RelB in lymphoid cells.EMBO J. 1994; 13: 4060-4069Crossref PubMed Scopus (84) Google Scholar Protein immunoblots and Northern blotting of RNA were performed as described previously.10Pfisterer P Zwilling S Hess J Wirth T Functional characterization of the murine homolog of the B-cell-specific coactivator BOB.1/OBF.1.J Biol Chem. 1995; 270: 29870-29880Crossref PubMed Scopus (61) Google Scholar, 15Zwilling S Dieckmann A Pfisterer P Angel P Wirth T Inducible expression and phosphorylation of coactivator BOB.1/OBF.1 in T cells.Science. 1997; 277: 221-225Crossref PubMed Scopus (78) Google Scholar Stimulation of fractionated B cells with antibodies to CD40 (10 μg/ml) and recombinant interleukin-4 (IL-4; 100 U/ml) was performed as described.28Rolink A Melchers F Andersson J The SCID but not the RAG-2 gene product is required for S mu-S epsilon heavy chain class switching.Immunity. 1996; 5: 319-330Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar Lipopolysaccharide (Sigma) was used in a final concentration of 50 μg/ml. The Oct1-specific antibody has been described previously,10Pfisterer P Zwilling S Hess J Wirth T Functional characterization of the murine homolog of the B-cell-specific coactivator BOB.1/OBF.1.J Biol Chem. 1995; 270: 29870-29880Crossref PubMed Scopus (61) Google Scholar and the RelA and IκBα-specific antibodies were bought from Santa Cruz. Wild-type control mice, TNFRp55−/−,29Pfeffer K Matsuyama T Kundig TM Wakeham A Kishihara K Shahinian A Wiegmann K Ohashi PS Kronke M Mak TW Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection.Cell. 1993; 73: 457-467Abstract Full Text PDF PubMed Scopus (1564) Google Scholar or LTβR−/− mice30Fütterer A Mink K Luz A Kosco-Vilbois M Pfeffer K The lymphotoxin β receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues.Immunity. 1998; 9: 59-70Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar were immunized by i.p. injection of 200 μg (4-hydroxy-3-nitrophenyl-acetyl) chicken γ globulin (molar ratio 19:1) adsorbed to alum per mouse. Spleens were removed and snap-frozen 10 days after immunization. The lack of germinal centers in BOB.1/OBF.1-deficient mice prompted us to investigate BOB.1/OBF.1 protein expression in germinal centers of normal human individuals. We therefore characterized BOB.1/OBF.1 expression in vivo in normal reactive human tonsils. This was done by immunohistochemistry using an affinity-purified antibody and cryostat sections of normal tonsils. Strong nuclear and weak cytoplasmic BOB.1/OBF.1 expression was found in the majority of normal tonsillar germinal center B cells. Nuclear expression was highest in the dark zone and decreased in the apical light zone of the germinal center (Figure 1a). In contrast to this, only scattered mantle zone and marginal zone lymphocytes were stained, but no plasma cells (Figure 1b and data not shown). Double staining experiments demonstrated that BOB.1/OBF.1 was confined to CD19-positive B cells, almost all of which coexpressed Bcl6 in normal tonsillar tissues (Figure 1c). Neither dendritic cells nor macrophages expressed BOB.1/OBF.1 or Bcl6. To strengthen the immunohistochemistry results we purified different B cell populations from human tonsils and analyzed BOB.1/OBF.1 expression by protein immunoblotting. In a first step, CD19-positive cells (B cells) were purified. These cells were then further subdivided into the CD38-positive fraction (germinal center B cells) and CD38-negative fraction. The CD38-negative cells were further sorted for expression of IgD on the surface (naive B cells). When protein extracts from these different populations were characterized for BOB.1/OBF.1 expression, the strongest signal was evident in the CD38-positive germinal center B cell fraction (Figure 2A). The other fractions also showed BOB.1/OBF.1 expression, albeit at a clearly reduced level. We also analyzed these fractions for Bcl6 expression as an additional marker for germinal center B cells. Consistent with the immunohistochemistry results, Bcl6 was also strongly up-regulated in the germinal center B cell fractions. The observed differences were, however, specific for these two proteins, in that RelA expression was rather uniform in the various samples (Figure 2A). The immunostainings shown above had revealed some cytoplasmic staining for BOB.1/OBF.1. Given the known function of BOB.1/OBF.1 as a nuclear regulator, this staining pattern came as a surprise. To investigate whether BOB.1/OBF.1 could be found in the cytoplasm of established B cell lines, cell fractionation followed by protein immunoblotting experiments were performed. This analysis revealed that BOB.1/OBF.1 can be found in the nuclear as well as the cytoplasmic fraction. In contrast, proteins known to be in the cytoplasm of PD31 cells like RelA and IκBα were found exclusively in the cytoplasmic fraction,31Schreck R Kistler B Wirth T The NF-κB/Rel system of transcriptional activators.in: Papavassiliou AG Transcription Factors in Eukaryotes. RG Landes Company, Austin, TX1997: 153-188Google Scholar whereas the Oct1 transcription factor was found exclusively in the nucleus (Figure 2B). The germinal center reaction requires a complex interplay of antigen-specific B cells, antigen-specific T cells, and antigen-presenting follicular dendritic cells. As a consequence, specific B cells are activated, proliferate, perform isotype-switching and somatic hypermutation, and finally differentiate into either plasma cells or memory B cells. We were, therefore, interested to determine whether the activation status of B cells determines the level of BOB.1/OBF.1 expression. To this end, we separated murine splenic B cells on discontinuous Percoll density gradients into different fractions, which grossly represent distinct activation stages of B lymphocytes. The high density fraction (HD) largely represents naive resting B cells, whereas the low density fraction (LD) is comprised predominantly of large activated B cells. In the high-density B cell fraction there was only a low level of BOB.1/OBF.1 protein, whereas expression levels were significantly higher in the fraction representing large activated B cells (Figure 3A). Consistent with an earlier report,16Qin XF Reichlin A Luo Y Roeder RG Nussenzweig MC OCA-B integrates B cell antigen receptor-, CD40L-, and IL 4-mediated signals for the germinal center pathway of B cell development.EMBO J. 1998; 17: 5066-5075Crossref PubMed Scopus (96) Google Scholar these low levels of BOB.1/OBF.1 expression in the high density B cell fraction could be increased by costimulation with CD40 and IL-4 at both the RNA and protein levels (Figure 3B). To address the question whether most or all B cells express at least baseline levels of BOB.1/OBF.1 protein and expression is up-regulated in germinal center B cells, or, alternatively, that the low expression levels detected in non-germinal center B cells might be due to contaminating germinal center B cells, we analyzed BOB.1/OBF.1 expression in spleens from mice that showed a defect in germinal center formation. Two such mouse mutants were investigated, one carrying a mutation in the TNFRI (p55 gene)32Le Hir M Bluethmann H Kosco-Vilbois MH Muller M di Padova F Moore M Ryffel B Eugster HP Differentiation of follicular dendritic cells and full antibody responses require tumor necrosis factor receptor-1 signaling.J Exp Med. 1996; 183: 2367-2372Crossref PubMed Scopus (190) Google Scholar, 33Matsumoto M Mariathasan S Nahm MH Baranyay F Peschon JJ Chaplin DD Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers.Science. 1996; 271: 1289-1291Crossref PubMed Scopus (344) Google Scholar and the second carrying a mutation in the gene encoding the lymphotoxin (LT) β receptor.30Fütterer A Mink K Luz A Kosco-Vilbois M Pfeffer K The lymphotoxin β receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues.Immunity. 1998; 9: 59-70Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar Both mutant mice have previously been demonstrated to lack germinal centers; this defect is not intrinsic to the B lymphocytes, but rather affects accessory cells. When spleen extracts from these mutant mice were compared with wild-type mice for their levels of BOB.1/OBF.1 expression, a reduction of the BOB.1/OBF.1 signal was evident (about two- to fourfold). Nevertheless, these mice still express significant levels of BOB.1/OBF.1 (Figure 3C). This finding suggests that B cells not engaged in germinal center reactions constitutively express a lower level of BOB.1/OBF.1. The Bcl6 protein plays an important role in regulating B cell differentiation within germinal centers.34Dent AL Shaffer AL Yu X Allman D Staudt LM Control of inflammation, cytokine expression, and germinal center formation by BCL-6.Science. 1997; 276: 589-592Crossref PubMed Scopus (789) Google Scholar, 35Fukuda T Yoshida T Okada S Hatano M Miki T Ishibashi K Okabe S Koseki H Hirosawa S Taniguchi M Miyasaka N Tokuhisa T Disruption of the Bcl6 gene results in an impaired germinal center formation.J Exp Med. 1997; 186: 439-448Crossref PubMed Scopus (311) Google Scholar, 36Ye BH Cattoretti G Shen Q Zhang J Hawe N de Waard R Leung C Nouri-Shirazi M Orazi A Chaganti RS Rothman P Stall AM Pandolfi PP Dalla-Favera R The BCL-6 proto-oncogene controls germinal-centre formation, and Th2-type inflammation.Nat Genet. 1997; 16: 161-170Crossref PubMed Scopus (691) Google Scholar In addition, its expression is often deregulated in germinal center-derived lymphomas.37Ye BH Lista F Lo Coco F Knowles DM Offit K Chaganti RS Dalla-Favera R Alterations of a zinc finger-encoding gene, BCL-6, in diffuse large-cell lymphoma.Science. 1993; 262: 747-750Crossref PubMed Scopus (635) Google Scholar, 38Bastard C Deweindt C Kerckaert JP Lenormand B Rossi A Pezzella F Fruchart C Duval C Monconduit M Tilly H LAZ3 rearrangements in non-Hodgkin's lymphoma: correlation with histology, immunophenotype, karyotype, and clinical outcome in 217 patients.Blood. 1994; 83: 2423-2427Crossref PubMed Google Scholar, 39Lo Coco F Ye BH Lista F Corradini P Offit K Knowles DM Chaganti RS Dalla-Favera R Rearrangements of the BCL6 gene in diffuse large cell non-Hodgkin's lymphoma.Blood. 1994; 83: 1757-1759Crossref PubMed Google Scholar, 40Migliazza A Martinotti S Chen W Fusco C Ye BH Knowles DM Offit K Chaganti RS Dalla-Favera R Frequent somatic hypermutation of the 5′ noncoding region of the BCL6 gene in B-cell lymphoma.Proc Natl Acad Sci USA. 1995; 92: 12520-12524Crossref PubMed Scopus (325) Google Scholar, 41Chen W Iida S Louie DC Dalla-Favera R Chaganti RS Heterologous promoters fused to BCL6 by chromosomal translocations affecting band 3q27 cause its deregulated expression during B-cell differentiation.Blood. 1998; 91: 603-607PubMed Google Scholar The observation that BOB.1/OBF.1 and Bcl6 showed a high degree of coexpression in normal germinal center B cells prompted us to investigate BOB.1/OBF.1 expression in a variety of human B cell lymphomas. To do so, 99 different B cell lymphomas were selected randomly from the German lymph node repository. The tumors had been classified according to the REAL guidelines and represented different types of B cell lymphomas, which correlate well with distinct stages of normal B cell development.20Harris NL Jaffe ES Stein H Banks PM Chan JK Cleary ML Delsol G De Wolf-Peeters C Falini B Gatter KC A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar In addition, 18 different T cell lymphomas such as T-lymphoblastic lymphomas (5 cases), large granular lymphocytic leukemia of T type (2 cases), angioimmunoblastic T cell lymphoma (AILD, 6 cases), and intestinal T cell lymphomas (5 cases) were also included in this study. All tumors were analyzed for the presence of BOB.1/OBF.1 as well as Bcl6 by immunohistochemistry. A representative example is shown in Figure 4. A summary of the results of the immunohistochemistry is given in Table 1. Overall, 54% (54/99) of the B cell lymphomas analyzed were BOB.1/OBF.1-positive. In contrast, all T cell lymphomas investigated were negative for BOB.1/OBF.1. All Burkitt's lymphomas, most follicular lymphomas (17/18), and 94% of high-grade diffuse large cell B cell lymphomas (29/31) stained strongly, whereas all low-grade lymphomas (B-CLL, mantle cell lymphomas, MALT-type lymphomas, plasmacytomas; 42/42) were negative for BOB.1/OBF.1 and Bcl6. Within low-grade lymphomas, only a few scattered large cells as well as reactive non-neoplastic germinal centers were positive for BOB.1/OBF.1 and Bcl6 (Figure 4, c and d). Strikingly, BOB.1/OBF.1 staining was paralleled by Bcl-6 staining in most high-grade B cell lymphomas (Figure 4, a and b). However, two diffuse large B cell lymphomas (DLBL. stained for neither BOB.1/OBF.1 nor Bcl6, and three other cases were positive for BOB.1/OBF.1 but negative for Bcl6.Table 1Immunohistochemical Stainings of Normal and Malignant Lymphatic Tissues for BOB.1/OBF.1 and Bcl6Normal tonsillar lymphoid tissueExpression of BOB.1/OBF.1 I Bcl6Malignant compartment (n)BOB.1/OBF.1 pos./Bcl6 pos.Early naive B celln.d.B-CLL (5)(0/0)Mantle cell (naive B cell)± /−Mantle cell lymphoma (16)(0/0)Germinal center B cell (dark zone)+++ /+++Burkitt (8)(8/8)Germinal center B cell++ /++Follicular lymphoma (18)(17/16)Germinal center B cell++ /++DLBL (31)(29/26)Marginal zone B cell (memory B cell)± /−MALT-type lymphoma (14)(0/0)Plasma cell− /−Plasmacytoma (7)(0/0)Sections of the indicated normal and malignant tissues were stained with antibodies specific for BOB.1/OBF.1 and Bcl6 as indicated.−, no stainings; ±, <10% of positive cells; +, ≥10% <30% of positive cells; ++, ≥30% <70% of positive cells; +++, ≥70% of positive cells. DLBL, diffuse large cell B cell lymphoma. Open table in a new tab Sections of the indicated normal and malignant tissues were stained with antibodies specific for BOB.1/OBF.1 and Bcl6 as indicated. −, no stainings; ±, <10% of positive cells; +, ≥10% <30% of positive cells; ++, ≥30% <70% of positive cells; +++, ≥70% of positive cells. DLBL, diffuse large cell B cell lymphoma. The most obvious phenotype of the BOB.1/OBF.1 knockout mice was a complete lack of germinal centers in spleen and lymph nodes. Consistent with this defect, we see a significant up-regulation of BOB.1/OBF.1 expression in normal germinal center B cells. This up-regulation can be mimicked by stimulating the B cells in culture with the potent mitogen LPS or by triggering CD40 signaling in the presence of IL-4. Similar results have recently been obtained by Qin and colleagues.16Qin XF Reichlin A Luo Y Roeder RG Nussenzweig MC OCA-B integrates B cell antigen receptor-, CD40L-, and IL 4-mediated signals for the germinal center pathway of B cell development.EMBO J. 1998; 17: 5066-5075Crossref PubMed Scopus (96) Google Scholar In addition, we found that in contrast to established B lymphoma cell lines, which all expressed significant levels of BOB.1/OBF.1 regardless of their differentiation state, such tumors in situ show striking differences with respect to BOB.1/OBF.1 expression. Like their normal counterparts, germinal center B cell-derived B cell lymphomas stained strongly positive for BOB.1/OBF.1 in immunohistochemistry, whereas lymphomas representing other stages of B cell development were negative. Although these results do not prove a role for BOB.1/OBF.1 in the pathogenesis of germinal center-derived tumors, the high level expression can at least be regarded as a consistent marker for the classification of this type of lymphomas. The earlier studies on expression of BOB.1/OBF.1 in the B lymphoid lineage had relied largely on the analysis in established tumor cell lines. The most detailed study along this line had revealed a rather constant expression level throughout B lymphoid development.13Schubart DB Sauter P Massa S Friedl EM Schwarzenbach H Matthias P Gene structure and characterization of the murine homologue of the B cell-specific transcriptional coactivator OBF-1.Nucleic Acids Res. 1996; 24: 1913-1920Crossref PubMed Scopus (44) Google Scholar Interestingly, expression levels in transformed pre-B cell lines was shown to be unaffected by LPS treatment.13Schubart DB Sauter P Massa S Friedl EM Schwarzenbach H Matthias P Gene structure and characterization of the murine homologue of the B cell-specific transcriptional coactivator OBF-1.Nucleic Acids Res. 1996; 24: 1913-1920Crossref PubMed Scopus (44) Google Scholar Indeed, we also did not see an induction of BOB.1/OBF.1 expression on LPS treatment of the WEHI231 cell line representing the stage of an immature B cell (Boehm J, Wirth T, unpublished observations). These results indicate a significant difference between regulation of BOB.1/OBF.1 expression in primary B cells as compared to established B cell lines. Recently, a critical role for BOB.1/OBF.1 up-regulation in B cell transformation has been suggested.16Qin XF Reichlin A Luo Y Roeder RG Nussenzweig MC OCA-B integrates B cell antigen receptor-, CD40L-, and IL 4-mediated signals for the germinal center pathway of B cell development.EMBO J. 1998; 17: 5066-5075Crossref PubMed Scopus (96) Google Scholar The in-depth analysis of BOB.1/OBF.1 expression in B lymphomas in situ performed in the present study indicates that deregulation of BOB.1/OBF.1 expression is not a general event in B cell tumorigenesis. Rather, the human lymphomas analyzed reflected the situation observed in their normal counterparts, namely low (undetectable by immunohistochemistry. expression of BOB.1/OBF.1 in tumors not derived from germinal center B cells and high expression in germinal center-derived tumors. Therefore, our results do not allow us to conclude whether high level expression of BOB.1/OBF.1 contributes to the transformation of these germinal center-derived lymphomas. It remains unclear why the initial difference in BOB.1/OBF.1 expression in different types of B lymphomas is lost when B cell lines are established. A potential explanation is that selection for continuous growth in tissue culture may result in the up-regulation of BOB.1/OBF.1 expression. This could be a consequence of permanent stimulation of BOB.1/OBF.1 expression by some component in the tissue culture medium or a stable genetic/epigenetic alteration acquired during the cell culture adaptation process. Bcl6 (also called Laz3) is a member of the family of zinc-finger transcription factors which contains an amino-terminal POZ/ZIN domain.37Ye BH Lista F Lo Coco F Knowles DM Offit K Chaganti RS Dalla-Favera R Alterations of a zinc finger-encoding gene, BCL-6, in diffuse large-cell lymphoma.Science. 1993; 262: 747-750Crossref PubMed Scopus (635) Google Scholar, 42Kerckaert JP Deweindt C Tilly H Quief S Lecocq G Bastard C LAZ3, a novel zinc-finger encoding gene, is disrupted by recurring chromosome 3q27 translocations in human lymphomas.Nat Genet. 1993; 5: 66-70Crossref PubMed Scopus (411) Google Scholar It functions as a sequence-specific transcriptional repressor.36Ye BH Cattoretti G Shen Q Zhang J Hawe N de Waard R Leung C Nouri-Shirazi M Orazi A Chaganti RS Rothman P Stall AM Pandolfi PP Dalla-Favera R The BCL-6 proto-oncogene controls germinal-centre formation, and Th2-type inflammation.Nat Genet. 1997; 16: 161-170Crossref PubMed Scopus (691) Google Scholar, 43Chang CC Ye BH Chaganti RS Dalla-Favera R BCL-6, a POZ/zinc-finger protein, is a sequence-specific transcriptional repressor.Proc Natl Acad Sci USA. 1996; 93: 6947-6952Crossref PubMed Scopus (392) Google Scholar Chromosomal translocations involving the bcl6 gene have been found in about 40% of diffuse large cell lymphomas and to a lesser extent (approximately 14%) in follicular lymphomas.37Ye BH Lista F Lo Coco F Knowles DM Offit K Chaganti RS Dalla-Favera R Alterations of a zinc finger-encoding gene, BCL-6, in diffuse large-cell lymphoma.Science. 1993; 262: 747-750Crossref PubMed Scopus (635) Google Scholar, 38Bastard C Deweindt C Kerckaert JP Lenormand B Rossi A Pezzella F Fruchart C Duval C Monconduit M Tilly H LAZ3 rearrangements in non-Hodgkin's lymphoma: correlation with histology, immunophenotype, karyotype, and clinical outcome in 217 patients.Blood. 1994; 83: 2423-2427Crossref PubMed Google Scholar, 39Lo Coco F Ye BH Lista F Corradini P Offit K Knowles DM Chaganti RS Dalla-Favera R Rearrangements of the BCL6 gene in diffuse large cell non-Hodgkin's lymphoma.Blood. 1994; 83: 1757-1759Crossref PubMed Google Scholar, 42Kerckaert JP Deweindt C Tilly H Quief S Lecocq G Bastard C LAZ3, a novel zinc-finger encoding gene, is disrupted by recurring chromosome 3q27 translocations in human lymphomas.Nat Genet. 1993; 5: 66-70Crossref PubMed Scopus (411) Google Scholar As a consequence of these translocations, expression of the bcl6 gene is controlled by heterologous promoters.41Chen W Iida S Louie DC Dalla-Favera R Chaganti RS Heterologous promoters fused to BCL6 by chromosomal translocations affecting band 3q27 cause its deregulated expression during B-cell differentiation.Blood. 1998; 91: 603-607PubMed Google Scholar Interestingly, in at least one case the translocation of bcl6 resulted in the fusion to the BOB.1/OBF.1 gene promoter.44Galieque Zouitina S Quief S Hildebrand MP Denis C Lecocq G Collyn-d'Hooghe M Bastard C Yuille M Dyer MJS Kerckaert JP The B cell transcriptional coactivator BOB1/OBF1 gene fuses to the LAZ3/BCL6 gene by t(3;11)(q27;q23.1) chromosomal translocation in a B cell leukemia line (Karpas 231).Leukemia. 1996; 10: 579-587PubMed Google Scholar, 45Yuille MA Galiegue-Zouitina S Hiorns LR Jadayel D De Schouwer PJ Catovsky D Dyer MJ Kerckaert JP Heterogeneity of breakpoints at the transcriptional co-activator gene, BOB-1, in lymphoproliferative disease.Leukemia. 1996; 10: 1492-1496PubMed Google Scholar Although normal expression of Bcl6 is not confined to the lymphocyte lineage, within the B cell lineage Bcl6 is specifically expressed in germinal center B cells.46Cattoretti G Chang CC Cechova K Zhang J Ye BH Falini B Louie DC Offit K Chaganti RS Dalla-Favera R BCL-6 protein is expressed in germinal-center B cells.Blood. 1995; 86: 45-53Crossref PubMed Google Scholar, 47Onizuka T Moriyama M Yamochi T Kuroda T Kazama A Kanazawa N Sato K Kato T Ota H Mori S BCL-6 gene product, a 92- to 98-kD nuclear phosphoprotein, is highly expressed in germinal center B cells and their neoplastic counterparts.Blood. 1995; 86: 28-37Crossref PubMed Google Scholar Interestingly, like the defect observed in the BOB.1/OBF.1-deficient mice, Bcl6-deficient B cells fail to generate germinal centers.34Dent AL Shaffer AL Yu X Allman D Staudt LM Control of inflammation, cytokine expression, and germinal center formation by BCL-6.Science. 1997; 276: 589-592Crossref PubMed Scopus (789) Google Scholar, 35Fukuda T Yoshida T Okada S Hatano M Miki T Ishibashi K Okabe S Koseki H Hirosawa S Taniguchi M Miyasaka N Tokuhisa T Disruption of the Bcl6 gene results in an impaired germinal center formation.J Exp Med. 1997; 186: 439-448Crossref PubMed Scopus (311) Google Scholar, 36Ye BH Cattoretti G Shen Q Zhang J Hawe N de Waard R Leung C Nouri-Shirazi M Orazi A Chaganti RS Rothman P Stall AM Pandolfi PP Dalla-Favera R The BCL-6 proto-oncogene controls germinal-centre formation, and Th2-type inflammation.Nat Genet. 1997; 16: 161-170Crossref PubMed Scopus (691) Google Scholar It was recently demonstrated that Bcl6 protein expression is unaffected by the BOB.1/OBF.1 mutation.16Qin XF Reichlin A Luo Y Roeder RG Nussenzweig MC OCA-B integrates B cell antigen receptor-, CD40L-, and IL 4-mediated signals for the germinal center pathway of B cell development.EMBO J. 1998; 17: 5066-5075Crossref PubMed Scopus (96) Google Scholar However, it is possible that expression of the two genes is coregulated by some so far unknown mechanism in the B cell lineage. The analysis of BOB.1/OBF.1 expression in normal lymphoid tissues and primary tumors suggests that BOB.1/OBF.1 might be a good marker for germinal center-related B cell transformation as described for bcl6 expression recently.48Allman D Jain A Dent A Maile RR Selvaggi T Kehry MR Staudt LM BCL-6 expression during B-cell activation.Blood. 1996; 87: 5257-5268Crossref PubMed Google Scholar In most tumor cases Bcl6 and BOB.1/OBF.1 were coexpressed at a high level, but we noted three cases of DLBL positive for BOB.1/OBF.1 but lacking detectable Bcl6 expression. However, we could not find a tumor positive for Bcl6 expression that lacked expression of BOB.1/OBF.1. Therefore, BOB.1/OBF.1 seems to be an appropriate marker for high grade malignant germinal center derived B cell lymphomas. Interestingly, we found two cases of DLBL negative for both Bcl6 and BOB.1/OBF.1. These two tumors were morphologically indistinguishable from the other DLBL cases and the reasons for their lacking BOB.1/OBF.1 expression are currently not known. We thank B. Kistler for the anti-CD40 antibodies and IL-4 as well as for many helpful suggestions. We thank M. Reichert, H. Haber, and A. Zant for invaluable technical assistance and A. Schimpl for many helpful comments and critical reading of the manuscript.

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