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Requirement for Cyclin D3 in Germinal Center Formation and Function

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Requirement for Cyclin D3 in Germinal Center Formation and Function Cell Research (2010) :1-16. © 2010 IBCB, SIBS, CAS All rights reserved 1001-0602/10 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr Requirement for cyclin D3 in germinal center formation and function Jonathan U Peled1, J Jessica Yu1, Jeganathan Venkatesh2, Enguang Bi1, B Belinda Ding1, 5, Melissa Krupski-Downs1, Rita Shaknovich3, Piotr Sicinski4, Betty Diamond2, Matthew D Scharff1, B Hilda Ye1 1Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; 2The Center for Autoimmune and Musculoskeletal Disease, The Feinstein Institute for Medical Research, Manhasset, NY 11030, USA; 3Depart- ments of Medicine and Pathology, Weill Cornell College of Medicine, New York, NY 10021, USA; 4Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA Germinal centers (GC) of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antibody genes. Although it is generally believed that GC B cells employ a unique proliferative program to accommodate these pro- cesses, little is known about how the GC-associated cell cycle is orchestrated. The D-type cyclins constitute an im- portant component of the cell cycle engine that enables the cells to respond to physiological changes. Cell type- and developmental stage-specific roles of D-type cyclins have been described but the cyclin D requirement during GC reaction has not been addressed. In this study, we report that cyclin D3 is largely dispensable for proliferation and Ig class switching of in vitro activated B cells. In contrast, GC development in Ccnd3–/– mice is markedly impaired, as is the T cell-dependent antibody response. Within the GC, although both switched and unswitched B cells are affected by cyclin D3 inactivation, the IgM– pool is more severely reduced. Interestingly, despite a compensatory increase in cyclin D2 expression, a significant number of Ccnd3–/– GC B cells accumulate in quiescent G0 state. Lastly, although cyclin D3 inactivation did not disrupt BCL6 expression in GC B cells, it completely blocked the GC promoting effect of BCL6 overexpression, suggesting that cyclin D3 acts downstream of BCL6 to regulate GC formation. This is the first demonstration that cyclin D3 plays an important and unique oler at the GC stage of B cell development. Keywords: B cell development, germinal center, cell cycle, cyclin D3 Cell Research advance online publication 20 April 2010; doi: 10.1038/cr.2010.55 Introduction high-affinity humoral immune response. After immuni- zation with T cell-dependent antigens, naïve B cells that Germinal centers (GCs) are dynamic functional niches have been activated by cognate T cells migrate to the within secondary lymphoid tissues that are critical to a boundary between the B and T cell areas and initiate rap- id focal expansion which quickly leads to the formation of GCs. Within the GC, B cells diversify their Ig genes Correspondence: B Hilda Ye by two distinct genetic mechanisms: class switch recom- Tel: +1-718-430-3339; Fax: +1-718-430-8574 bination (CSR) and somatic hypermutation (SHM). CSR E-mail: [email protected] 5Current address: Department of Anatomy and Cell Biology, University of enables production of antibodies other than the IgM and Kansas Medical Center, Kansas City, KS 66160, USA IgD isotypes while SHM introduces point mutations into Abbreviations: CSR (class switch recombination); Cdk (cyclin dependent the variable regions of Ig genes, which leads to Ig affin- kinase); DAPI (diamidino-2-phenylindole); DLBCL (diffuse large B cell ity maturation when higher-affinity clones are positively lymphoma); DN (double negative); FDC (follicular dendritic cell); Fo (fol- licular B cells); GC (germinal center); IHC (immunohistochemistry); KO selected [1, 2]. Within the GC, B cells are segregated (knock-out); LN (lymph node); MZ (marginal zone); NP (nitrophenyl); into dark zone and light zone compartments, where they PNA (peanut agglutinin); PI (propidium iodide); SHM (somatic hypermu- are referred to as centroblasts and centrocytes, respec- tation); Tfh (T follicular helper cells) tively. The transit of B cells between these two compart- Received 16 February 2010; revised 9 March 2010; accepted 10 March 2010 ments and the specific features of GC B cells in these npg Role of cyclin D3 in GC formation and function 2 areas have been subjects of significant interest [3, 4]. In ther Cdk4 or Cdk6. In turn, activated Cdk4/6 hyperphos- a standard model, SHM and most of the cell proliferation phorylates Rb and related pocket proteins, i.e. p107 and occur in the dark zone while CSR and positive/negative p130, leading to their release from the E2F factors and selection occur in the light zone as a result of B cell transcriptional activities required for G1-S progression interactions with follicular dendritic cells (FDCs) and [10]. Unlike other cyclins whose expression oscillates T follicular helper (Tfh) cells. Unswitched, IgM+ GC B with the cell cycle phases, D-type cyclins are primar- cells are exclusively found in the FDC (light) zone while ily regulated by mitogenic and oncogenic stimuli and the majority of the switched, IgM– GC B cells occupy the are expressed in developmentally programmed tissue- non-FDC (dark) zone [3]. After exiting the GC, selected specific patterns [11]. They are particularly important B cells further differentiate to become memory or plasma to hematopoietic cells as deficiency in all three (D1, D2 cells. The GC stage of B cell development has a number and D3) leads to a profound defect in expansion of he- of unique features. For instance, because CSR and SHM matopoietic stem cells and lineage-committed progeni- generate several types of DNA damage that are intrinsi- tors, while apparently sparing other cell lineages in the cally linked to the DNA damage response, it has been developing mouse embryo [12]. In certain cell types, a suggested that GC B cells execute a modified DNA dam- switch from one D-type cyclin to another coincides with age signaling pathway that dampens the threshold for a change in control of cell proliferation during differen- cell cycle checkpoint activation [5]. tiation. In T cells, a switch from cyclin D2 to cyclin D3 Available evidence also suggests that the cell cycle is important during their transition from cytokine-depen- engine itself may operate differently in GC B cells dent proliferation in the double negative stage-3 (DN- compared to other mitotic cells. A hallmark of the GC 3) to the pre-TCR-dependent proliferative burst in DN-4 response is the extremely fast proliferation of GC B [13]. Similarly, as pro-B cells differentiate into large pre- cells, with cell division time estimated to be as short as B cells in the bone marrow, the newly assembled pre- 6-12 h, placing them among the fastest-cycling cells in BCR not only triggers an increase in cyclin D3 protein higher eukaryotes [4]. Curiously, c-Myc and NF-κB, but also shifts the expansion of the pre-B cell pool to a two transcription factors thought to be required for all cyclin D3-dependent mode [14]. The D-type cyclins also proliferating cells, are specifically down-regulated when play prominent roles in tumorigenesis. Their expression B cells enter the GC [5, 6]. A close examination of the is often upregulated by major oncogenic pathways. In ad- relationship between cell proliferation and BCL6, the dition, gene amplification and chromosomal translocation master regulator of GC formation, also raises the pos- can also lead to their aberrant expression and activity in sibility that GC B cells may use different cell cycle regu- many types of tumors. With respect to GC-derived B cell lators. A recent genome-wide BCL6 promoter binding malignancies, the cyclin D3 gene CCND3 is rearranged study revealed that in normal GC B cells, BCL6 inhibits and over-expressed in some patients with diffuse large B a number of genes known to promote cell proliferation, cell lymphomas (DLBCL), and its overexpression pre- including c-Myc, cyclin D1, cyclin D2, Pim1, and JunB dicts poor clinical outcome [15, 16]. [7]. We have previously reported that by suppressing c- Gene expression profiling and histological analyses Myc, cyclin D2, and the IL-6/STAT3 signaling pathway, have demonstrated that B cells undertake a D2 to D3 BCL6 exerts a strong negative effect on the G1-S transi- switch upon entering the GCs [17-19]. It is unclear, how- tion in macrophages [8]. Thus, it is likely that in GC B ever, whether this phenomenon simply reflects the GC- cells as well as in macrophages, BCL6-mediated silenc- associated upregulation of BCL6, a strong inhibitor of ing of proliferation related genes will have a significant cyclin D2 [20], or serves a biological mandate due to impact on cell cycle control. As many of these BCL6 a specific requirement for cyclin D3 function. Here we target genes function in the G1 phase of the cell cycle, an report that while cyclin D3 is largely dispensable for the attractive hypothesis is that the G1 phase is differentially development and proliferation of follicular B cells, GC regulated in GC B cells compared to other types of cells. formation and T cell-dependent antibody responses are To our knowledge, the only direct study that compared notably impaired in cyclin D3 knock-out (KO) mice. the role of cell cycle regulators in GC and non-GC B Moreover, genetic analyses reveal that cyclin D3 func- cells found that the cyclin-dependent-kinase (Cdk) in- tions at a step downstream of BCL6 in GC formation. hibitor p18INK4c is dispensable for GC but is required for post-GC plasma cell differentiation [9].
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