Expression, Regulation, and Function of B Cell-Expressed CD154 in Germinal Centers Amrie C. Grammer, Richard D. McFarland, Jonathan Heaney, Bonnie F. Darnell and Peter E. Lipsky This information is current as of September 25, 2021. J Immunol 1999; 163:4150-4159; ; http://www.jimmunol.org/content/163/8/4150 Downloaded from References This article cites 74 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/163/8/4150.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 *average by guest on September 25, 2021 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Expression, Regulation, and Function of B Cell-Expressed CD154 in Germinal Centers1 Amrie C. Grammer,* Richard D. McFarland,† Jonathan Heaney,* Bonnie F. Darnell,† and Peter E. Lipsky2* Activated B cells and T cells express CD154/CD40 ligand in vitro. The in vivo expression and function of B cell CD154 remain unclear and therefore were examined. Tonsillar B and T cells expressed CD154 at a similar density both in situ and immediately ex vivo, whereas a significantly higher percentage of the former expressed CD154. CD154-expressing B cells were most frequent in the CD38positiveIgD1 pre-germinal center (GC)/GC founder, CD38positive GC and CD382IgD2 memory populations, and were also found in the CD382IgD1 naive and CD38brightIgD1 plasmablast subsets, but not in the CD38brightIgD2 plasma cell subset. B cell expression of CD154 was induced by engaging surface Ig or CD40 by signals that predominantly involved activation of AP-1/NF-AT and NF-kB, respectively. The functional importance of CD154-mediated homotypic B cell interactions in vivo was Downloaded from indicated by the finding that mAb to CD154 inhibited differentiation of CD38positiveIgD2 GC B cells to CD382IgD2 memory cells. In addition, mAb to CD154 inhibited proliferation induced by engaging sIg or CD40, indicating the role of up-regulation of this molecule in facilitating B cell responsiveness. Of note, CD154 itself not only functioned as a ligand but also as a direct signaling molecule as anti-CD154-conjugated Sepharose beads costimulated B cell responses induced by engaging surface Ig. These results indicate that CD154 is expressed by human B cells in vivo and plays an important role in mediating B cell responses. The Journal of Immunology, 1999, 163: 4150–4159. http://www.jimmunol.org/ cells express CD154/CD40 ligand following activation in cient in CD40 (12–14) or its ligand (CD154) (14–16) do not form vitro by ionomycin and phorbol ester (1–3) as well as by functional GCs following immunization with T-dependent (TD) B the polyclonal B cell activators Staphyloccus aureus Ag. Additionally, humans with X-linked hyper-IgM syndrome Cowan I (1, 2) or LPS (4, 5). The finding that CD154 expressed by (HIgMXL) syndrome, who cannot express a functional CD154 (re- activated B cells mediates homotypic CD154-CD40 interactions viewed in Refs. 17 and 18) do not form functional GCs in response resulting in aggregation, clonal expansion, and differentiation into to TD Ag (19, 20). Ig-secreting cells (1, 2, 4, 5) suggests that up-regulation of this The GC is one of the structures in which maturation of the by guest on September 25, 2021 molecule might play an essential role in propagating humoral re- humoral response to Ag occurs, fostering somatic hypermutation, sponses in vivo. Further support for this possibility is the finding selection, and isotype switching of activated B cells (reviewed in that circulating B cells from patients with autoimmune diseases, Refs. 21–23). Although CD154-CD40 interactions are essential for such as systemic lupus erythematosus, characterized by polyclonal initiation and propagation of the GC reaction, there are stages of B cell activation express CD154 (5–7). Moreover, certain B cell the GC reaction that appear to proceed in the absence of CD1541 lymphomas express CD154 in a functional manner (8–11). These T cells. CD1541 T cells are absent from the dark zone (DZ), where considerations prompted an examination of whether CD154 is ex- rapid B cell proliferation and somatic hypermutation occur, and are pressed physiologically in vivo at sites of B cell activation in sec- found infrequently if at all in the basal light zone (LZ) of the GC ondary lymphoid tissue as well as a delineation of the regulation of (24–28), where high avidity Ag-binding B cells are rescued from B cell CD154 expression and its functional activity. apoptosis (29, 30). Despite the paucity of CD154-expressing T Signaling through CD40 is important in the formation and per- cells, an established GC rapidly disassembles when CD154-CD40 petuation of the germinal center (GC)3 reaction since mice defi- interactions are blocked (31). One possible explanation for this finding is that cells other than T cells express CD154 in the DZ and LZ of GCs in secondary lymphoid tissues. Activated CD154-ex- *Harold C. Simmons Arthritis Research Center and Departments of Internal Medicine pressing B cells are prime candidates to provide the essential † and Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235 CD40-mediated signals in these regions. Received for publication February 24, 1999. Accepted for publication July 29, 1999. To test this hypothesis requires an examination of CD154 ex- The costs of publication of this article were defrayed in part by the payment of page pression by B cells in secondary lymphoid organs. In humans, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tonsils have been employed extensively to understand GC behav- 1 This research was supported by National Institutes of Health (NIH) Grant AI-31229. ior, despite their chronic inflammatory and often infected character A.C.G. was supported in part by NIH Postdoctoral Training Grant AR-18550. (reviewed in Ref. 32). Therefore, the current experiments were 2 Address correspondence and reprint requests to Dr. Peter E. Lipsky, Harold C. undertaken to determine whether CD154 is expressed in situ by Simmons Arthritis Research Center, University of Texas Southwestern Medical Cen- tonsillar B cells, to examine the nature of signals that regulate B ter, 5323 Harry Hines Boulevard, Dallas, TX 75235-8884. E-mail address: [email protected] cell CD154 expression, and to investigate the functional activity of 3 Abbreviations used in this paper: GC, germinal center; MFI, mean fluorescence CD154 expressed by human B cells. The data clearly indicate that intensity; TD, T-dependent; DZ, dark zone; LZ, light zone; sIg, surface Ig; hIg, human CD154 is expressed by tonsillar B cells, is up-regulated by en- Ig; Cy, cyclosporine; MNC, mononuclear cell; MKK1, mitogen-activated protein ki- nase kinase 1; LAC, lactacystein; HIgMXL syndrome, X-linked hyper-IgM syn- gagement of surface Ig (sIg) or CD40 itself, and is likely to be of drome. great importance in propagating GC reactions. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 4151 Materials and Methods Ridgefield, CT) or biotinylated h2431 followed by either streptavidin Cells conjugated to PE (Becton Dickinson), 613, or 670 (Life Technologies). Alternatively, CD154 expression was analyzed with 8976-PE (Becton Tonsils were minced and digested in RPMI medium (Life Technologies, Dickinson) or with 2443 (kind gift of Dr. Randolph J. Noelle) or uncon- Grand Island, NY) containing 210 U/ml collagenase type I (Worthington jugated 8976, followed by biotinylated rat anti-mouse Ig and streptavidin- Biochemical, Lakewood, NJ) and 90 KU/ml DNase (Sigma, St. Louis, PE. In some cases, B cells were acid washed as described (1) to remove MO) for 30 min at 37°C. Following filtration through a wire mesh, the cells bound CD40 before staining with anti-CD154 mAb. Subsets of tonsillar B were washed twice in 20% NHS-RPMI and once with 10% NHS-RPMI. cells were delineated with Ab specific for CD19 (PerCP, APC, Becton Mononuclear cells (MNC) were obtained by centrifugation of heparinized Dickinson; biotinylated, Coulter), IgD (FITC, Caltag; PE, Southern Bio- venous blood or digested tonsil tissue over diatrizoate/Ficoll gradients technology, Birmingham, AL), CD38 (HB7, APC or PE, Becton Dickin- (Sigma). Blood was obtained from healthy adult volunteers or an HIgMXL son; HIT2, FITC, Caltag), CD23 (biotinylated, The Binding Site, San Di- patient previously demonstrated to lack functional CD154 expression. The ego, CA; FITC, Becton Dickinson), TdT (FITC, Supertech, Rockville, mutations in CD154 of this HIgMXL patient have been reported (Refs. 1, MD), Ki67 (Biogenics, Ramon, CA) followed by anti-mouse IgG1 (FITC, 33; EMBL accession number X96710). In some cases, MNCs were de- The Binding Site), CD44 (A3D8, biotinylated, ATCC), and CD77 (Biode- 9 pleted of NK cells and monocytes and separated into T cell-enriched and sign, Kennebunk, ME) followed by goat F(ab )2 anti-rat IgM FITC (Jack- B cell-enriched populations as described (1). The B cell population was son ImmunoResearch). Isotype-matched mAb were used as controls. Anal- further purified by positive selection on a Ceprate streptavidin column ysis was performed using CellQuest and Paint-a-Gate Software (Becton (CellPro, Bothell, WA) following staining of cells with biotinylated anti- Dickinson).