The Identification and Characterization of a Ligand for Bovine CD5 Karen M. Haas and D. Mark Estes This information is current as J Immunol 2001; 166:3158-3166; ; of September 27, 2021. doi: 10.4049/jimmunol.166.5.3158 http://www.jimmunol.org/content/166/5/3158 Downloaded from References This article cites 46 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/166/5/3158.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 27, 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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Identification and Characterization of a Ligand for Bovine CD51 Karen M. Haas* and D. Mark Estes2*† CD5, a type I glycoprotein expressed by T cells and a subset of B cells, is thought to play a significant role in modulating Ag receptor signaling. Previously, our laboratory has shown that bovine B cells are induced to express this key regulatory molecule upon Ag receptor cross-linking. To date, a ligand has not been described for bovine CD5. Given the importance ligand binding presumably plays in the functioning of CD5 on this B cell subset and on T cells, we sought to characterize the ligand for this protein using a bovine CD5-human IgG1 (CD5Ig) fusion protein produced by both mammalian and yeast cells. As determined by CD5Ig binding, expression of this ligand is negative to low on freshly isolated lymphocytes, with low-density expression being limited to activated B cells. Activation with LPS, PMA, and calcium ionophore, or ligation of CD40 alone or in combination with anti-IgM, resulted in B cell-specific expression of this ligand. Interestingly, activation through B cell Ag receptor cross-linking alone, Downloaded from although able to induce CD5 expression, did not result in expression of CD5 ligand (CD5L). In addition, we demonstrate a functional role for CD5L as a costimulatory molecule that augments CD40L-stimulated B cell proliferation. Finally, immuno- precipitation with CD5Ig suggests that the ligand characterized in this study has a molecular mass of ϳ200 kDa. The data reported herein, as well as future studies aimed at further characterizing this newly identified bovine CD5L, will undoubtedly aid in understanding the role that the CD5-CD5L interaction plays in immune responses. The Journal of Immunology, 2001, 166: 3158–3166. http://www.jimmunol.org/ D5 is a 67-kDa type I monomeric glycoprotein belonging (22), and HIV infection (23) is evidenced by reports demonstrating to the ancient family of scavenger receptor cysteine-rich disease-related CD5ϩ B cell-specific expansion. CD5ϩ B cells C proteins (1). It has remained highly conserved throughout have also been implicated in the development of certain autoim- evolution and, therefore, is thought to play a conserved role in both mune conditions, such as Sjogren’s syndrome and rheumatoid ar- lymphocyte development and function among species (2–5). CD5 thritis (24), as well as various B cell lymphomas and leukemias, is found to be expressed by all T cells and a subset of B cells, such as B cell chronic lymphocytic leukemia, which are often of a previously termed B-1 cells (6). Although the origination of this CD5ϩ phenotype (25). ϩ CD5 B cell subset remains controversial, it has been clearly es- Recent studies have suggested that the function of CD5 may by guest on September 27, 2021 tablished by our laboratory and others that CD5Ϫ B cells, or con- differ with respect to specific cell population. For example, on ventional B (B-2) cells, from multiple species can be activated to peripheral T cells, CD5 appears to play a costimulatory role (26– express this marker (7–12). However, whether a B-1 cell is devel- 29), yet on Jurkat cells, CD5 was recently shown to negatively opmentally distinct from a B-2 cell induced to express CD5 as a regulate TCR signaling (30). Although it has been suggested that result of activation (such as Ag receptor cross-linking) remains to CD5 may function to provide continual stimulation through the B 3 be clearly established. cell Ag receptor (BCR) via its interaction with VH framework CD5ϩ B cells have been reported to exist in a number of spe- regions (31), studies involving CD5 knockout mice have suggested cies, although the proportion of B cells expressing CD5, as well as that CD5 negatively regulates Ag receptor signaling in both thy- their anatomical localization, varies among species. CD5 is ex- mocytes and B cells (32, 33). In addition, a recent study has dem- pressed by subpopulations of human (hu), mouse, and bovine B onstrated that ligation of CD5 on tonsillar B cells results in apo- cells (6, 13–16), while in other species, such as the rabbit (17) and ptosis, while ligation on resting peripheral T cells does not (34). chicken (2), nearly all B cells express CD5. The importance of this That CD5 negatively regulates signaling through the BCR is fur- B cell population during the immune response to a variety of ther supported by a recent biochemical study that has shown CD5 pathogenic infections, such as trypanosomosis (16, 18), schistoso- to recruit SHP-1, a protein tyrosine phosphatase, in B cells (35). In miasis (19, 20), bovine leukemia virus (21), chronic hepatitis C further support of the negative regulatory role of CD5 on B cells, a recent study by Hippen et al. (36) has suggested that CD5 plays a role in maintaining B cell anergy. Although the role CD5 ex- *Department of Molecular Microbiology and Immunology, School of Medicine, and pression plays on various cell populations remains to be com- † Department of Veterinary Pathobiology, College of Veterinary Medicine, University pletely understood, the interaction between CD5 and its ligand(s) of Missouri, Columbia, MO 65211 in all likelihood plays a critical role in regulating the function of Received for publication August 14, 2000. Accepted for publication December 28, 2000. CD5. Thus, the identification of the ligand(s) for CD5 is key to The costs of publication of this article were defrayed in part by the payment of page further elucidating the function of CD5 on T and B cell charges. This article must therefore be hereby marked advertisement in accordance populations. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the U.S. Department of Agriculture National Research Initiative Competitive Grants Program Project 96-35204-3584. 3 Abbreviations used in this paper: BCR, B cell Ag receptor; L, ligand; sIgM, surface 2 Address correspondence and reprint requests to Dr. D. Mark Estes, Department of IgM; TD, T cell-dependent; TI, T cell-independent; CD5Ig, bovine CD5-huIgG1; Veterinary Pathobiology, University of Missouri, 201 Connaway Hall, Columbia, MO mCD5Ig, MOP-8-expressed bovine CD5Ig; yCD5Ig, yeast-expressed bovine CD5Ig; 65211. E-mail address: [email protected] DTSSP, 3,3Јdithiobis-(sulfosuccinimidylpropionate); bo, bovine; hu, human. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 3159 The constitutive B cell marker, CD72, was described by Van de ture, transfected cells and nontransfected DAP3 cells were treated with 50 Velde et al. (37) as the first ligand for CD5. However, recent stud- ␮g/ml mitomycin C for 30 min at 37°C, washed three times in HBSS, and ies have suggested that CD5 may bind to one or more alternative allowed to adhere to culture dishes for 1 h and residual unbound fibroblasts were removed. PMA and calcium ionophore A23187 (Sigma, St. Louis, ligand(s). Using a CD5-huIgG1 fusion protein, Biancone et al. (38) MO) stimulation of B cells was performed at final concentrations of 10 described an activation-induced CD5 ligand (CD5L) expressed by ng/ml and 1 ␮g/ml, respectively. Endotoxin (from Escherichia coli O55: activated murine B cells, as well as T cell clones. Similarly, Bikah B5; Sigma) and pokeweed mitogen (Phytolacca americana lectin; Sigma) et al. (39) have described a ligand expressed by murine peritoneal were used at concentrations of 20 EU/ml and 10 ␮g/ml, respectively. B cells, activated splenic B cells, and B lymphoma cell lines. More Proliferation assay recently, Calvo et al. (40) described a CD5L expressed by cell types of lymphoid, myeloid, and epithelial origin. In addition to B cell proliferation assays were conducted on B cells cultured in triplicate at a concentration of 2 ϫ 105 B cells per well in a 96-well plate. Twenty- these studies, which suggest that CD5 may potentially interact with four hours after culture, CD5Ig or isotype control (huIgG1) was added to more than ligand, CD5 has been reported to interact with VH Ig cultures at concentrations of 25, 12.5, or 6.25 ␮g/ml. B cells were pulsed framework regions in both rabbit and human studies (41, 42). with 1 ␮Ci [3H]thymidine (DuPont/NEN, Boston, MA) after 72 h culture Given the variation in function CD5 appears to serve with respect and harvested 18 h later onto Skatron filter mats (Skatron Instruments, Lier, to cell-specific expression, as well its structural similarity to scav- Norway) using a cell harvester (Skatron Instruments).