Ligand for Bovine CD5 the Identification and Characterization of A

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

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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, immunoprecipitation 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.

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). Thymidine incorpo- ration was determined by scintillation counting (Beckman Coulter, enger receptors (known to bind multiple ligands), it is plausible to Fullerton, CA). hypothesize that CD5 may indeed have multiple ligands that per- form unique functions. Further characterization of these proposed Flow cytometry ligands and the interactions that they form with CD5 is required to The following Abs specific for bovine Ags were used for staining: CC17 Downloaded from begin to understand the functional significance that the CD5-CD5L recognizing CD5 (provided by Chris Howard, Institute for Animal Health), interaction may have in vivo. MM1A (anti-CD3), and GB25A (anti-CD21) (WSU mAb Center); goat Although a variety of ligands have been described for human, anti-bovine IgM-FITC (Kirkegaard & Perry Laboratories) and mouse anti- bovine IgM (BM23; Sigma). Secondary detection reagents used include rat mouse, and rabbit CD5, a bovine CD5L has not yet been described. anti-mouse IgG1-PE (BD Becton Dickinson, San Jose, CA), streptavidin- In the following study, we have addressed the ability of a recom- PE, and streptavidin-APC (BD PharMingen, San Diego, CA). Abs used as

binant bovine CD5-huIgG1 fusion protein to interact with the pu- negative staining controls include the secondary detection Abs listed above http://www.jimmunol.org/ tative ligand for CD5 on the surface of bovine PBMCs. The data used alone and in conjunction with isotype-matched mouse IgG1 and huIgG1, and goat Ig-FITC (Sigma). Cells were stained and washed in cold demonstrate that CD5Ig expressed in either yeast or mammalian PBS containing 1% BSA and 0.1% sodium azide and were fixed in 2% cells interacts with an activation-induced ligand (ϳ200 kDa) ex- buffered paraformaldehyde. Cells were analyzed using a FACSVantage pressed by B cells, which functions as a costimulatory molecule to flow cytometer and CellQuest acquisition and analysis programs (BD Bec- enhance B cell proliferation. Furthermore, we demonstrate that ton Dickinson). To characterize CD5Ig binding, cells were allowed to bind unlike CD5 expression, which is up-regulated on B cells after sur- CD5Ig in the presence of PBS alone or PBS containing 25 mM EDTA, 1 M glucose, 1 M fructose, or 1 M mannose. Protease pretreatment was face IgM (sIgM) cross-linking but not CD40 ligation, its ligand conducted by incubating cells in 0.05% trypsin-EDTA (Sigma) or 1 mg/ml appears to be regulated in a reciprocal manner, in which CD40 proteinase K (Sigma) in PBS for 20 min before CD5Ig staining. ligation, but not Ag receptor cross-linking, results in expression of by guest on September 27, 2021 the ligand for CD5. The potential implications for the counterregu- Production and purification of chimeric fusion proteins lation of CD5 and its ligand with respect to T cell-dependent (TD) Yeast-expressed bovine CD5Ig (yCD5Ig). The cDNA encoding the ex- and T cell-independent (TI) B cell responses are discussed. tracellular domain of bovine CD5 lacking its native signal sequence was fused to the cDNA encoding the Fc portion of huIgG1 (CH2 and CH3 domains minus hinge region) and cloned into the yeast expression vector, Materials and Methods picZ␣A (Invitrogen, Carlsbad, CA). Pichia pastoris was transformed with Animals CD5Ig-picZ␣A via electroporation, according to manufacturer’s instructions (Gene Pulser; Bio-Rad, Richmond, CA). Transformed yeast was se- Blood donors used for these studies were primarily male steers between 2 lected for growth on yeast extract peptone dextrose plates containing (100 and 12 mo of age maintained in an indoor housing facility. ␮g/ml) Zeocin. Individual yeast clones transformed with CD5Ig-picZ␣A Purification of bovine PBMCs and B cells were further identified by PCR screening. PCR-positive clones were grown in methanol-inducing medium and supernatants were analyzed at various Blood was collected in 10ϫ sodium citrate anticoagulant solution (0.15 M time points for the presence of secreted CD5Ig by Western blot analysis. sodium citrate, 80 mM citric acid, 0.16 M dextrose) and centrifuged 20 min Large-scale production of yCD5Ig followed. yCD5Ig was purified by Ami- at 2000 rpm (900 ϫ g) to obtain a buffy coat. Buffy coats were harvested con filtration (XM50; Amicon, Beverly, MA) followed by protein A pu- and residual RBCs were lysed in ACK RBC lysis buffer (0.15 M NH4Cl, rification (Pierce) in the presence of 0.05% Tween 20. 1 mM KHCO3, 0.1 mM EDTA (pH 7.3)) 7 min at room temperature, ϫ MOP-8-expressed bovine CD5Ig (mCD5Ig) and CD40Ig. The extracel- centrifuged 7 min at 300 g, and washed two times in HBSS to obtain lular domain of bovine CD5 including its native signal sequence and an PBMCs. B cells were selected by passive panning, as previously described ϩ ϩ ϩ added Kozak’s consensus sequence was fused to cDNA encoding the (7). Residual CD3 , CD5 , and CD14 cells were removed by treatment huIgG1 Fc portion (CH2 and CH3 domains lacking hinge region) and with Abs to bovine CD3 (MMIA; Washington State University Monoclo- cloned into the mammalian expression vector, pcDNA3.1/neo(ϩ) (Invitro- nal Antibody Center, Pullman, WA), CD5 and CD14 (CC17 and CCG33; gen). CD5Ig-pcDNA3.1(ϩ) was introduced into MOP8 NIH3T3 cells generously provided by Chris Howard, Institute for Animal Health, Comp- (CRL-1709; American Type Culture Collection, Manassas, VA) using Li- ton, U.K.) followed by magnetic depletion with sheep anti-mouse IgG- pofectamine (Life Technologies, Gaithersburg, MA) according to manu- coated magnetic beads (Dynal, Lake Success, NY). Cells were routinely Ͼ ϩ Ͻ ϩ facturer’s instructions. Stable transfectants were selected by limited dilu- 90% IgM and 5% CD5 as determined by flow cytometric analysis. tion cloning and selection in complete DMEM-10 containing 200 ␮g/ml B cell culture conditions G418 (Life Technologies). Supernatants were analyzed for mCD5Ig via dot blot and Western blot analysis. CD5Ig was purified from supernatants by B cells were cultured in cRPMI containing 10% FCS at a concentration of dialysis against PBS (pH 8) using 50000 MWCO cellulose dialysis tubing 2 ϫ 106 cells/ml. Cross-linking of bovine IgM was conducted using (SpectraPor; Spectrum Laboratories, Los Angeles, CA). Similarly, a Ј F(ab )2 of goat anti-bovine IgM (Kirkegaard & Perry Laboratories, Gaith- CD40Ig fusion protein containing the extracellular portion of bovine CD40 ersburg, MD) generated using pepsin cleavage (Immobilized Pepsin, and the CH2 and CH3 domains of huIgG1 was purified from stably trans- Pierce, Rockford, IL) according to manufacturer’s instructions. Culture of fected MOP8 cells. CD40Ig was found to bind boCD40L transfectants. B cells with a murine fibroblast cell line, DAP3, stably transfected with Purified human CTLA-4Ig, derived from American Type Culture Collec- bovine CD40L (boCD40L-DAP3), was conducted at a ratio of 1 CD40L- tion CRL-10762 (generously supplied by David R. Lee) did not demon- DAP3 cell to every four B cells, as previously described (7). Before cul- strate appreciable binding to bovine PBMCs and, thus, was used in addition 3160 THE IDENTIFICATION AND CHARACTERIZATION OF A LIGAND FOR BOVINE CD5 to huIgG1 as a negative control for nonspecific fusion protein binding attributable to human Fc interactions. Fusion proteins were biotinylated for use in flow cytometric analysis. Immunoprecipitation In experiments which the thiol-cleavable cross-linking agent, 3,3Јdithiobis- (sulfosuccinimidylpropionate) (DTSSP; Pierce) was used, cells were allowed to bind CD5Ig or isotype control (huIgG1 or CTLA4Ig) for 30 min, washed three times in cold PBS (pH 8), and simultaneously surface biotinylated and cross-linked with equal amounts of EZ-Link Sulfo-NHS-LC biotin (Pierce) and DTSSP in PBS (pH 8) for1honice. Cells were then washed three times in PBS and lysed in CHAPS lysis buffer (10 mM CHAPS, 150 mM NaCl, 20 mM Tris-Cl (pH 8), 0.2 mM PMSF, 10 mM iodoacetamide, 1 mM EDTA, 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin) for1honice. Cell lysate was then centrifuged for 10 min at 10,000 ϫ g at 4°C. Lysate supernatant was then incubated with protein A-Sepharose at 4°C overnight. Bound immune complexes were washed three times in lysis buffer and once in 0.05 M Tris-HCl (pH 6.8), reduced, and removed from protein A by heating in 2ϫ SDS sample buffer containing 2-ME followed by 10 s centrifugation at 10,000 ϫ g. Protein samples were then subjected to SDS-PAGE followed by electrotransfer via semidry blotting (Trans-Blot SD cell; Bio-Rad, Hercules, CA) to Hybond-P membrane (Amersham, Pis- cataway, NJ). Biotinylated proteins were detected by streptavidin-peroxi- Downloaded from dase (Vector Laboratories, Burlingame, CA) and enhanced chemilumines- cence (ECLϩ kit; Amersham). In experiments in which the cross-linking agent, DTSSP, was not used, harvested cells were washed three times and biotinylated as described above, lysed, and precleared overnight with protein A-Sepharose. Precleared lysate was then incubated with CD5Ig or huIgG1 for2hat4°Candthen incubated overnight with protein A-Sepha-

rose. Samples were then treated and analyzed as described above. http://www.jimmunol.org/ Results CD5L expression, as assessed by CD5Ig binding, is activation- state dependent To begin to characterize and identify the ligand for bovine CD5, we constructed a chimeric fusion protein consisting of the extracellular bovine CD5 domain fused to the Fc portion of huIgG1 (CD5Ig) and expressed this protein in both yeast (yCD5Ig) and FIGURE 1. Binding of CD5Ig by bovine PBMCs. A, PBMCs isolated mammalian cells (mCD5Ig). Similar to CD5, which exists as a from various donors were cultured in medium alone or in the presence of by guest on September 27, 2021 2ϩ monomer, the CD5Ig fusion protein was designed to be secreted as PMA and Ca ionophore for the indicated period and assayed for binding a monomer by eliminating the hinge region in the IgG1 construct, to mCD5Ig by flow cytometry. B, PBMCs were activated for 3 days in the presence of medium (dashed line), PMA (gray shaded area), Ca2ϩ iono- which allows for dimerization. CD5Ig was verified to be produced ϩ phore (bold solid line), or Ca2 ionophore and PMA (solid line) and as- in monomeric form by nonreducing PAGE analysis. Using this sayed for mCD5Ig binding. HuIgG1 and huCTLA4Ig are shown as nega- fusion protein, we sought to characterize the cellular expression of tive staining controls. Results are representative of three experiments. the ligand for CD5 by FACS analysis. PBMCs freshly isolated from five healthy donors demonstrated low to undetectable levels of mCD5Ig binding (Fig. 1A, d0, and data not shown) relative to for their ability to inhibit CD5Ig binding. To determine the re- the negative staining controls, huIgG1 and huCTLA4Ig. However, quirement for divalent cations for CD5Ig binding to CD5L, cells activation of PBMCs with PMA and ionophore resulted in the activated for 3 days with PMA and ionophore were stained with gradual increase in CD5Ig binding over a 3-day period, followed CD5Ig in the presence or absence of 25 mM EDTA and analyzed by a decrease in binding by day 4 (Fig. 1A). In some cases, culture by flow cytometry. As indicated in Table I, EDTA had no effect on of cells in medium alone over a 3-day period resulted in increased CD5Ig binding. The lack of inhibition of EDTA on CD5Ig binding CD5Ig binding, albeit at lower levels than PMA plus ionophore- indicates that the CD5-CD5L interaction is cation independent. activated cells. In the interest of determining whether PMA or The involvement of selected carbohydrate moieties in CD5Ig bind- calcium ionophore could act independently to induce CD5L ex- ing was investigating by carrying out CD5Ig binding in the presence pression, we treated cells for 3 days with calcium ionophore, PMA, or the combination of PMA and ionophore, and compared the level of CD5Ig binding between treatments. As shown in Fig. 1B, PMA Table I. Characterization of CD5Ig binding to activated PBMCsa alone is unable to enhance CD5L expression over that observed for cells cultured in medium alone. However, activation of cells with Treatment mCD5Ig Bindingb ionophore alone results in a CD5Ig binding pattern that is nearly EDTA ϩ identical with that observed for cells activated with both PMA and Fructose ϩ ionophore. Thus, activation of cells with ionophore alone is suf- Mannose ϩ ficient to induce CD5L expression, as assessed by CD5Ig binding. Glucose ϩ Moreover, PMA does not appear to enhance this effect. Trypsin – Proteinase K –

Characteristics of the CD5Ig-CD5L binding interaction a PBMCs were activated for 3 days with PMAϩ ionophore and analyzed for CD5Ig binding under the indicated conditions (see Materials and Methods). In an attempt to further characterize the CD5Ig-CD5L interaction, b Relative to untreated (ϩ indicates no change in binding; – indicates complete various binding conditions and cell pretreatments were examined abrogation of binding). The Journal of Immunology 3161 of several monosaccharides (D-glucose, D-fructose, and D-mannose). Concentrations of 1 M D-glucose, D-fructose, and D-mannose had no inhibitory effect on PBMC CD5Ig binding (Table I). Thus, these carbohydrates are not likely to contribute significantly to the CD5- CD5L interaction. Finally, pretreatment of activated cells with either trypsin or proteinase K totally abrogated CD5Ig binding (Table I). CD5L expression is detected on activated B cells Independent studies aimed at characterizing the ligand for CD5 have demonstrated the ligand to be expressed on a variety of cell types (37–42). Analysis of freshly isolated bovine PBMCs indicated that cells expressing low levels of CD5L in vivo were of an activated phenotype based on forward angle light scatter and were CD3Ϫ, CD5Ϫ, and IgMϩ (data not shown). To determine which cell population(s) could be induced to express the CD5L, small resting (Percoll-fractionated) PBMCs were activated for 3 days with PMA and Ca2ϩ ionophore and analyzed by flow cytometry using Abs against various bovine surface markers. Concordant Downloaded from with the results observed for freshly isolated PBMCs, mCD5Ig binding was primarily confined to B cells, according to CD21 (Fig. 2A) and IgM (data not shown) dual staining results. However, PMA and ionophore-activated CD3ϩ T cells did not appear to express the ligand that binds the CD5Ig fusion protein. Impor-

tantly, preincubation of cells with huIgG1 or CTLA4Ig before http://www.jimmunol.org/ staining did not block CD5Ig binding. Similar to the results obtained for resting lymphocytes activated in vitro, bulk (nonfractionated) PBMCs activated to express CD5L were primarily IgMϩ cells (Fig. 2B). In some experiments, a small fraction of non-IgMϩ cells was observed to bind CD5Ig. Although the exact identity of this cell population is presently unknown, these cells could potentially be non-IgM-expressing B cells that have undergone isotype switching. Alternatively, these CD5L-expressing cells could be- long to a non-B, non-T cell type. Importantly, as shown in Fig. 2B, by guest on September 27, 2021 yCD5Ig bound to activated cells in a pattern analogous to FIGURE 2. Cell populations demonstrating CD5Ig binding. A, Small mCD5Ig. In addition, preincubation of activated cells with unla- resting Percoll-fractionated PBMCs activated for 3 days with PMA plus ϩ beled yCD5Ig was found to partially inhibit mCD5Ig binding, Ca2 ionophore were analyzed for mCD5Ig binding and CD3 or CD21 2ϩ thereby demonstrating that yCD5Ig and mCD5Ig compete for dual staining. B, Three days poststimulation, PMA plus Ca ionophore- binding to a similar site (23% vs 11%; Fig. 2C). This result sug- activated PBMCs (nonfractionated) were analyzed for binding to mCD5Ig or yCD5Ig and IgM expression. C, Preincubation of PMA plus Ca2ϩ iono- gests that any differences in protein modifications occurring be- phore-activated PBMCs with unlabeled yCD5Ig inhibits binding of labeled tween yeast and mammalian-cell expression of CD5Ig do not sub- mCD5Ig. D, Bovine macrophages, BL3 and K562 cell lines do not bind stantially affect binding of CD5Ig to its ligand. CD5Ig. Macrophages were adhered in culture for 5 days, harvested, Additional cell populations analyzed did not demonstrate CD5Ig blocked with huIgG1 (3 ␮g/ml), and analyzed for CD5Ig binding by flow ϩ binding. CD14 adherent bovine macrophages were not observed cytometry. HuCTLA4Ig, boCD40Ig, huIgG1, and mouse IgG1 were used to bind the CD5Ig fusion protein under proper blocking conditions as negative staining controls. (Fig. 2D). Interestingly, a CD5ϩ bovine B lymphoblastoid cell line (BL3) was unable to bind to CD5Ig. Finally, K562 cells, shown by Calvo et al. (40) to bind huCD5Ig, were not observed to bind B cells activated with PMA and ionophore (data not shown). Un- CD5Ig (Fig. 2D), nor were murine fibroblast cell line DAP3, liver like endotoxin activation, PWM stimulation did not result in an cell line BNLSV A.8, or monkey kidney cell line COS-7 (data not appreciable increase in CD5Ig binding. Although the percentages shown). These results indicate the ligand for bovine CD5, as mea- of B cells induced to express CD5L were observed to vary from sured by both mCD5Ig and yCD5Ig binding, is primarily ex- experiment to experiment and from donor to donor, ligand expres- pressed by activated B cells. This finding is similar to recent de- sion on B cells was typically increased twofold or greater after scriptions of the alternative murine CD5L as characterized by activation with ionophore or endotoxin. huCD5Ig binding (38, 39). In addition to PMA and ionophore, other cellular activators were Ligation of CD40, but not B cell receptor cross-linking, results examined for their ability to induce CD5L expression on B cells. in CD5Ig binding As shown in Fig. 3, stimulation of PBMCs with endotoxin also Recently, we have demonstrated that CD5 expression can be in- resulted in increased CD5L expression on IgMϩ cells compared duced on bovine B cells after sIgM cross-linking, but not CD40 with cells cultured in medium alone. Whereas only 20% of B cells ligation, and furthermore, that activation of B cells through CD40 were found to express CD5L after 3 days of culture in medium functions to inhibit sIgM-mediated CD5 expression (7). Given the alone (as assessed by mCD5Ig and yCD5Ig binding), 40% of B differences in the regulation of CD5 expression with regard to cells expressed the ligand after activation with endotoxin (Fig. 3). CD40 and sIgM signaling, we were interested in determining This result is similar to the observed increase in CD5Ig binding by whether the expression of CD5L was affected by these distinct 3162 THE IDENTIFICATION AND CHARACTERIZATION OF A LIGAND FOR BOVINE CD5 Downloaded from

FIGURE 3. Endotoxin-activated B cells up-regulate CD5L expression, as assessed by CD5Ig binding. PBMCs were cultured in medium alone or activated with PWM (10 ng/ml) or endotoxin (20 EU/ml) for 3 days and assayed for MOP8-CD5Ig (or yCD5Ig, shown for medium) binding and

IgM expression via flow cytometry. http://www.jimmunol.org/ activation pathways. To determine the effect of CD40 ligation or sIgM cross-linking on CD5L expression, highly purified nonfractionated CD5Ϫ IgMϩ B cells were cultured for 2 days in the presence of nontransfected DAP3 cells, CD40L transfectants, goat anti- Ј ␮ bovine IgM F(ab )2 (25 g/ml) and DAP3 cells, or a combination Ј of CD40L-DAP3 cells and goat anti-bovine IgM F(ab )2. These cells were subsequently analyzed for CD5Ig binding. The result of by guest on September 27, 2021 CD5Ig binding by IgMϩ-gated cells is shown in Fig. 4A. One-third of the B cell population was observed to express CD5L in the absence of activation (35%, culture with DAP3 cells alone). How- ever, activation of B cells via BCR cross-linking did not result in an increase in the percentage of B cells expressing CD5L (38%). Interestingly, CD40 ligation did cause an increase in the number of B cells that bound CD5Ig (48%). Finally, CD40 ligation and sIgM activation synergistically resulted in a higher percentage of B cells FIGURE 4. CD40 ligation, but not IgM cross-linking alone, results in ϩ expressing CD5L (57%). However, activation of B cells with Ca2 up-regulation of CD5Ig binding. Non-Percoll-fractionated (A) or percoll- ionophore was the most efficient stimulus, resulting in induction of fractionated small resting (B) highly purified B cells (Ͼ90% CD5ϪIgMϩ) B cell expression of CD5L in 70% of B cells. were cultured in the presence of nontransfected DAP3 cells, CD40L-DAP3 Ј ␮ One potential explanation for the relatively high level of CD5Ig transfectants, goat anti-bovine IgM F(ab )2 (25 g/ml) and DAP3 cells, CD40L-DAP3 cells and goat anti-bovine IgM F(abЈ) (25 ␮g/ml) (A and binding by B cells not artificially activated in vitro (35%; Fig. 4A; 2 B), or Ca2ϩ ionophore (A) for 2 days and analyzed for mCD5Ig binding DAP3) was that there was a pre-existing population of naturally (solid line) and IgM expression. CD5Ig binding results are shown for activated B cells at the onset of culture. To determine whether this ϩ Ϫ ϩ IgM -gated cells. HuCTLA4Ig binding (gray shaded area) is shown as a was indeed the case, small resting CD5 IgM B cells were iso- negative control. C, B cells purified similar to that described in part B were lated by percoll fractionation and stimulated as described above for cultured for 2 days in the presence of CD40L transfectants or in the pres- ϩ Ј 2 days and IgM -gated cells were analyzed for CD5Ig binding. As ence of both CD40L-DAP3 cells and goat anti-bovine IgM F(ab )2 (25 predicted for a population of unstimulated resting B cells, only 3% ␮g/ml) and analyzed for mCD5Ig binding, CD5, and IgM expression via of the IgMϩ B cell population cultured with control DAP3 cells flow cytometry. CD5Ig and CD5 dual-staining results are shown for IgMϩ- expressed the ligand for CD5 (Fig. 4B). Similar to the observation gated cells. reported for nonfractionated cells stimulated with goat anti-bovine Ј IgM F(ab )2, no significant increase was observed in the percent- Ј ␮ age of B cells expressing CD5L after sIgM cross-linking (5%). centrations of goat anti-bovine IgM F(ab )2 (50, 75, and 100 g/ However, CD40 ligation dramatically increased the percentage of ml) were analyzed for the ability to increase CD5Ig binding, yet IgMϩ cells demonstrating CD5Ig binding (42%). Furthermore, while observed to result in increased CD5 expression, were in- dual activation of B cells with both CD40L transfectants and anti- effective at causing expression of its ligand to be enhanced (data IgM treatment appeared to synergize to result in an even greater not shown). Notably, B cells which were observed to express percentage of B cells expressing CD5L compared with cells acti- CD5 after BCR cross-linking were not found to coexpress vated through CD40 alone (57% vs 42%). Importantly, higher con- CD5L, although in cultures of nonPercoll-fractionated B cells, The Journal of Immunology 3163

CD5ϪCD5Lϩ B cells were indeed present. Given the finding that stimulation of B cells via both Ag receptor cross-linking and CD40 ligation display enhanced CD5Ig binding, we sought to determine whether B cells stimulated to express CD5 in these cultures were also induced to coexpress CD5L. To determine this, purified B cells were stimulated in the presence of CD40L transfectants alone or in combination with anti-IgM. As shown in Fig. 4C, the percentage of B cells (IgMϩ-gated cells) coexpressing CD5 and CD5L after 2 days stimulation with CD40L transfectants is only 2%. A greater percentage of B cells that are activated via BCR cross-linking and CD40 ligation are induced to express CD5 compared with those cultured in the presence of CD40L transfectants alone, as expected. However, although both CD5 and its ligand are induced on B cells after CD40 ligation and anti-IgM activation, CD5 and CD5L appear to be dual-expressed on only a small fraction of costimulated B cells (4%; Fig. 4C). Although a minor fraction of CD5ϩ B cells may be capable of coexpressing CD5L, the majority of B cells expressing CD5L after coculture with CD40L transfectants and BCR cross-linking Ab do not express CD5 Downloaded from (26%), despite the presence of a significant number of CD5ϩ B cells (25%). Thus, most CD5ϩ B cells in cultures stimulated by anti-IgM and CD40L are not observed to express CD5L. This finding is further supported by the observation that freshly isolated PBMCs (which include CD5ϩ B cells) expressing low levels of ϩ

CD5L are CD5-negative. In addition to this, the CD5 bovine B http://www.jimmunol.org/ lymphoblastoid cell line, BL3, was not observed to bind CD5Ig (Fig. 2D). Thus, most B cells expressing CD5L do not appear to coexpress CD5. Overall, these results indicate that CD5L is indeed, an activation Ag, whose expression is induced through ligation of CD40, or the combination of CD40 ligation and sIgM cross-linking. Importantly, sIgM cross-linking solely, even at high concentrations, was unable to modulate expression of CD5L. In- terestingly, these observations are the reciprocal of what we have reported for the regulation of CD5 expression by CD40 and B cell by guest on September 27, 2021 receptor signaling. CD5Ig augments B cell proliferation mediated by CD40 ligation Upon determining that CD5L was induced by ligation of CD40, we sought to determine whether CD5Ig had an effect on B cell proliferation mediated by stimulation of CD40. To determine the effect of CD5Ig on proliferation, panned B cells were cultured in the presence of CD40L-DAP3 cells and various concentrations of CD5Ig or isotype control (huIgG1) for 4 days and assayed for proliferation in the ﬁnal 18 h of culture via [3H]thymidine uptake. As shown in Fig. 5A, proliferation of CD40L-activated B cells was enhanced nearly 2.5-fold in the presence of 25 ␮g/ml CD5Ig compared with that observed for B cells activated by CD40L-DAP3 cells alone or CD40L transfectants and isotype control. This en- FIGURE 5. CD5Ig augments CD40L-stimulated B cell proliferation. B hancement of proliferation by CD5L stimulation appeared to be cells puriﬁed by passive panning were cultured with CD40L transfectants dose dependent, because decreasing concentrations of CD5Ig re- alone (A) or CD40L-DAP3 cells combined with goat anti-bovine IgM Ј ␮ sulted in reduced enhancement of proliferation. In addition to in- F(ab )2 (25 g/ml) (B), along with varying concentrations of MOP8-CD5Ig vestigating the effect of CD5Ig on proliferation elicited by ligation or huIgG1 for 4 days and assayed for proliferation, measured via [3H]thy- of CD40, we were interested in determining whether CD5Ig would midine uptake. C, Proliferation of panned B cells cultured with CD40L- Ϫ ␮ also have an effect on CD40L- and anti-IgM-stimulated prolifer- DAP3 cells alone ( ), or 25 g/ml huIgG1, MOP8-CD5Ig, or yCD5Ig was ation. As demonstrated in Fig. 5B, in the presence of CD5Ig, B cell analyzed 4 days poststimulation. D, Proliferation of B cells cultured with yCD5Ig (25 ␮g/ml) or mCD5Ig (12.5 ␮g/ml) in the presence or absence of proliferation was slightly enhanced over that of B cells activated CD40L transfectants. A–D, Data are shown as means of triplicate cultures. with CD40L and anti-IgM, in the absence or presence of huIgG1 Error bars represent the SD of the mean. Results are representative of two control. In addition to investigating the effect mCD5Ig had on B to three separate experiments. cell proliferation, the effect of yCD5Ig on B cell proliferation was examined. Not surprisingly, yCD5Ig enhanced CD40L-mediated B cell proliferation to a level similar to that observed with mCD5Ig (Fig. 5C). Notably, in the absence of additional stimuli, CD5Ig CD5Ig immunoprecipitates a 200-kDa protein alone was not observed to have a noticeable effect on background To further characterize the putative CD5L bound by CD5Ig, an B cell proliferation (Fig. 5D). attempt was made to immunoprecipitate the ligand from activated 3164 THE IDENTIFICATION AND CHARACTERIZATION OF A LIGAND FOR BOVINE CD5

B cells. B cells activated with PMA and Ca2ϩ ionophore for 3 days BL3 cells were used as a negative control cell population, because were allowed to bind CD5Ig or huIgG1, washed, and simulta- these cells do not demonstrate CD5Ig binding (Fig. 2D). As shown neously biotinylated and cross-linked using DTSSP. Cross-linking in Fig. 6B, a similar 200-kDa band was immunoprecipitated by of CD5Ig to CD5L was performed in initial experiments in an mCD5Ig from activated PBLs, but not from BL3 cells. However, attempt to stabilize an interaction that potentially would not be the isotype control did not immunoprecipitate proteins of this size. maintained under conditions of lysis. A similar procedure was used A similar result was obtained using activated bovine spleen cells, to successfully identify the ligand for CD6, ALCAM (43). The in which a 200-kDa protein was immunoprecipitated by CD5Ig, CD5Ig-CD5L complexes were isolated by protein A-Sepharose but not huIgG1 (data not shown). Although attempts have been immunoprecipitation and after denaturation (and hydrolysis of made to isolate sufficient quantities of the 200-kDa protein for cross-linking agent), resolved by denaturing SDS-PAGE. The pu- sequence analysis, sequence information for this protein has not tative ligand was detected by Western blotting with avidin-perox- yet been attainable due to the poor efficiency of immunoprecipi- idase. The immunoblots in Fig. 6A show that a protein of ϳ200 tation by the CD5Ig fusion protein in the absence of cross-linking. kDa is immunoprecipitated by mCD5Ig and yCD5Ig. However, However, these preliminary results indicate that the CD5L may immunoprecipitation of B cells using huIgG1 as an isotype control indeed be a protein of 200 kDa, although further investigation of did not result in the detection of a band of this size, nor did this putative protein is required to confirm that it is, in fact, the huCTLA4Ig (data not shown). Upon demonstrating that CD5Ig ligand for CD5. immunoprecipitated a 200-kDa protein, we next determined whether CD5Ig, in the absence of cross-linking, could immuno- Discussion precipitate its ligand from cell surface biotinylated B cell lysates. In this report, we describe the cellular distribution, as well as the Downloaded from induction, of bovine CD5L based on the binding characteristics of a chimeric bovine CD5Ig fusion protein. As determined by CD5Ig binding, expression of this ligand was typically negative to low on freshly isolated PBMC from various donors, with low-density expression being limited to B cells. We demonstrate that CD5L is

transiently up-regulated 1–4 days after stimulation with PMA and http://www.jimmunol.org/ Ca2ϩ ionophore, with maximal expression occurring 2–3 days poststimulation. Notably, Ca2ϩ ionophore alone was capable of inducing CD5L, independent of PMA stimulation. Importantly, this finding indicates that a [Ca2ϩ] increase stimulated by Ca2ϩ ionophore, but not activation of pathways targeted by PMA (PKC, etc.), is sufficient to up-regulate CD5L expression. Similar to murine CD5Ls described recently (38, 39), we demonstrate that the ligand for CD5 is not constitutively expressed, but inducible on B cells after various modes of activation (ie., Ca2ϩ by guest on September 27, 2021 ionophore, LPS, or CD40 ligation). Although Bikah et al. (39) have described a ligand on murine spleen cells induced by IgM cross-linking, our data demonstrate that BCR cross-linking alone on resting peripheral blood B cells is not sufficient to induce ligand expression. This observed difference can perhaps be attributed to differences in cellular activation state or the source of cells (peripheral blood vs spleen) or even differences in species. Our results regarding CD5L induction are similar to those reported by Bian- cone et al. (38), which demonstrated that the induction on murine splenic B cells requires the presence of T cells activated by anti- CD3 and anti-CD28, although no role for CD40 ligation was shown. Our data indicate that whereas CD40 ligation alone results in the up-regulation of CD5L, activation of B cells by both CD40 ligation and sIgM cross-linking results in even greater cell surface expression. Interestingly, activation through BCR cross-linking alone, although able to induce CD5 expression, did not result in expression of CD5L. The apparent discrepancy in CD5L up-regulation between B cells activated by BCR cross-linking, known to result in Ca2ϩ mobilization (44), and B cells activated by Ca2ϩ ionophore, is perhaps explained by differences in the signaling pathways that become activated as a result of ionophore-stimulation vs BCR ligation, as has previously been reported (45). More- FIGURE 6. CD5Ig immunoprecipitates a protein of ϳ200 kDa. A, Im- over, the level of intracellular Ca2ϩ increase is likely greater in munoprecipitation of lysates from cell surface biotinylated B cells acti- response to Ca2ϩ ionophore than to BCR cross-linking via soluble vated with PMAϩCa2ϩ ionophore for 3 days (A and B) or BL3 cells (B)by anti-IgM, and this could potentially account for the differences CD5Ig and huIgG1. In A, cell surface protein cross-linking (using DTSSP) and cell surface biotinylation was performed simultaneously before immu- observed in CD5L up-regulation. noprecipitation. Immunoprecipitated proteins were reduced, resolved on a The lack of CD5L induction by BCR cross-linking is an inter- 6% (A) or 5–20% gradient (B) SDS-PAGE gel, and detected by enhanced esting finding, because it indicates that B cell-specific expression chemiluminescent immunoblotting. The putative ligand is indicated by the of CD5 and its ligand are reciprocally regulated by CD40 and BCR arrow. signaling (Fig. 7). The implications for the counterregulation of The Journal of Immunology 3165

potentially serve as a checkpoint for CD5ϩ (or B-1) cell activation, such that in the absence of other activation or danger signals, CD5L is not expressed, and CD5ϩ B cell responsiveness is sup- pressed. Loss of this regulatory mechanism would presumably contribute to the uncontrolled expansion and transformation, as well as to the production of pathogenic autoantibodies by this B cell subset. The CD5-CD5L interaction might also play a role in the process of Ag presentation that may potentially occur between a CD5ϩ B cell and a CD5ϪCD5Lϩ (B-2) B cell. Due to their expression of low affinity, polyspecific Ag receptors, CD5ϩ B cells could potentially present TI-2 Ags captured by their Ab receptors to CD5Ϫ B cells expressing high affinity receptors, as has been previously proposed (46). During such an encounter, CD5, through its interaction with its ligand, could potentially costimulate acti- ϩ FIGURE 7. Proposed reciprocal regulation of CD5 and its ligand by vation of a CD5L B cell. Although much speculation can be CD40 and BCR signaling and hypothetical role of the CD5-CD5L inter- made, many questions remain to be answered regarding the role of action in B cells responses. Based on our results, B cells stimulated via Ag the CD5-CD5L interaction in immune responses, both protective receptor cross-linking (i.e., upon TI-2 Ag encounter) are induced to express and pathogenic.

CD5, which may subsequently function to negatively inhibit additional Similar to murine and human studies, which have suggested that Downloaded from signals transmitted through the Ag receptor. This process of CD5 up-reg- CD5 binds to a ligand other than CD72 (38–40), it is unlikely that ulation is inhibited by signals generated upon ligation of CD40. However, the ligand we have characterized in this report is bovine CD72. CD40 ligation, as reported here, is able to instead, stimulate B cell expres- First, the size of the protein immunoprecipitated by CD5Ig (180– sion of the ligand for CD5. Similarly, additional signals received by B 200 kDa) argues against a CD5Ig-CD72 interaction, because im- cells, such as LPS, would likely result in expression of CD5L. Although BCR cross-linking alone appears to be unable to result in the expression of munoprecipitation of CD72 would have been expected to result in

CD5L, it synergizes with signals generated by ligation of CD40 to enhance much smaller sized protein (45 kDa). However, it must be noted http://www.jimmunol.org/ CD5L up-regulation. Interaction between a CD5ϩ B cell and a CD5Lϩ B that immunoprecipitation of the 200-kDa protein by CD5Ig does cell may result in inhibition of the negative regulatory effect CD5 may not confirm that this protein is itself CD5L, because it may be a otherwise have on BCR signaling through physical sequestration of CD5. protein that associates with the true ligand. Sequence information Similarly, positive costimulatory signals received through CD5L may re- for this protein will assist in designing experiments directed to- ϩ sult in the augmentation of CD5L B cell activation. ward confirming whether it is indeed the ligand for CD5. Assum- ing CD72 follows a similar expression pattern in ruminants, if CD72, as expressed on resting B cells, acts as a ligand for CD5, CD5 and its ligand with regard to TD and TI B cell responses is resting bovine B cells would have been expected to bind CD5Ig. presently unknown. However, it can be hypothesized that the cog- However, this was not observed. These findings are in agreement by guest on September 27, 2021 nate interaction between an Ag-specific B cell and an activated T with recent studies that suggest murine and human CD5 bind to an cell (expressing CD40L) would result in the expression of CD5L activation-induced B cell-expressed ligand other than CD72 (38, (Fig. 7). On T cells, interaction of CD5 with its ligand could po- 39). However, to definitely prove that bovine CD5 does not inter- tentially augment signals received through the TCR, as previously act with CD72, bovine CD72 must first be cloned in order that reported (26–29). Similarly, according to the data presented in this experiments that are aimed at examining the interaction CD72 may study, costimulatory signals transmitted through CD5L (expressed form with CD5 can be performed. by a CD40L-activated B cell) would likely enhance CD40L-me- An important observation reported here is that the CD5Ig fusion diated B cell activation and proliferation. Thus, it is plausible to proteins produced by MOP8 cells (mammalian) and Pichia pas- suspect that the CD5-CD5L interaction that occurs between a T toris (yeast) demonstrated nearly identical binding patterns and and B cell plays an important role in driving TD immune were capable of competing for a similar binding site as determined responses. by blocking inhibition experiments. This suggests that potential The potential outcome of the CD5-CD5L interaction occurring differences in processing or modifications in the fusion protein that between a CD5ϩ B cell and a CD5Lϩ B cell is less clear. Based exist between mCD5Ig and yCD5Ig have no apparent effect on the on a study that demonstrated that the sequestration of CD5 away binding of CD5Ig to the CD5L. Furthermore, as assessed by their from IgM before BCR cross-linking blocks the inhibitory effect of ability to augment proliferation, these two fusion proteins appear CD5 on BCR signaling (32), engagement of CD5, through inter- to demonstrate equal functional activity. Additionally, unlike re- action with its ligand, may similarly block the negative influence cent studies in which huCD5Ig constructs have been used to char- CD5 potentially exerts on signals transmitted through the BCR. acterize the murine CD5L (38, 39), we have used a species-specific Such a B-B cell interaction might be expected to occur during TI-2 construct to avoid potential complications resulting from differ- Ag responses. Our data suggest that surface IgM cross-linking, ences in CD5 species cross-reactivity. Overall, this lends further mimicking TI-2 Ag activation, although able to result in CD5 ex- credence to the data presented within this study, which for the first pression, is not effective at up-regulating CD5L in the absence of time, characterize the expression pattern, binding requirements, other stimuli (i.e., CD40 ligation). The interaction of a B cell ex- activation conditions for induction, putative size (ϳ200 kDa), and pressing CD5 in response to the cross-linking of its polyreactive costimulatory role, for the first ligand to be identified for bovine Ab receptor (via encounter with a TI-2 Ag) with a B cell express- CD5. The data reported herein, as well as future studies aimed at ing CD5L (via activation by LPS, CD40L, or other signal) may further characterizing this newly identified bovine CD5L, will un- allow for further activation of the CD5ϩ B cell. In the absence of doubtedly aid in understanding the role that the CD5-CD5L inter- the CD5-CD5L interaction, CD5 may continue to negatively reg- action plays in immune responses. In addition, the characterization ulate BCR signaling, and thereby inhibit further B cell activation of this newly identified bovine CD5L may assist in elucidating the or responses to TI-2 Ags. Thus, the CD5-CD5L interaction could role that CD5 expression plays on bovine B cells during natural 3166 THE IDENTIFICATION AND CHARACTERIZATION OF A LIGAND FOR BOVINE CD5 infections, such as bovine leukemia virus and trypanosomosis in 22. Curry, M. P., L. Golden-Mason, N. Nolan, N. A. Parfrey, J. E. Hegarty, and C. ϩ which the CD5ϩ B cell population becomes expanded. O’Farrelly. 2000. Expansion of peripheral blood CD5 B cells is associated with mild disease in chronic hepatitis C virus infection. J. Hepatol. 32:121. 23. Sampalo, A., M. Lopez-Gomez, J. Jimenez-Alonso, F. Ortiz, F. Samaniego, and Acknowledgments F. Garrido. 1993. CD5ϩ B lymphocytes in HIV infection: relationship to immu- nological progression of disease. Clin. Immunol. Immunopathol. 66:260. We thank Louise Barnett for her technical assistance with flow cytometric 24. Murakami, M., and T. Honjo. 1995. Involvement of B-1 cells in mucosal immu- analysis. We also thank Dr. David Lee for supplying huCTLA4-Ig and Dr. nity and autoimmunity. Immunol. Today 16:534. Chris Howard for supplying CC17 and CCG33 Ab. 25. Schroeder, H. W., Jr., and G. Dighiero. 1994. The pathogenesis of chronic lymphocytic leukemia: analysis of the antibody repertoire. Immunol. Today 15:288. 26. Imboden, J. B., C. H. June, M. A. McCutcheon, and J. A. Ledbetter. 1990. References Stimulation of CD5 enhances signal transduction by the T cell antigen receptor. 1. Resnick, D., and P. A. Krieger. 1994. The SRCR superfamily: a family reminis- J. Clin. Invest. 85:130. cent of the Ig superfamily. Trend. Biochem. Sci. 19:5. 27. Ceuppens, J. L., and M. L. Baroja. 1986. Monoclonal antibodies to the CD5 2. Koskinen, R., T. W. Gobel, C. A. Tregaskes, J. R. Young, and O. Vainio. 1998. antigen can provide the necessary second signal for activation of isolated resting The structure of avian CD5 implies a conserved function. J. Immunol. 160:4943. T cells by solid-phase-bound OKT3. J. Immunol. 137:1816. 3. Fabb, S. A., K. J. Gogolin-Ewens, and J. F. Maddox. 1993. Isolation and nucle- 28. Spertini, F., W. Stohl, N. Ramesh, C. Moody, and R. S. Geha. 1991. Induction of otide sequence of sheep CD5. Immunogenetics 38:241. human T cell proliferation by a monoclonal antibody to CD5. J. Immunol. 146: 4. Yu, Q., M. Reichert, T. Brousseau, Y. Cleuter, A. Burny, and R. Kettmann. 1990. 47. Sequence of bovine CD5. Nucleic Acids Res. 18:5296. 29. June, C. H., P. S. Rabinovitch, and J. A. Ledbetter. 1987. CD5 antibodies increase 5. Huang, H. J., N. H. Jones, J. L. Strominger, and L. A. Herzenberg. 1987. Mo- intracellular ionized calcium concentration in T cells. J. 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