Proc. Nati. Acad. Sci. USA Vol. 87, pp. 5031-5035, July 1990 Immunology T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-1 PETER S. LINSLEY*, EDWARD A. CLARKt, AND JEFFREY A. LEDBETTER* *Oncogen, 3005 First Avenue, Seattle, WA 98121; and tDepartment of Microbiology, University of Washington, Seattle, WA 98195 Communicated by Seymour J. Klebanoff, March 30, 1990 ABSTRACT Studies using monoclonal antibodies (mAbs) intercellular adhesion mediated by major histocompatibility have implicated the homodimeric glycoprotein CD28 as an complex (MHC) class I (13) and class II (14) molecules with important regulator of human T-cell activation, in part by the CD8 and CD4 accessory molecules, respectively. We posttranscriptional control ofcytokine mRNA levels. Although have expressed the CD28 antigen to high levels in Chinese the CD28 antigen has functional and structural characteristics hamster ovary (CHO) cells and have used these transfected of a receptor, a natural ligand for this molecule has not been cells to develop a CD28-mediated cell adhesion assay. By identified. Here we show that the CD28 antigen, expressed in using this assay as a screening method, we have demon- Chinese hamster ovary (CHO) cells, mediated specific inter- strated an interaction between CD28 and a natural ligand cellular adhesion with human lymphoblastoid and leukemic expressed on activated B lymphocytes, the B7/BB-1 antigen. B-cell lines and with activated primary murine B cells. CD28- mediated adhesion was not, dependant upon divalent cations. Several mAbs were identified that inhibited CD28-mediated MATERIALS AND METHODS adhesion, including mAb BB-1 against the B-cell activation mAbs. mAb 9.3 (anti-CD28, ref. 15) was purified from antigen B7/BB-1 and some mAbs against major histocompat- ascites fluid before use. A number of mAbs to B-cell asso- ibility complex class I antigens. B7/BB-1 expression correlated ciated antigens were tested for their abilities to inhibit CD28- closely with CD28-mediated adhesion, but class I expression mediated adhesion. mAbs 60.3 (CD18); iF5 (CD20); G29-5 did not. Transfected COS cells expressing the B7/BB-1 antigen (CD21); G28-7, HD39, and HD6 (CD22); HD50 (CD23); adhered to CD28+ CHO cells; this adhesion was blocked by KB61 (CD32); G28-1 (CD37); G28-10 (CD39); G28-5 (CD40); mAbs to CD28 and B7/BB-1. The specific recognition by CD28 HERMES1 (CD44); 9.4 (CD45); LB-2 (CD54); and 72F3 of the B-cell activation antigen B7/BB-1 represents a hetero- (CD71) have been described and characterized (16-18). philic interaction between members of the immunoglobulin These mAbs were purified before use. &TA4-1 (ref. 19, anti- superfamily that may serve to regulate T-cell cytokine levels at IgD); 2C3 (ref. 20, anti-IgM); Nambl, H1DE, p10.1, and sites of B-cell activation. W6/32 (refs. 20 and 21, anti-human class I); and HBiOa (ref. 20, anti-MHC class II) were also purified before use. mAbs The generation of a T-lymphocyte immune response is a B43 (CD19); BL-40 (CD72); AD2, 1E9.28.1, and 7G2.2.11 complex process involving cell-cell interactions (1) and pro- (CD73); EBU-141, LN1 (CDw75); CRIS-1 (CD76); 424/4A11 duction of soluble immune mediators (cytokines or lympho- and 424/3D9 (CD77); Leu 21, Ba, 1588, LO-panB-1, FN1, kines) (2). This response is regulated by several T-cell surface and FN4 (CDw78); and M9, G28-10, HuLymlO, 2-7, F2B2.6, molecules, including the T-cell receptor complex (3) and 121, L26, HD77, NU-B1, BLAST-1, BB-1, anti-BL7, anti- other "accessory" surface molecules (1). HC2, and L23 were used as coded samples provided to One such accessory molecule is the CD28 antigen, a participants in the Fourth International Conference on Hu- homodimeric glycoprotein of the immunoglobulin superfam- man Leukocyte Differentiation Antigens (September 1989, ily (4) found on most mature human T cells (5). Current Vienna) (22). Most of these were used in ascites fluid form. evidence suggests that this molecule functions in an alterna- mAbs BB-1 and LB-1 (23) were also purified from ascites tive T-cell activation pathway distinct from that initiated by fluid before use. Anti-integrin receptor mAbs, P3E3, P4C2, the T-cell receptor complex (for review, see ref. 6). Mono- and P4G9 (24), were used as hybridoma culture supernatants. clonal antibodies (mAbs) to CD28 can augment T-cell re- Plasmids and Transfections. cDNA clones encoding the sponses initiated by various polyclonal stimuli (5-7). These T-cell antigens CD4, CD5, and CD28 (4) in the expression stimulatory effects may result from mAb-induced cytokine vector TH3M (4) were kindly provided by Sandro Aruffo and production (8) as a consequence of increased mRNA stabi- Brian Seed (Massachusetts General Hospital, Boston). An lization (9). Anti-CD28 mAbs can also have inhibitory effects; expressible cDNA clone encoding the B7/BB-1 antigen (25) i.e., they can block autologous mixed lymphocyte reactions was provided by Gordon Freeman (Dana-Farber Cancer (10) and activation of antigen-specific T-cell clones (11). Institute, Boston). Dihydrofolate reductase-deficient CHO The in vivo function of CD28 is not known, although its cells were cotransfected as described (26) with a mixture of structure (4) suggests that like other members of the immu- plasmids, irH3M-CD28 (4) and pSV2dhfr (27). COS cells noglobulin superfamily (12) it might function as a receptor. were transfected with B7/BB-1, CD4, or CD5 cDNAs in the CD28 could conceivably function as a cytokine receptor, presence of DEAE-dextran using a protocol supplied by B. although this seems unlikely since it shares no homology with Seed and A. Aruffo. other lymphokine or cytokine receptors (4). CD28-Mediated Adhesion Assay. Cells to be tested for Alternatively, CD28 might be a receptor that mediates adhesion were labeled with 51Cr, washed, and preincubated cell-cell contact. In this paper, we describe experiments in complete RPMI medium [RPMI 1640 medium containing designed to test this possibility. For this purpose, we have 10% (vol/vol) fetal bovine serum, penicillin (100 units/ml), taken an approach based on experiments used to demonstrate and streptomycin (100 ,g/ml)] containing 10 mM EDTA The publication costs of this article were defrayed in part by page charge Abbreviations: mAb, monoclonal antibody; MHC, major histocom- payment. This article must therefore be hereby marked "advertisement" patibility complex; FITC, fluorescein isothiocyanate; LPS, lipopoly- in accordance with 18 U.S.C. §1734 solely to indicate this fact. saccharide. 5031 Downloaded by guest on September 26, 2021 5032 Immunology: Linsley et al. Proc. Natl. Acad. Sci. USA 87 (1990) unless otherwise indicated. mAbs to be tested for adhesion CD28 positive CHO inhibition were then added to 10 tig/ml, and cells were incubated for -1 hr at 230C. In some experiments, a mouse No Addition mAb having irrelevant specificity was added to the labeling MAb 9.3 and preincubation reaction mixtures to saturate Fc receptors. Labeled cells (1-10 x 106 cells per well in 0.2 ml of complete EDTA RPMI medium containing EDTA and mAbs, where indi- cated) were then added to the CHO monolayers. When MAb 9.3 + EDTA adhesion inhibition by mAb 9.3 was measured, the mAb or its 0 20 40 60 80 F(ab')2 were added to the CHO cells 1 hr prior to addition of labeled cells. Adhesion was initiated by centrifugation in a CD28 negative CHO plate carrier and continued at 370C for 1 hr. Monolayers were No Addition then washed five times with ice-cold complete RPMI medium and solubilized by addition of 0.5 M NaOH, and radioactivity MAb 9.3 was measured in a y counter. Numbers of bound cells were calculated by dividing total bound radioactivity (cpm) by the EDTA specific activity (cpm per cell) of labeled cells. When COS MAb 9.3 + EDTA A.~~~~~~~~~~~~~~~~~~~~~~~I cells were used, results are expressed as cpm bound since 0 20 40 60 80 their viability at the end of the experiment was generally Cells Bound <50%. FIG. 1. T51 lymphoblastoid cells adhere specifically to CD28- transfected CHO cells. Monolayers of CD28' and CD28- CHO cells RESULTS (1-1.2 x 105 cells percm2 in 48-well plastic dishes) were fixed in 0.5% paraformaldehyde for 20 min at 230C, washed, blocked in complete Isolation of CHO Cells Expressing the CD28 Antigen. If RPMI medium, and then preincubated alone or with mAb 9.3 or mAb CD28 binds to a cell surface ligand, then cells expressing the 9.3 F(ab')2 at 10,ug/ml in complete RPMI for 1 hr at 370C. T51 cells ligand should adhere more readily to cells expressing CD28 were labeled with 51Cr, preincubated alone or with 10 mM EDTA, than to cells that do not. To test this hypothesis, we first and added to CHO cells, and cellular adhesion was measured. Data cotransfected an expressible cDNA clone encoding CD28 (4) are the number of cells bound (x 10-3; mean ± SD; error bars) of plus a selectable marker (pSV2dhfr, ref. 27) into dihydrofo- three replicate determinations. late-reductase-deficient CHO cells. Transfectants were iso- lated, and the dihydrofolate reductase marker was amplified munohistological staining of T51 cells (Fig. 2). A similar, but by growth in increasing concentrations of methotrexate. Cell slightly less significant increase in adhesion specificity was lines expressing high (CD28+) and low (CD28-) levels of the also measured in the presence of the calcium-specific chela- CD28 antigen were isolated from amplified populations by tor, EGTA (data not shown). fluorescence-activated cell sorting after immunostaining with CD28 Ligand Is a B-Cell Activation Marker. The increased mAb 9.3.