Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 2603-2607, April 1990 Immunology Role of interaction of CD2 molecules with lymphocyte function- associated antigen 3 in T-cell recognition of nominal antigen (cell adhesion/cytotoxic T cells/T-cell activation/T-cell receptor) SHIGEO KOYASU*t*, TREBOR LAWTON*, DAVID NovICK*, MICHAEL A. RECNY*§¶, ROBERT F. SILICIANO11, BARBARA P. WALLNER**, AND ELLIS L. REINHERZ*tt *Laboratory of Immunobiology, Dana-Farber Cancer Institute, and Departments of tPathology, ttMedicine, and 1Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; §Procept, Inc., Cambridge, MA 02139; 'Division of Molecular and Clinical Rheumatology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and **Biogen Research Corporation, Cambridge, MA 02142 Communicated by Max D. Cooper, January 3, 1990 (receivedfor review December 1, 1989) ABSTRACT The role of the interaction of CD2 molecules expressing human CD2 have suggested that CD2-LFA-3 with lymphocyte function-associated antigen 3 (LFA-3) in interaction is important for the T-cell recognition process (7, facilitating nominal antigen recognition by T lymphocytes was 8). However, virtually nothing is known about the mecha- studied by utilizing an HLA-DR4-restricted CD4+ cytotoxic nism by which CD2-LFA-3 interaction augments T-cell rec- human T-cell clone specific for human immunodeficiency virus ognition-in particular, whether the interaction influences envelope glycoprotein gpl20 as a responder and murine flbro- recognition of nominal antigen and whether this conjugate blasts transfected with human class II major histocompatibility pair interacts with TCR during the antigen recognition pro- complex (MHC) and/or human LFA-3 molecules as antigen- cess. To analyze the contribution of CD2-LFA-3 interaction presenting cells (APC). Although expression of the DR4 re- to T-cell recognition of nominal antigen, we have examined striction element in fibroblasts is sufficient for T-cell recogni- the nature of physiologic interactions between the CD4' tion of a gpl20 peptide asjudged by induction ofproliferation, HLA-DR4-restricted human cytotoxic T-cell clone Een- coexpression of human LFA-3 on DR4+ APC decreases the 217.5, which is specific for the H3DCG human immunode- molar requirement of nominal antigen by greater than one ficiency virus 1 envelope glycoprotein gp120 (9), and murine order ofmagnitude. Both LFA-3 and the relevant class H MHC fibroblasts into which human class II MHC restricting ele- molecules are necessary for antigen-independent conjugate ments and LFA-3 were selectively transfected. Such a sys- formation, but the binding is further enhanced by specific tem, in which only defined human molecules are expressed nominal antigen. CD2-LFA-3 interaction is independent of on antigen-presenting cells (APC), allows for a controlled T-cell receptor-MHC interaction and contributes directly to analysis of individual receptor-ligand pairs. the stabilized conjugate between the T cell and LFA-3-bearing APC; soluble CD2 and monoclonal antibodies to LFA-3 and CD2 reduce T-cell-APC binding to the level mediated by MATERIALS AND METHODS nominal antigen and MHC. During conjugate formation, CD2 Materials. A peptide corresponding to residues 410-429 of but not CD3 molecules are reorganized into the cell-cell gp120 ("gpl20 peptide") was synthesized on an Applied interaction site in an antigen-independent manner. Thus, re- Biosystems 431A automated peptide synthesizer and purified organization and/or coassociation of CD2 with CD3 molecules by reverse-phase HPLC using a Waters Deltapak C18 column. is not essential for T-cell activation. The secreted membrane anchor-lacking forms of CD2 and CD4 molecules were prepared in a baculovirus system as The ability ofT lymphocytes to recognize specific antigens in described (10, 11). The following monoclonal antibodies the context of class I and class II major histocompatibility (mAbs) were used in this study: Leu4 (anti-CD3); l9Thy5D7 complex (MHC) molecules is determined by the unique (anti-CD4); 3T4-8B5 (anti-T111); 1OLD24CI (anti-T112); 9-49 clonotypic T-cell receptor (TCR)-CD3 complex which they (directed against the common determinant of human class II individually bear. The process of antigen recognition in- MHC molecule); 11.4.1 (anti-murine H-2Kk); TS2/9.1.1 (an- volves the physical interaction of a TCR with a nominal ti-human LFA-3); fluorescein isothiocyanate (FITC)- peptide antigen bound to a specific MHC molecule referred conjugated 1OLD24C1; and tetramethylrhodamine isothio- to as a restricting element. This process is not singularly cyanate (TRITC)-conjugated Leu4. dictated by the TCR complex itself, but rather is dependent Cells and Growth Conditions. The CD4' CD8- human on other structures, including CD4, CD8, CD2, and lympho- T-lymphocyte clone Een217.5 is a subclone ofEen217 that is cyte function-associated antigen 1 (LFA-1) (reviewed in refs. specific for gpl20 in a DR4-restricted manner (9). Nomen- 1-3). CD4 and CD8 structures bind to monomorphic regions clature and phenotype of the murine L cell fibroblast trans- of class II and class I MHC structures, respectively, thereby fectants used as APC in this study are shown in Table 1. The facilitating the interaction of the TCR with the MHC restrict- preparation of murine L cell fibroblasts transfected with ing element (reviewed in ref. 4). In addition to the MHC full-length cDNA encoding human class II DR a and / chains binding accessory structures, a set of adhesion structures was described previously (9, 12). Those cells were further facilitates non-antigen-specific interactions between T lym- transfected with Xmn I-linearized animal expression plasmid phocytes and their cognate partners. This set includes the BG8/LFA-3, which contains the full-length cDNA for the CD2/lymphocyte function-associated antigen 3 (LFA-3) re- transmembrane form ofLFA-3, and Sca-I-linearized plasmid ceptor-ligand pair (5, 6). Studies employing antibodies directed against CD2 or Abbreviations: APC, antigen-presenting cell(s); FITC, fluorescein LFA-3 as well as murine T-cell hybridoma transfectants isothiocyanate; LFA-1, lymphocyte function-associated antigen 1; LFA-3, lymphocyte function-associated antigen 3; mAb, monoclonal antibody; MHC, major histocompatibility complex; TCR, T-cell The publication costs of this article were defrayed in part by page charge receptor; TRITC, tetramethylrhodamine isothiocyanate. payment. This article must therefore be hereby marked "advertisement" *On leave from: Tokyo Metropolitan Institute of Medical Science, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Tokyo 113, Japan. 2603 Downloaded by guest on October 2, 2021 2604 Immunology: Koyasu et al. Proc. Natl. Acad. Sci. USA 87 (1990) Table 1. Phenotypes of L cell transfectants the human Een217.5 T-cell clone is cultured with the DR4+ Cell line Class II MHC LFA-3 LFA-3- murine L cell transfectant L89.2, it is stimulated to proliferate in expanding T-cell colonies and readily lyses L* - _ L89.2 only in the presence of specific peptide antigen derived L-HT16 - ++ from gpl20 of the human immunodeficiency virus H3DCG L17.8 DRw53t _ strain (Fig. 1). Using various L cell transfectants as APC, we L12.2 DR7 - examined the effect of CD2-LFA-3 interaction on the pro- L12.2.16 DR7 ++ liferative response of Een217.5 to gpl20 peptide at a range of L89.2 DR4 - concentrations. As shown in Fig. 2a, L89.2.1 could stimulate L89.2.11 DR4 proliferation of Een217.5 to the same degree as L89.2 but L89.2.1 DR4 ++ required <1/10th the molar concentration of peptide. *L cells were established from C3H/He mice (H-2k). L89.2.11, which expresses 1/5th the LFA-3 of L89.2.1, tExpression of class II MHC molecules among different positive stimulated Een217.5 at a lower concentration ofpeptide than transfectants was virtually identical except for L17.8, which ex- presses 1/5th the surface copy number. L89.2 but was less efficient than L89.2.1. As anticipated by tAll LFA-3 transfectants expressed virtually equivalent copy num- the MHC restriction of Een217.5, neither L cells nor L cell bers of surface LFA-3 except for L89.2.11, which expresses 1/5th transfectants L-HT16, L17.8, L12.2, and L12.2.16 induced the surface copy number. Een217.5 to proliferate even in the presence of 1,uM specific peptide (data not shown). Although not shown, Een217.5 also pOPF, which carries the thymidine kinase gene (13). Trans- lysed L89.2.1 at a decreased peptide concentration relative to fected cells were selected in Dulbecco's modified Eagle's L89.2. medium (DMEM)/10% (vol/vol) fetal calf serum (FCS) con- The enhanced proliferation of Een217.5 in response to a taining 100 ,uM hypoxanthine/0.4 mM aminopterin/16 ,M suboptimal concentration of peptide presented by L89.2.1 thymidine (HAT) and were further analyzed for expression of versus L89.2 is clearly a consequence of CD2-LFA-3 inter- surface LFA-3 as well as class II molecules by indirect action. As shown in Fig. 2b, when anti-LFA-3 mAb was immunofluorescence. added to the culture stimulated by antigen and L89.2.1, the Proliferation Assays. Proliferation assays using fibroblast proliferative response was reduced to the same level as the cells as APC were performed as follows. Fibroblasts were response induced by L89.2. In contrast, the same antibody incubated with DMEM/10% FCS containing mitomycin C at did not affect the proliferative response to peptide plus L89.2. 10 ,g/ml and 1 mM thymidine for 4 hr. After extensive mAbs against the endogenous L cell H-2Kk murine class I washing, the cells were trypsinized and plated into 96-well MHC molecules did not show any inhibitory effect. These flat-bottom plates (2 x 104 per well). Een217.5 cells (5 x 104 results indicate that expression of the relevant class II MHC per well) were stimulated with those fibroblasts in the pres- molecules on APC is sufficient for T-cell proliferation but that ence ofgpl20 peptide at various concentrations for 24 hr.