[CANCER RESEARCH 48, 37-40, January 1, 1988] Molecular Pathways of Adhesion in Spontaneous Resetting of T-Lymphocytes to the Hodgkin's Cell Line L428 Martin E. Sanders,1 M. William Makgoba, Eileen H. Sussman, Gale E. G. Luce, Jeffrey Cossman, and Stephen Shaw The Immunology Branch [M. E. S., M. W. M., G. E. G. L., S. SJ and the Laboratory of Pathology [E. H. S., J. C.J, National Cancer Institute, NIH, Bethesda, Maryland 20892 ABSTRACT Spontaneous resetting of T-lymphocytes to Reed-Sternberg cells has been observed both in vitro and in vivo but its molecular mechanism has not been defined. We have investigated such resetting using the Hodgkin's cell line L428. L428 expresses high levels of LFA-3 and ICAM-1, both of which are ligands for T-cell adhesion. Monoclonal antibody inhibition of spontaneous resetting indicated that it is not dependent on the T-cell receptor complex but is largely mediated by interaction of T-cell CD2 (Tll/E-rosette receptor) with its ligand LFA-3 on I 428 cells. Studies using an alternate assay of adhesion (conjugate formation) confirm the roles of CD2/LFA-3 and also implicate a second mode of binding via LFA-1 on T-cells to ICAM-1 on L428. These data explain the previously reported finding of T-cell resetting with Reed-Sternberg cells as an exaggeration of normal antigen-independent T-cell adhesion. INTRODUCTION RS2 cells are a malignant cell type of uncertain origin that are found in diseased lymphoid tissues of patients with Hodg- kin's disease. A unique characteristic of RS cells is that they spontaneously form rosettes with T-lymphocytes (1-3). Such rosettes form rapidly in vitro with autologous lymphocytes in single cell suspensions of fresh splenic tissue or lymph nodes from patients with Hodgkin's disease; rosettes also form with allogeneic lymphocytes (4). In vivo adhesion of lymphocytes to RS cells is evidenced by the frequent finding of similar clusters of lymphocytes around RS cells in Giemsa-stained imprints of Hodgkin's tissue sections (2), as well as electron microscopic studies of Hodgkin's tissues showing tight apposition and uro pod formation between lymphocytes and RS cells (5, 6). The pathophysiological relevance of such lymphocytes sur rounding RS cells in tissue sections to tumor immunity has remained unclear. One study suggested that the finding of large numbers of autologous lymphocytes adherent to RS cells in tissue suspension was a favorable prognostic indicator (7). Furthermore, one electron microscopic study (6) noted ultra- structural changes in the RS cells suggesting a cytotoxic effect of the surrounding lymphocytes. However, other studies have demonstrated that autologous lymphocytes may adhere to RS cells in culture for up to several wk without leading to cytolysis (1, 2). Progress has been made in elucidating the molecular mech anisms involved in other models of T-cell adhesion. Studies have shown that cytotoxic T-lymphocytes may adhere to other cells via antigen-independent mechanisms (8, 9). Since such adhesion does not involve the specific antigen receptor, cytolysis does not occur. Two different molecular pathways of antigen- independent adhesion have been identified (9). The CD2 mol ecule (also known as Til, LFA-2, or the sheep erythrocyte Received 6/25/87; revised 9/23/87; accepted 9/28/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at NIH, Bldg. 10, Room 4BI7, Bethesda, MD 20892. 2The abbreviations used are: RS, Reed-Sternberg; MAB, monoclonal antibody. receptor) on T-lymphocytes mediates adhesion via binding to lymphocyte function-associated antigen 3 (LFA-3), a ubiqui tously distributed cell surface glycoprotein (10, 11). CD2 bind ing to LFA-3 is the mechanism of adhesion in the phenomenon of rosette formation of T-lymphocytes with human erythrocytes ( 12,13) and in human thymocyte adhesion to thymic epithelium (14). The other molecular pathway of T-cell adhesion involves interaction of T-cell surface LFA-1 with one or more target cell surface ligands. Functional evidence indicates that intercellular adhesion molecule 1 (ICAM-1) is one ligand for LFA-1; in some experimental systems, LFA-1-dependent adhesion of B- cells and T-cells can be inhibited by anti-ICAM-13 (15). L428 is a Hodgkin's cell line which has morphological char acteristics and cell surface marker patterns identical to those of freshly obtained RS cells and shows the typical RS cell phenom enon of spontaneous rosette formation with T-lymphocytes (4, 16, 17). In the present study we demonstrate that the observed spontaneous adhesion of T-lymphocytes to the Hodgkin's cell line L428 occurs primarily via binding of T-lymphocyte CD2 to L428 cell LFA-3, with some lesser contribution to this adhesion by T-cell LFA-1 and L428 cell ICAM-1. MATERIALS AND METHODS Cells and Reagents. L428 was kindly provided by Dr. V. Diehl, University of Cologne, Federal Republic of Germany, and was grown in RPMI 1640 with 10% fetal calf serum. For rosette assays human T- lymphocytes were purified from peripheral blood mononuclear cells of normal donors by resetting with 2-aminoethylisothiouronium bromide hydrobromide-treated sheep erythrocytes. Conjugate assays were per formed using a cytotoxic T-lymphocyte clone 8.2 (specific for DPw2 and noncytolytic for L428) (18). Monoclonal antibody TS2/9 to LFA- 3 and RR1/1 to ICAM-1 were kindly provided by Dr. T. Springer (Dana FärberCancer Institute, Boston, MA), (15, 19). MAB 84H10 to ICAM-1 was kindly provided by Dr. P. Mannoni (Institute J. Paoli-I. Calmette, Marseille, France). MAB MHM23, specific for the ß chain of LFA-1, was kindly provided by Dr. J. Hildreth (The Johns Hopkins University, Baltimore, MD). Anti-CD2 MAB 95-5-49 was kindly pro vided by Dr. R. Quiñonesand Dr. R. Gress (National Cancer Institute, NIH, Bethesda, MD). MAB W6/32, to a framework epitope of human class I HLA, was kindly provided by Dr. S. Jacobson (National Institute of Neurological and Communicative Disorders and Stroke, NIH, Be thesda, MD). The CD3 MAB OKT3 was kindly provided by Dr. G. Goldstein (Ortho Pharmaceuticals, Raritan, NJ). All MABs were used as purified immunoglobulin. Fab fragments for MAB to LFA-1 and LFA-3 were prepared by papain digestion. Flow Microfluorometry. One x 10' cells per sample were incubated with saturating amounts of relevant antibody or culture supernatant for 30 min at 4°C.After two washes, cells were further incubated with a saturating amount of goat anti-mouse IgG F(ab')z for 30 min at 4'C. Following two final washes, 50,000 cells were analyzed on a Becton Dickinson FACS II flow cytometer and results expressed as linear unit millivolts. Rosette Assay. One x IO6 L428 cells and 10 x 10' T-lymphocytes were added to test tubes containing 1.0 ml of RPMI 1640 with 10% fetal calf serum and 10 pg of a purified MAB. Tubes were centrifuged 1 M. W. M. ft al., submitted for publication. 37 Research. on December 4, 2015. © 1988 American Association for Cancer cancerres.aacrjournals.org Downloaded from