Eur. J. Immunol. 1990. 20: 2259-2268 Regulation of T-Tcell interactions 2259 Staley A. BrodV, Meghan Purvee, Deborah Benjamin and David A. Hafler Center for Neurologic Diseases, Division of Neurology, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston T-T cell interactions are mediated by adhesion molecules* The mechanism by which Tcells signal other Tcells is not well defined. This was investigated by studying the ability of circulating T cells to induce the proliferation of autologous T cell clones. Peripheral blood T cells activated by cross-linking of the CD3/Tcell receptor complex, which increased the expression of cell adhesion molecules LFA-1, LFA-3 and ICAM-1, induced the proliferation of autologousT cell clones. Irradiated antigen-activated peripheral blood T cells could also induce the proliferation of Tcell clones which could not recognize that antigen. T-Tcell activation required cell contact, was not major histocompatibility complex (h4HC) restricted and was blocked by monoclonal antibodies directed against adhesion molecules CD2 and LFA-3 but was not blocked by antibody to class I1 MHC determinants. As CD2 is the natural ligand for LFA-3, increased expression of Tcell surface adhesion molecules LFA-1, ICAM-1 and particularly LFA-3 during an inflammatory response may rapidly recruit T cells that are activated through the CD2 pathway. These results allow a simplified model to explain how relatively few antigen/MHC-specificTcells can recruit large numbers of non-antigen-specificTcells in the generation of an inflammatory response and postulates a novel role of the CD2 molecule in T cell immune function. 1 Introduction Regulation of cellular immune response occur through T cell interactions with self Tcells [l-51. The ability of Tcells to signal other Tcells appears in many instances to require cell to cell contact. The mechanisms controlling these interactions between autologous T cells have remained largely unknown. Recent studies of T cell-induced signaling by other T cells have focused on either putative recognition of TcR [2,5] or T cell recognition of antigen presented in the context of MHC by autologous T cells [3]. Identifying other cell surface molecules that are involved in T-T cell interactions and signaling may be important in understand- ing cellular immunoregulation. T cells can be induced to proliferate by stimulation of the CD3/TcR complex [6, 71, which is mediated by antigen presented in the context of autologous MHC, allogeneic MHC or by autologous MHC alone in the autologous mixed lymphocyte reaction (AMLR). T cells can also be stimu- lated through the CD2 determinant, a molecule of M, 50 000 expressed on all human thymocytes and T cells [8]. The ligand for CD2 is a lymphocyte function-associated antigen 3 (LFA-3), a widely distributed cell surface glyco- protein. The role of CD2-LFA-3 interactions in immune regulation particularly outside of the thymus is not known. The high densities of LFA-3 and its ligand CD2 expression after Tcell activation suggest a potential role of these cell [I 82181 * Supported by National Institutes of Health Grant NS 24247 and NS 00981. Postdoctoral fellow of the National Multiple Sclerosis Society. Correspondence: Staley A. Brod, Center for Neurologic Diseases, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA surface molecules in T-T cell signaling during an immune response. In the present study, a model was devised to investigate T-T cell interactions by examining the ability of peripheral blood T cells to induce the proliferation of resting T cell clones.We show that Tcells activated by cross-linking of the CD3RcR complex were a potent stimulus signaling the proliferation of autologous Tcell clones. Moreover, T-Tcell signaling was mediated in part through CD2-LFA-3 and LFA-1-ICAM-1 ligands. These results suggest a novel role of the CD2 adhesion molecule in T cell function. 2 Materials and methods 2.1 Subjects and preparation of the cells Peripheral blood was obtained by venous puncture from normal subjects after informed consent. PBMC were isolated from heparinized venous blood by means of a Ficoll-Hypaque density gradient (Pharmacia Fine Chemi- cals, Piscataway, NJ), washed twice with HBSS (Gibco, Grand Island, NY), counted and resuspended in standard media consisting of 10% pooled human AB in RPMI (Whitaker Bioproducts, Walkersville, MD), 2% L-glutami- ne (Gibco), and 1% penicillidstreptomycin (Gibco) over- night at 37°C in 5% COz in a humidified atmosphere. The following day the cells were washed twice with experimen- tal medium at a final concentration of 20 x 106/ml. Purified Tcells were prepared in one of two ways: (a) PBMC were rosetted with SRBC (Microbiological Associates, Bioprod- ucts, Walkersville, MD) for 1 h and then separated on another Ficoll-Hypaque gradient. The rosetting T cell population was recovered after lysis of the SRBC by a Tris-buffered ammonium chloride solution. T cell popula- tions consisted of > 95% T cell as measured by anti-T11 mAb (Coulter Immunology, Hialeah, FL) or (b) PBMC in medium were incubated on a nylon wool column for 1 h at 37 "C and eluted by passing 30-40 ml medium through the column and collecting the effluent. 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990 0014-2980/90/1010-2259$3.50 + .25/0