The maximal T-cell response to its antigen requires presentation of the antigen by a major histocompatibility complex class II molecule as well as the delivery of one or more costimulatory signals provided by the antigenpresenting cell (APC). Although a number of candidate molecules have been identified that are capable of delivering a costimulatory signal, increasing evidence suggests that one such critical pathway involves the interaction of the T-cell surface antigen CD28 with its ligand B7, expressed on APCs.In view of the number of potential costimulatory molecules that might be expressed on the cell surface of APCs, artificial APCs were constructed by stable transfection of NIM 3T3 cells with HLA-DR7, B7, or both. Here, we show that in a human antigen-specific model system, when tetanus toxoid peptide antigen is presented by cells cotransfected with HLA-DR7 and B7, optimal T-cell proliferation and interleukin 2 production result. In contrast, antigen presentation, in the absence of B7 costimulation, results in T-cell clonal anergy. These results demonstrate that it is possible to induce antigen-specific clonal tolerance in human T cells that have been previously sensitized to antigen. The artificial antigen-presenting system provides a useful model for the investigation of the biochemical events involved in the generation of tolerance and for the study of signals necessary to overcome tolerance.The maximal T-cell response to its antigen requires presentation of the antigen by a major histocompatibility complex (MHC) class II molecule and the delivery of one or more costimulatory signals provided by heterotypic adhesion between receptor ligand pairs on the T cell and the antigenpresenting cell (APC) (1-3). Signaling through the T-cell receptor (TCR) complex alone can result in a downregulatory signal for human T-cell growth (4, 5). In vitro systems in the mouse have demonstrated that in the absence of a costimulatory signal, occupancy of the TCR by peptide fragments in the context of MHC class II is capable of inducing a long-lasting antigen-specific unresponsiveness, termed anergy (1, 3, 6). Increasing in vitro evidence in murine and human systems suggests that one such critical costimulatory pathway involves the interaction of the T-cell surface antigen CD28 with its ligand B7 on the APC (7-10). CD28 is constitutively expressed on 95% of resting CD4+ and 50% of CD8+ human T lymphocytes (11,12). After T-cell activation, CTLA4, a second higher-affinity ligand for B7 (13), is expressed and CD28 expression increases. B7 expression is limited to cells capable of presenting antigen, including activated B cells (14), activated monocytes (15), and dendritic cells (16). After T-cell activation, ligation of CD28 by anti-CD28 monoclonal antibody (mAb) or by B7 induces maximal proliferation and interleukin 2 (IL-2) secretion (7,8,17,18). Two recent studies in murine systems suggest that the inhibition of the CD28-B7The publication costs of this article were defrayed in part by page charge payment. This articl...

Occupancy of the T-cell receptor complex does not appear to be a sufficient stimulus to induce a T-cellmediated immune response. Increasing evidence suggests that cognate cell-cell interaction between an activated T cell and an antigen-presenting cell may provide such a stimulus. A candidate T-cell surface molecule for this costimulatory signal is the T-cell-restricted CD28 antigen. Following crosslinking with anti-CD28 mAb, suboptimally stimulated CD28' T cells show increased proliferation and markedly increased secretion of a subset of lymphokines. Recently, the B-cell surface activation antigen B7 was shown to be a natural ligand for the CD28 molecule, and both B7 and CD28 are members of the immunoglobulin superfamily. Here we report that B7-transfected CHO cells can induce suboptimally activated CD28+ T cells to proliferate and secrete high levels of interleukin 2. The response is identical whether T cells are submitogenically stimulated with either phorbol myristate acetate or anti-CD3 to activate the T cells. This response is specific and can be totally abrogated with anti-B7 monoclonal antibody. As has previously been observed for anti-CD28 monoclonal antibody, B7 ligation induced secretion of interleukin 2 but not interleukin 4. We have previously demonstrated that B7 expression is restricted to activated B lymphocytes and interferon V-activated monocytes. Since these two cellular populations are involved in antigen presentation as well as cognate interaction with T lymphocytes, B7 is likely to represent a central costimulatory signal that is capable of amplifying an immune response.

SummaryFollowing occupancy of the T cell receptor by antigen, T cell proliferation and lymphokine production are determined by a second costimulatory signal delivered by a ligand expressed on antigen presenting cells. The human B cell activation antigen B7, which is expressed on antigen presenting cells including activated B cells and 'Y interferon treated monorytes, has been shown to deliver such a costimulatory signal upon attachment to its ligand on T cells, CD28. We have cloned and sequenced the murine homologue of the human B7 gene . The predicted murine protein has 44% amino acid identity with human B7 . The greatest similarity is in the IgV and Ig-C like domains . Murine B7 mRNA was detected in murine hematopoietic cells of B cell but not T cell origin . Cells transfected with murine B7 provided a costimulatory signal to human CD28+ T lymphocytes. These results demonstrate the costimulatory activity of murine B7 and provide evidence that the ligand attachment site is conserved between the two species .Although occupancy of the TCR complex by antigen in 13, association with the MHC is necessary for the initiation of T cell activation, several lines of evidence suggest that a second costimulatory signal is essential for the induction of proliferation and lymphokine secretion (1-4) . In murine and human systems, this costimulatory signal is delivered by APC and requires cell to cell contact (2, 4) . Cells which can deliver this costimulatory signal include activated, but not resting B lymphocytes (5), INF-y activated monorytes, and dendritic cells (2, 6) .Several recent studies in human systems have provided compelling evidence that the B cell activation antigen B7 can provide one such costimulatory signal (7-9) . B7, a member of the Ig supergene family, has been shown to be a ligand for another member of this family, the T cell surface protein CD28, (10-13) . CD28 is constitutively expressed on 95% of human CD4+ T cells, 50% of CD8+ T cells, and on thymocytes which coexpress CD4 and CD8 (14-16) . Following suboptimal activation of T cells with anti-CD3 mAb (16), anti-CD2 mAb, or phorbol ester (17), crosslinking of CD28 by anti-CD28 mAb results in enhanced T cell proliferation and greatly augments synthesis of multiple lymphokines (18). B7 is likely to be an important regulator of T cell proliferation and lymphokine production as evidenced by its pattern of expression and functional activity. B7 is not expressed on resting B cells (19) but appears following crosslinking of surface Ig (10, 19) or class II MHC (9) . Moreover, B7 is not expressed on unstimulated monorytes and is specifically induced by INF-y but not other stimuli which activate monocytes (20). Human B7 (hB7) 1 transfected cells or recombinant B7-Ig fusion protein augment proliferation and induce IL2, but not IIT4, synthesis in T cells which have been treated with phorbol ester or anti-CD3 mAb (7-9) .In murine systems, a homologue for CD28 has recently been cloned (21); however, a conserved functional activity has not yet been demonstrated . In ...

We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex.Stimulation of highly purified CD4+ T lymphocytes with peptide-major histocompatibility complex (MHC) complexes, monoclonal antibodies (mAbs) against T-cell receptor (TCR)/CD3 complexes, lectins, or mAbs against the glycosyl-phosphatidylinositol (GPI)-anchored proteins Thy-1 and Ly-6A.2 does not lead to cell proliferation or lymphokine secretion. The above activation pathways require a second, costimulatory, signal for lymphokine gene expression and proliferation that is provided by an antigen-presenting cell (APC) or by pharmacological reagents such as phorbol 12-myristate 13-acetate (PMA) (1, 2).The nature of the costimulatory signal appears to be dependent upon the subset of CD4+ T cells that is activated. On the basis of their capacity to secrete specific lymphokines, two types of murine CD4+ T-helper cells were defined originally. Th-1 clones synthesize interleukin (IL)-2, interferon y, and lymphotoxin, whereas Th-2 clones secrete IL-4, IL-5, and IL-6 (3). In the case of Th-2 type CD4+ T cells, this costimulatory signal appears to be IL-1 (4). In contrast, the nature of the costimulatory signal for Th-1-type CD4+ T cells is unknown. Recent data in the human system suggest that B7, a 45-to 60-kDa cell surface glycoprotein present on activated B cells and on interferon-y-stimulated monocytes/macrophages (5, 6), may be costimulatory for Th-1 CD4+ T cells. Transfected cell lines that express human B7 or recombinant B7-immunoglobulin fusion protein can synergize with anti-CD3 or phytohemagglutinin-driven T-cell activation (7-9). Furthermore, binding of mAbs to CD28, a 44-kDa homodimeric T-cell glycoprotein that is a receptor for B7 (10), can augment the proliferation or lymphokine secretion of T cells stimulated with suboptimal doses of anti...