The murine B7 (mB7) protein is a potent costimulatory molecule for the T-cell receptor (TCR)-mediated activation of murine CD4+ T cells. We have previously shown that stable mB7-transfected Chinese hamster ovary (CHO) cells but not vector-transfected controls synergize with either anti-CD3 monoclonal antibody-induced or concanavalin A-induced T-cell activation, resulting ultimately in lymphokine production and proliferation. We now have generated a hamster anti-mB7 monoclonal antibody. This reagent recognizes a protein with an apparent molecular mass of 50-60 kDa. The mB7 antigen is expressed on activated B cells and on peritoneal exudate cells (PECs). Antibody blocking experiments demonstrate that mB7 is the major costimulatory molecule expressed by PECs for the activation of murine CD4+ T cells. This suggests an important role for mB7 during immune-cell interactions. We have also surveyed a panel of murine cell lines capable of providing costimulatory activity. Our results indicate that mB7 is the major costimulatory molecule on some but not all cell lines and that there may be additional molecules besides mB7 that can costimulate the activation of murine CD4+ T cells.Stimulation of highly purified murine CD4+ T lymphocytes through the T-cell receptor (TCR) with peptide bound to major histocompatibility complex (MHC) proteins, anti-TCR-CD3 complex monoclonal antibodies (mAbs), or lectins 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 a phorbol ester. This costimulatory signal has been postulated to play a central role in the regulation of T-cell activation and in thymic development (1, 2). Its characterization is therefore of great interest.In the case of interleukin 4 (IL-4)-secreting, Th-2 type CD4+ T cells, the costimulatory signal appears to be IL-1 (3, 4). In contrast, the nature of the costimulatory signal for IL-2-secreting, Th-1 type CD4+ T cells has not yet been definitely identified. We and others have recently shown that the B7 antigen (5) can provide a potent costimulatory signal for IL-2-secreting CD4+ T cells. Transfected cell lines that express human (6, 7) or murine B7 (mB7) (8) can synergize with anti-CD3-or lectin-driven T-cell activation. Furthermore, in the human, a recombinant B7 immunoglobulin fusion protein (7) or mAbs to CD28 (9, 10), a 44-kDa homodimeric T-cell glycoprotein that is a receptor for B7 (11), can augment the proliferation or lymphokine secretion of T cells stimulated with suboptimal doses of anti-CD3 mAbs.In the present report, we describe a hamster-anti-mB7 mAb. With this reagent we have characterized the expression and function of mB7 by normal and transformed cells. Our results indicate that mB7 is the major costimulatory molecule expressed by peritoneal exudate cells (PECs) and by certain cell lines. Our results also raise the possibility that there may be other molecules b...
The murine B7 (mB7) protein is a potent co-stimulatory molecule for the activation of murine CD4+ T cells. We have previously shown that stable mB7-transfected Chinese hamster ovary (CHO) cells synergize with either anti-CD3 monoclonal antibodies (mAb) or concanavalin A to stimulate T cell activation. In addition, mB7 can synergize with phorbol 12-myristate 13-acetate (PMA) to induce T cell activation in an alternative pathway. In the present report we describe the effects of three immunosuppressive drugs, cyclosporin A (CsA), FK 506, and mycalamide A on mB7-mediated T cell activation. The immunophilin ligands CsA and FK 506 block activation of murine CD4+ T cells by the combination of anti-CD3 mAb and CHO-mB7 cells but do not affect activation by CHO-mB7 cells and PMA. These results support the relevance of pharmacological studies of immunosuppressive compounds in the murine model prior to their application in man. In contrast to the effects of CsA and FK 506, mycalamide A blocks activation of murine CD4+ T cells by anti-CD3 mAb and mB7 as well as activation by mB7 and PMA. On a molar basis, mycalamide A appears to be at least 10-fold more potent than FK 506 which is 100-fold more potent than CsA. Because of its effects on multiple activation pathways and because of its potency, mycalamide A could have considerable therapeutic potential.
Activation of T lymphocytes requires the recognition of peptide-ajor histocompatibility complex complexes and costimulatory signals provided by antigenpresenting ceDls (APCs). The best-characterized costimulatoy molecule to date is the B7 antigen, a member of the immunoglobulin family that binds two receptors, CD28 and CTLA-4, expressed on the T-cell surface. Using the anti-mouse B7 (mB7) monoclonal antibody (mAb) Activation of CD4+ T cells occurs upon recognition, by the T-cell receptor (TCR), of peptide-major histocompatibility complex complexes (MHC), which are expressed on the surface of an antigen-presenting cell (APC). TCR triggering, however, is not sufficient to impart complete T-cell activation. The induction of lymphokine secretion and cell proliferation requires a second, costimulatory signal, which is also provided by an APC (1, 2). The CD28 molecule is the best-characterized costimulatory receptor. Binding of monoclonal antibodies (mAbs) to CD28 (3), a 44-kDa homodimeric T-cell glycoprotein that is a receptor for B7 (4), can augment the proliferation or lymphokine secretion of T cells stimulated with anti-CD3 mAb or with antigen (5-8) and overcome T-cell anergy in T-cell clones (8). CTLA-4 is an additional TCR with affinity for B7, but the function of CTLA-4 in T-cell activation remains to be determined (9-12).We and others have shown that B7 (13-20) can provide a costimulatory signal for CD4+ T cells (20)(21)(22)(23)(24)(25), but it is currently unknown whether there exist additional ligands, besides B7, with affinity for the CD28 and CTLA-4 receptors. In the present report, we have addressed this issue in the murine system, using the anti-mouse (mB7) mAb and CTLA4Ig, a soluble fusion protein between human CTLA-4 and an Igy constant region, which inhibits T-cell activation in vitro (10,26) and in vivo (27)(28)(29). This reagent binds both human B7 and mB7 and has the potential to detect additional ligands besides B7 with affinity for the CD28 and CTLA-4 receptors. In the present report, we provide eviThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.dence that there exists indeed at least one additional ligand, distinct from B7, with affinity for the CTLA-4 receptor. MATERIALS AND METHODSAntigens and Fusion Proteins. Rabbit anti-mouse immunoglobulin (RAMG) was obtained from rabbits immunized with normal mouse immunoglobulin and was purified on normal mouse immunoglobulin-conjugated Sepharose columns.F(ab')2 fragments of RAMG were prepared by pepsin digestion using a commercially available kit (Pierce). The fusion protein CTLA4Ig, composed of the extracellular domains of human CTLA-4 and the hinge-CH2-CH3 domains derived from a human IgGl gene, was derived by polymerase chain reaction using a strategy similar to that previously reported (10). The genetic fusion was expressed in CHO cells, and the fusion protein was purified from cul...
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