Identification of a monoclonal antibody specific for a murine T3 polypeptide.

Abstract

A monoclonal antibody (145-2C11) specific for the murine T3 complex was derived by immunizing Armenian hamsters with a murine cytolytic T-cell clone. The antibody is specific for a 25-kDa protein component (T3-e) of the antigen-specific T-cell receptor. It reacts with all mature T cells and can both activate and inhibit T-cell function. These results identify T3-E as a cell surface protein involved in the transduction of activation signals.Activation of mature T cells is a process that requires both receptor occupancy by foreign antigen in the context of major histocompatibility complex (MHC) proteins and transduction of a transmembrane signal. The specific antigen recognition has been shown to be dependent on a disulfide-linked heterodimer containing two integral membrane glycoprotein chains, a (45-50 kDa) and / (40-44 kDa) (1). The a and /3 chains are encoded by families of genes, giving rise to a polymorphic set of cell membrane receptors clonally expressed on T lymphocytes. Dembic et al. (2) and Saito et al. (34) have demonstrated that transfection and expression of the genes encoding the aB heterodimer results in the acquisition of MHC-restricted antigen recognition, establishing the direct relationship of antigen specificity to aB expression. The second step of T-cell activation (transmembrane signaling) is presently thought to be mediated by an invariant complex of proteins (T3) that by immunoprecipitation has been shown to be noncovalently associated with the af3 heterodimer in the T-cell receptor (TCR) complex (3, 4). The human T3 complex consists of at least three polypeptides, two glycosylated proteins of 25 kDa and 20 kDa (T3-8 and T3-y, respectively) and an endoglycosidase F-insensitive protein of20 kDa (T3-E) (5). The murine T3 structure has been identified as a similar complex of low molecular mass proteins, including T3-8, T3-e, T3-y, and a 16-kDa polypeptide present as a 32-kDa homodimer (T3-t) (6)(7)(8). The role of the T3 proteins in transmembrane signaling is substantiated by the finding that several of the T3 components have a long intracellular polypeptide portion (9-11) that is phosphorylated upon af3 heterodimer occupancy (12). In addition, monoclonal antibodies (mAbs) binding to the human T3-8 molecule can result in a rise in cytoplasmic free calcium (13), metabolism of phosphatidylinositol phosphates (14), T-cell proliferation (15), lymphokine production (16), and cytolysis (17,18). Recently, mAbs to T3 have allowed the identification of a subpopulation of human T cells that express T3 but not the a,8 heterodimer (19,20). In these cells, the T3 components are associated on the cell surface with polypeptides other than a and 83, including one that appears to be the product of the y gene (19). Anti-T3 mAbs have been shown to activate this T-cell subset, indicating that T3 might also be involved in the signaling of activation signals initiated by the recently described TCRY receptor complex (20).Unlike the study of human systems, the study of murine T-cell activation and ontogeny...

Concanavalin A-mediated T cell proliferation is regulated by herpes virus entry mediator costimulatory molecule

“…The cells were co-cultured in RPMI1640 containing 10% FBS supplemented with gentamycin (10 µg/ml) and stimulated by ConA (4 µg/ml, Sigma-Aldrich), PMA (2 ng/ml, Sigma-Aldrich) plus ionomycin (20 ng/ml, Sigma-Aldrich) or anti-CD3 mAb (1 µg/ml). Anti-CD3 mAb was obtained from hybridoma 145-2C11 culture (Leo et al 1987).…”

Differences in Expression, Affinity, and Function of Soluble (s)IL-4Rα and sIL-13Rα2 Suggest Opposite Effects on Allergic Responses

“…The following antisera, Abs, and mAbs were prepared as described: goat antisera to mouse IgD (GaMD) and keyhole limpet hemocyanin (GaKLH) (50); EM-95 (rat IgG2a anti-mouse IgE (51); and 145-2C11 (hamster IgG antimouse CD3 ) (52). Affinity-purified goat anti-mouse IL-13R␣2 Ab was produced by immunizing a goat with mouse IL-13R␣2-human IgGFc fusion protein (a gift from D. Donaldson, then at Wyeth Research) in CFA (Difco), boosting with the same Ag in IFA (Difco), adsorbing with human IgG coupled to CNBr-activated Sepharose (Pharmacia), and affinity purifying by adsorption to and 3.5 M MgCl 2 elution from mouse IL-13R␣2-mouse IgGFc fusion protein (D. Donaldson, then at Wyeth Research) coupled to cyanogen bromideactivated Sepharose.…”

Administration of anti-CD3 monoclonal antibody during experimental Chagas’ disease induces CD8+ cell-dependent lethal shock

“…The hamster MoAb 145-2C11 directed against mouse CD3 was produced as supernatant of 145-2C11 hybridoma [16].…”

“…The hamster MoAb 145-2C11 that specifically recognizes the chain of the mouse CD3 complex [16] induces an acute polyclonal T cell activation resulting in massive secretion of several cytokines. In vivo, injection of the 145-2C11 MoAb into normal mice induces a rapid release of IFN-and tumour necrosis factoralpha (TNF-) in the bloodstream and a transient non-lethal shock syndrome [17][18][19].…”