Use of bispecific heteroconjugated antibodies (anti-T cell antigen receptor x anti-MHC class II) to study activation of T cells with a full length or truncated antigen receptor zeta-chain

Abstract

Ligand-induced activation of T cells involves recognition of monovalent peptide Ag complexed with a cell surface MHC-encoded molecule. In contrast, antibody-induced activation of T cells typically requires external cross-linking of the TCR. To examine the mechanisms that underlie the ability of these different stimuli to signal, we have created bispecific chimeric antibody molecules (BA) that mimic Ag in several important aspects. Anti-TCR-alpha, -beta, or anti-CD3-epsilon Fab fragments were covalently coupled to an anti-MHC class II Fab fragment. These BA elicited IL-2 production or proliferation from Ag-specific T cell hybridoma cells or splenic T cells, respectively, in the presence, but not the absence, of accessory cells expressing the appropriate MHC class II molecule. This response was prevented by soluble blocking antibodies against the TCR or MHC class II. When "presented" by MHC class II-bearing accessory cells, anti-TCR x anti-MHC class II BA, like cell surface Ag, elicited IL-2 production from T cell transfectants expressing full length TCR zeta-chain but not from otherwise identical cells expressing truncated zeta; when immobilized on a plastic surface these BA were potent stimulators that induced equal amounts of IL-2 from the same cells. Purified Ag/MHC complexes immobilized on plastic were able to induce IL-2 production from T cells expressing the full length, but not the truncated, form of zeta. We hypothesize that TCR-mediated T cell activation requires stable aggregation of the TCR. In this model, activation by mobile cell surface Ag/MHC or BA occurs in two steps, occupancy-induced TCR clustering followed by stable aggregation facilitated by the presence of a full length zeta-chain. Immobilized high affinity anti-TCR antibodies, but not low affinity Ag/MHC complexes, directly promote stable receptor aggregates, and thus would not require a full length zeta-chain.