Biochemical characterization of antigen-specific glycosylation-inhibiting factor from antigen-specific suppressor T cells: I. Identification of a 55-kilodalton glycosylation-inhibiting factor peptide with TCR α-chain determinant

Stimulation of OVA-specific suppressor T cell (Ts) hybridoma and bee venom phospholipase A₂ (PLA₂)-specific Ts hybridoma with Ag-pulsed APC or by cross-linking of CD3 resulted in the formation of Ag-specific glycosylation-inhibiting factor (GIF). Affinity-purified Ag-specific GIF preparations, obtained by using Ag-coupled Sepharose or anti-TCR α-chain-coupled Affi-Gel, contained a 55-kDa peptide, which bound both polyclonal anti-GIF Abs and anti-TCR-α mAb in immunoblotting. The same hybridomas constitutively secrete 13-kDa bioactive GIF peptide that has no affinity for homologous Ag, but neither the Ag-specific GIF activity nor 55-kDa GIF peptide was detectable in culture supernatants of unstimulated cells. Northern blot analysis of mRNA from the anti-CD3-stimulaled hybridoma with ³²P-labeled GIF cDNA revealed only 0.6 kb mRNA, which encodes the 13-kDa nonspecific GIF. No mRNA of the 55-kDa GIF was detectable. A representative OVA-specific Th hybridoma, DO 11.10 cells contain the 0.6 kb GIF mRNA and constitutively secrete inactive GIF peptide. However, the Th hybridoma failed to secrete the 55-kDa peptide or any peptide with the TCR-α determinant upon stimulation with anti-CD3. It appears that the formation of the 55-kDa peptide with the TCR-α determinant is unique for a subset of T cells including Ts cells that form bioactive GIF.