916 Purification of Azobenzenearsonate - Binding Murine T Cells

Even though it has been relatively easy to demonstrate the immunogenicity of azobenzenearsonate compounds in the guinea pig (1-5) and the rat (6), the mouse has distinct advantages in terms of ease of T-cell identification and the characterization of idiotypic markers associated with anti-azobenzenearsonate (ABA) t specificity. A major cross-reactive idiotype (CRI) found on 20-70% of the anti-ABA antibodies produced by individual strain A/J mice has been extensively studied by Nisonoff et al. (7). We have recently shown that monofunctional ABA-tyrosine (RAT) induces Tcell responses in A/J mice, determined by the appearance of antigen-binding cells and the development of delayed hypersensitivity (8). Anti-ABA antibody could not be detected in animals immunized with the monofunctional compound, but a major fraction of ABA-binding T cells expressed the CRI, as determined by the blocking activity of anti-CRI antibody. Furthermore, A/J mice make T-dependent antibody responses to bifunctional conjugates of RAT and of ABA-histidine (8). These conjugates evoke a primary anti-dinitrophenyl (DNP) response peaking at 7 days with -3 × 10 :~ IgM plaque-forming cells (PFC)/spleen. This IgM response occurs without an apparent shift to IgG production, and is accompanied by only weak immunological memory. Thus, whereas the response of A/J mice to monoand bifunctional ABA antigens serves to demonstrate the existence of T-cell activity to this determinant and the expression of the CRI on a fraction of the T cells, their magnitude is insufficient to permit the isolation of numbers of T cells required for characterization of the antigen receptor. Recently, several laboratories have used hapten-coupled autologous immunoglobulins to generate substantial hapten-specific helper (9) and suppressor (10) activity in mice. We now describe this approach to generate a sizable pool of functional ABAspecific T cells in A/J mice, many of which express the CRI, as well as the subsequent purification and characterization of the cells.