Fine specificity of cloned insulin-specific T cell hybridomas: evidence supporting a role for tertiary conformation.

Two groups of cloned insulin-specific T cell hybridomas were derived from the fusion of (BALB/c X A/J)F1 T cells with the BW5147 tumor line. Group I responded only to the immunogen pork insulin and failed to respond to rat insulin, which differs only at amino acids A4 and B3. The second group of T cell hybridomas exhibited a broader pattern of reactivity to heterologous insulins because they could be stimulated with the B chain-identical pork, beef, sheep, and human insulins, as well as by isolated oxidized B chain. These hybridomas, however, displayed a much greater reactivity to pork insulin than to beef insulin or to the oxidized B chain. Comparison of the reactivity profiles of these two groups of hybridomas with the three-dimensional structure of the insulin molecule strongly suggested that group I cells were recognizing an immunogenic moiety composed of residues A4 and/or B3 in association with A chain loop determinants and that group II hybrids recognized a moiety composed of the amino acid sequence of the B chain and the A chain loop. Both groups of hybrids were restricted to the high-responder I-Ad allele but could be distinguished by their pattern of alloreactivity. We were unable to show any effect of a panel of monoclonal anti-insulin antibodies on the induction of interleukin 2 secretion by these hybridomas or to demonstrate the presence of idiotypic determinants on these T hybrids by using a panel of anti-idiotypic reagents raised against the monoclonal anti-insulin antibodies.