An insight into the T-cell recognition systemusing antigen-specific functional T-cell lines

Abstract

Summary For a better understanding of the molecular nature of the antigen-specific T-cell recognition system, continuous T-cell lines specific to the synthetic polypeptide (T,G)-A-L, have been established from murine and human activated T cells. These lines are constitutive secretors of antigen-specific T-cell replacing helper factors. Lines prepared from (T,G)-A-L activated C3H.SW (high-responder) T cells were cloned and characterized. The cells and their secreted products were identical in their fine antigenic specificity, as tested using synthetic polypeptides closely related to (T,G)-A-L. The secreted T-cell helper factor was shown to possess MHC determinants as well as V-region determinants or, more specifically, idiotypic determinants. Using the fluorescence-activated cell sorter (FACSII), we demonstrated the expression of cross-reactive idiotypic markers on the monoclonal helper T cells. Gel analysis of the twice affinity-purified eluate of a (T,G)-A-Lcolumn revealed the existence of iodinated bands with a molecular weight of 15 kd and 17 kd, in addition to a diffuse band of high molecular weight. The specific helper activity of the factors was associated with a 65–75% ammonium sulfate precipitate. Gel electrophoresis experiments indicated that both a high (less than 67 kd) and a low (15–17 kd) molecular weight fraction contained the biological activity of the factor. The two fractions were also shown to be synthesized by the T-cell lines, as indicated by internal labelling experiments using 35 S-methionine. A (T,G)-A-L-specific long-term T-cell line of human origin which constitutively secreted a helper factor, was established from antigen-activated T cells of a donor previously determined to be a responder to (T,G)-A-L. In addition to the antigenic specificity of the factor, it was found to possess V H determinants in addition to the relevant HLA-DR determinants. The latter finding was confirmed by the demonstration that the factor reacted with monoclonal anti-Ia antibodies. Thus, the molecular characterization of the active products of the antigen-specific T-cell lines of murine and human origin will hopefully lead to elucidation of the T-cell receptor.