The T‐cell receptor as analyzed by functional T‐cell lines specific to a synthetic polypeptide antigen

Abstract

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 antigen poly(Tyr,Glu)‐poly(DLAla)‐‐poly(Lys) [(T,G)–A‐‐L] were established from C3H.SW (high‐responder) activated T‐cells, cloned, and characterized. These lines and their derived clones are also constitutive secretors of antigen‐specific T‐cell replacing helper factors. The secreted T‐cell helper factor was shown to possess MHC determinants as well as V‐region determinants, or more specifically, idiotypic determinants that are cross‐reactive with those expressed on (T,G)–A‐‐L‐specific antibodies of the same mouse strain. Using the fluorescence‐activated cell sorter (FACS II) and individual C57BL/6 anti‐idiotypic sera produced against (T,G)–A‐‐L‐specific antibodies of C3H.SW origin, we have demonstrated the expression of the cross‐reactive idiotypic markers on the monoclonal helper T‐cells. Attempts were made to purify the active fraction of the T‐cell factors secreted by the (T,G)–A‐‐L continuous helper lines. Gel analysis of the twice affinity‐purified eluate of a (T,G)–A‐‐L column revealed the existence of iodinated bands with molecular weight of 17,000 and 15,000, 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 of the latter fraction, as well as of an eluate of a (T,G)–A‐‐L–Sepharose column indicated that a high‐molecular‐weight (< 67,000) and a low‐molecular‐weight (15,000–17,000) fraction contained the biological activity of the factor. Similar results were obtained following chromatography of the factor on Sephadex G‐100 columns. The two fractions were shown to be synthesized by the T‐cell lines, as indicated by internal labeling experiments using 35S‐methionine. Thus, it is suggested that a fraction of an apparent molecular weight of 15,000–17,000 preserves both the antigen specificity and the helper activity of the factor produced by the (T,G)–A‐‐L‐specific T‐cell lines.