The aims of this study were firstly to establish permanent T-cell lines from infiltrated NOD islets, by repeated stimulation of the antigen T-cell receptor with anti-V beta monoclonal antibodies (mAb) and, secondly, to characterize some of their cytotoxic and pathogenic properties. The use of anti-V beta antibodies was aimed at driving the expansion of the T cells in the absence of pancreatic antigen and, at the same time, at selecting lymphocytes expressing a given V beta gene product as an element of their TCR. Twelve lines were established as long-term cultures by regular stimulation with plastic-bound anti-V beta 6 or anti-V beta 8 mAb. The eight lines cultured with anti-V beta 6 mAb were phenotyped as early as one month after initiation and were all V beta 6+/V beta 8-. Three were CD8+ and five CD4+. Of the four lines established with anti-V beta 8 mAb, three were V beta 8+/V beta 6- and one (FD) was unexpectedly phenotyped as V beta 6+/V beta 8-. Clones derived from the FD line confirmed the expression of V beta 6. The cell-mediated cytolytic properties of the 12 lines were evaluated in two independent assays: an antibody-redirected assay to measure the lytic potential irrespective of antigen specificity and a direct cytolytic assay on YAC cells for assessing NK-like activity. The results indicate that practically all the lines (11 out of 12), irrespective of their CD4/CD8 phenotype or V beta expression, can exert cell-mediated cytotoxicity when their TCR/CD3 complex is linked to a target cell. On the other hand, anti-YAC activity is almost exclusively confined to CD8+ cell lines. Pathogenicity was evaluated in two CD4+ T cell lines, one which showed cytolytic activity in the redirected assay but not in the YAC assay (FD) and one which was totally devoid of cytotoxic activity (AH). The two lines were injected into newborn NOD mice with or without CD8+ polyclonal T cells. The results indicate that FD, the cytotoxic line, can induce severe lesions of insulitis when coinjected with polyclonal CD8+ T cells. In contrast, AH, the non-cytotoxic line, injected under the same conditions, induces no lesions. Altogether, the present data demonstrate the feasibility of establishing permanent T-cell lines on the basis of V beta expression.(ABSTRACT TRUNCATED AT 400 WORDS)