ESTABLISHMENT OF T-CELL LINES FROM INFILTRATED NOD ISLETS BY STIMULATION OF SPECIFIC T-CELL RECEPTOR V-BETA SEGMENTS

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-Vβ monoclonal antibodies (mAb) and, secondly, to characterize some of their cytotoxic and pathogenic properties. The use of anti-Vβ 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 Vβ gene product as an element of their TCR. Twelve lines were established as long-term cultures by regular stimulation with plastic-bound anti-Vβ6 or anti-Vβ8 mAb. The eight lines cultured with anti-Vβ6 mAb were phenotyped as early as one month after initiation and were all Vβ6+/Vβ8-. Three were CD8+ and five CD4+. Of the four lines established with anti-Vβ8 mAb, three were Vβ8+/Vβ6- and one (FD) was unexpectedly phenotyped as Vβ6+/Vβ8-. Clones derived from the FD line confirmed the expression of Vβ6. 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 Vβ 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 Vβ expression. In addition, they indicate that some of the lines established under such conditions may have relevancy with regard to the autoimmune pathological process as suggested by their in-vivo cytotoxic properties and their in-vivo capacity to produce intra-pancreatic lesions. © 1993 Academic Press, Inc.