Characterization of five cloned murine cell lines showing high cytolytic activity against YAC-1 cells.

Abstract

Abstract In an attempt to provide a selection procedure for the long-term growth and subsequent cloning of NK cells, mouse lymphocytes were treated with monoclonal anti-Thy-1.2 antibody and complement, incubated for 24 hr with poly l:C to boost residual NK cell activity, and then transferred into growth medium containing supernatant from Con A-stimulated mouse spleen cells. After cloning on appropriate feeder cells, two clones of C57BL/6 origin and three CBA clones were isolated and studied in detail. All five clones had several features in common with splenic NK cells. They had extremely high cytotoxic activity (100- to 1000-fold increases over normal spleen cells) against YAC-1 cells in a 4-hr 51Cr-release assay. Although they lacked the Mac-1 antigen, they bore a series of other surface antigens characteristic of NK cells, namely asialo-GM1, Qa-5, and NK-1.2. In addition, cytotoxicity by these cloned cell lines was blocked by anti-Ly-5 antibody, a reagent that inhibited lysis by splenic NK cells but not by cytotoxic T lymphocytes. Morphologically, the cells were large and contained prominent azurophilic granules. Although YAC-1 cells were the most sensitive target cells for the cloned lines, all the clones showed cytolytic reactivity somewhat different from that of normal or activated splenic NK cells. In particular, cytotoxicity by one clone, tested on a panel of 44 target cells, was generally restricted to murine lymphoid tumor target cells. With this cytotoxic clone there was, most strikingly, an almost complete lack of cytotoxicity against either solid-tumor-derived targets or against xenogeneic targets. Of major interest was the observation that two of the cloned cell lines bore high quantities of Lyt-2 antigen, whereas the other three lines had very small or undetectable (by flow cytofluorimetry) amounts of this antigen. In other respects (specificity, surface markers, cytochemistry), the Lyt-2-positive and Lyt-2-negative cell lines were extremely similar. The existence of two cloned cell lines expressing simultaneously characteristics of both NK cells and cytotoxic T lymphocytes is intriguing and suggests the possibility of a close relationship between these two lineages of cells.