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

Abstract

In an attempt to provide a selection procedure for the long-term growth and subsequent cloning of NK [natural killer] cells, mouse lymphocytes were treated with monoclonal anti-Thy-1.2 antibody and complement, incubated for 24 h with poly I:C to boost residual NK cell activity, and then transferred into growth medium containing supernatant from Con A[concanavalin A]-stimulated mouse spleen cells. After cloning on appropriate feeder cells, 2 clones of C57BL/6 origin and 3 CBA clones were isolated and studied in detail. All 5 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-h 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. 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 clones showed cytolytic reactivity somewhat different from that of normal or activated splenic NK cells. In particular, cytotoxicity by 1 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 solid-tumor-derived targets or against xenogeneic targets. Two of the cloned cell lines bore high quantities of Lyt-2 antigen, whereas the other 3 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 2 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 2 lineages of cells.