Cultivation and characterisation of avian lymphocytes with natural killer cell activity

Conditioned medium (CM), as a presumed source of lymphokines including interleukin-2, was prepared from chicken spleen cell cultures stimulated with concanavalin A (con A). When CM was used to cultivate spleen cells from 6- to 8-week-old P-2 chickens, eight of nine spleens yielded cell lines which grew continuously for at least 50 days. Six of the cultures were tested for natural killer (NK) cell activity against LSCC-RP9, a lymphoblastoid cell known to be susceptible to NK cells, and against several Marek's disease lymphoblastoid cell lines (MDCC-CU2, -CU36 and -MSB-1). All six cultures lysed the RP9 cells in a chromium release assay with high levels of specific release (30 to 50%) at effector cell to target cell ratios of 5:1 or 10:1. CU2 and CU36, which are NK-cell resistant, were not lysed, while there was a low level of specific activity against MSB-1. The cells were characterised for surface and internal antigens with monoclonal antibodies and were negative for thymocyte antigen, IgM, a T cell antigen also present on granulocytes and red blood cells, and two antigens found in macrophages. Two of the six lines examined had a low number of cells expressing la antigen, while the other four were negative. An antigen present on circulating T cells and a macrophage (sub)population was present on all lines. The majority of the cells had the morphological appearance of mammalian large granular lymphocytes (LGL) with a rather small kidney-shaped nucleus. Granules and vacuoles were present in the cytoplasm. However, there was a variable percentage of lymphoblastoid (LB) cells also present. Cell lines established with CM from con A-stimulated spleen lymphocytes in other studies were also shown to have high levels of NK activity regardless of the relative proportions of cells with the morphological appearance of LGLs or LBs, or the relative frequency of expression of the two T cell markers or la antigen, all of which varied markedly.