Figure 5.

GM-CSF is required for activation of LN/splenocytes for tumor cell killing in vitro by NAb–reovirus complexes and is dependent on NK cells and CD11b⁺ cells. (a) B16ova cells expressing GFP, which are resistant to direct reovirus killing, were cultured for 24 hours at a 1:10 ratio with LN/splenocyte cells ± 10 ng/ml GM-CSF. After 24 hours, reovirus, reovirus/anti-reovirus-NAb (100 µl immune serum) complexes or reovirus/anti-VSV-NAb complexes were added at MOI 1 and, after a further 6 hours, cells and supernatants were harvested. (a) (i)–(vii): The number of viable GFP-expressing cells was determined by FACS as a measure of immune-mediated cytotoxicity against the B16ova cells, and (d) TNF-α in the supernatants was determined by ELISA. (b) LN/splenocyte effector cells were first depleted of CD4⁺ T cells, CD8⁺ T cells, NK cells, or CD11b⁺ cells, as indicated, before culture as described in a. (c) Intact LN/splenocyte cells, or isolated NK cells, CD11b⁺, or combined NK + CD11b⁺ cells were cocultured with target tumor cells as in a, and the number of viable GFP-expressing cells was determined by FACS as a measure of cytotoxicity against the B16ova cells. FACS, fluorescence-activated cell sorting.