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. Author manuscript; available in PMC: 2009 Aug 20.
Published in final edited form as: Mol Ther. 2008 Apr 22;16(7):1217–1226. doi: 10.1038/mt.2008.83

Figure 5. PC61/interleukin-2 (IL-2)-activated natural killer/lymphokine-activated killer cells facilitate intratumoral viral replication and spread.

Figure 5

(a) Virus replication and spread throughout intact, dissociate B16 tumors was measured as described in Materials and Methods. Splenocytes recovered from mice treated with PC61/control immunoglobulin G (IgG)/± asialo GM-1 and then with phosphate-buffered saline (PBS) or with recombinant human IL-2 were added to the wells containing explanted B16 tumors along with vesicular stomatitis virus (VSV) [4 × 108 plaque forming units (pfu)/well]. One set of tumors was treated with splenocytes from mice that had received PC61 + IL-2 with added EDTA (1 mmol/l). Virus titers from freeze–thaw lysates of the tumors 48 hours later are shown (pfu/mg tumor; three/group). (b) The experiment described in a was repeated, except that, after co-culture with splenocytes (from mice treated as shown over each panel) and VSV, tumors were dissociated in vitro and analyzed for the extent of viral spread/infection, using flow cytometry for green fluorescent protein expression, 24 hours after plating. Dissociated cultures were almost exclusively tumor cells as assessed by fluorescence-activated cell sorting for the melanoma marker gp100. (c) 5 × 105 B16 cells were plated in vitro and 24 hours later, 5 × 105 splenocytes from mice treated with PC61/IL-2/asialo-GM-1 as in a were added along with VSV at a density of 5 × 104 pfu/well. After a further 48 hours, the supernatants and surviving cells were recovered. Virus titers from freeze–thaw lysates are shown as pfu/ml (three/group). (d) C57Bl/6 mice received an intraperitoneal injection of PC61 or control immunoglobulin G. One group of these mice received asialo GM-1 antibody 24 hours before treatment with PC61. After 24 hours, the mice were injected intraperitoneally with PBS or with rhIL-2 (75,000 U/injection for 10 injections). After a further 24 hours, splenocytes from these mice were recovered, cDNA was prepared, and the expression of the metalloproteinase-2 (MMP-2) gene was analyzed using PCR as shown. Equal loading of RNA was demonstrated using amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control. (e) The experiment described in a was repeated with 107 splenocytes from mice treated with PBS, IL-2, PC61, or PC61/IL-2 added to wells containing explanted B16 tumors along with VSV at a density of 4 × 108 pfu/well. One set of tumors (n = 2) was not treated with splenocytes; instead they were incubated with VSV (4 × 108 pfu/well) along with recombinant MMP-2 (12.5 ng/ml). After a further 48 hours, the tumors were recovered from the wells and dissociated, and virus titers were determined from freeze–thaw lysates (shown as pfu/mg tumor; two/group).