Figure 4.

Parental and MRD cells are differentially recognized by NK cells. B16 or MRD B16 cells (10⁵/well) were cocultured in triplicate with purified NK cells from either wild-type C57BL/6 (IL6⁺) or IL6 KO mice at an effector:target ratio of 20:1. Seventy-two hours later, supernatants were assayed for IFNγ (A) or IL6 (B) by ELISA. Mean and SD of triplicates are shown, *, P < 0.05; **, P < 0.001 (ANOVA). Representative of three separate experiments. C, Splenocytes and lymph node cells from wild-type C57BL/6 mice were plated with B16 or B16 MRD #2 cells and grown for 72 hours in TNFα at an effector:target ratio of 50:1. Seventy-two hours later, cells were harvested and analyzed for expression of NK1.1 and IL6. D, Three small primary B16ova tumors (<0.3 cm diameter, Pri#1-3) from PBS-treated C57BL/6 mice or three small recurrent B16ova tumors from mice were excised, dissociated, and plated in 24-well plates overnight and then supernatants were assayed for IL6 and TNFα by ELISA. Mean and SD of triplicates are shown; ***, P < 0.0001, for IL6 between primary and recurrent tumors (T-test). E and F, C57BL/6 mice (n = 5 mice/group) were challenged subcutaneously with parental B16ova cells (E) or B16ova MRD cells (F; expanded from a regressed B16ova tumor site for 72 hours in TNFα) at doses of 10³ or 10⁴ cells per injection. Included in E and F is a group of mice depleted of NK cells using anti-asialo GM-1 and challenged with 10³ B16 or B16 MRD cells. Representative of two separate experiments. Survival analysis was conducted using log-rank tests. The threshold for significance was determined by using the Bonferroni correction for multiple comparisons.