Fig. 3

High concentrations of smTRAIL inhibited the number and function of NK cells in BALB/c-Nude mice. A Experimental layout. BALB/c-Nude mice were subcutaneously (s.c.) inoculated with 5 × 10⁴ 4T1 cells or 2 × 10⁵ CT26 cells in the right flanks, and smTRAIL was injected intraperitoneally (i.p.) for eight consecutive days (n = 6). B Tumor growth curves. C At 3 d after the last treatment, CT26 tumors were collected and analyzed by flow cytometry to calculate the percentages of intratumoral NK cells (CD3⁻CD49b⁺), activated NK cells (CD3⁻CD49b⁺CD107a⁺) D TAMs (CD45⁺F4/80⁺CD11b⁺), M1-TAMs (CD45⁺F4/80⁺CD11b⁺CD86⁺), and M2-TAMs (CD45⁺F4/80⁺CD11b⁺CD206⁺) (n = 5). E Experimental layout and tumor growth curves in the presence and absence of the NK cell depletion antibody anti-asialo GM1 (n = 6). Each mouse was intravenously (i.v.) treated with 20 μL of anti-asialo GM1 every four days. F Schematic diagram of the experimental strategy used for the activation of NK cells in vitro. G NK cells (5 × 10⁴ per well) were treated with the indicated concentrations of smTRAIL for 16 h, and cell viability was determined by MTT assay. H Following incubation of the isolated NK cells with different concentrations of protein for 16 h, the functional marker of NK cells was assayed by flow cytometry. I NK cells incubated with the protein were cocultured with YAC-1 cells for 4 h in proportion, and NK cell cytotoxicity was assayed using a lactate dehydrogenase cytotoxicity assay kit. A two-way ANOVA test was used to compare tumor growth responses. One-way ANOVA was performed to calculate the significant differences between groups, followed by LSD analysis. *P < 0.05; ***P < 0.001; and ****P < 0.0001