Figure 2.

CD4⁺/CD8⁺-dependent antitumor response and increased production of pro-inflammatory cytokines are induced by active/inactive arazyme treatment. C57Bl/6 mice (n = 6) were challenged endovenously with 5 × 10⁵ B16F10-Nex2 cells and treated intraperitoneally with 3 mg/kg of active/inactive arazyme or PBS (Control) on alternate days for 2 weeks. Spleens were collected 24 h after the last dose, splenocytes were stimulated ex vivo with B16F10-Nex2 tumor lysate, and intracellular cytokine staining (ICCS) was then performed for CD4⁺ and CD8⁺ positive T cells. (A) The percentage of splenic IFNγ- or IL10-producing CD4⁺ or CD8⁺ T cells was determined by FACS, and is represented by the numbers in the upper right quadrants. Histograms represent six pooled animals. (B) C57BL/6 mice were depleted of CD4⁺ or CD8⁺ T lymphocytes by two doses (500 μg each) of specific monoclonal antibodies 72 and 48 h before challenge with tumor cells and treatment with active/inactive arazyme. Animals were sacrificed 24 h after the last dose, and the number of lung nodules is represented individually. (C) IFNγ, (D) TNF-α, and (E) IL-10 were quantified in lung homogenates, (F) IL-12 was quantified in culture supernatant of splenocytes stimulated with tumor lysate, and (G) IFNγ was quantified in 1:2 diluted serum from C57Bl/6 mice challenged and treated as described above. Cytokines were quantified in individual animals, and bars represent the average ± SD of at least three animals. *p < 0.05; ***p < 0.001; and ****p < 0.0001, analyzed by one-way ANOVA with Tukey's multiple comparisons (in B) and one-way ANOVA with Dunnett's multiple comparisons (in C–G).