Fig. 3: Tumor cells lysed by M1 mature iDCs and enable the iDCs to prime de novo T-cell responses ex vivo.

A Diagrammatic sketch of the coculture experiment performed ex vivo. B16F10 cells were treated with ctrl, M1, UV-inactivated M1, or physical lysis on day 0, and then the supernatant of each group was collected and fed to immature DCs (iDCs) on day 2. One batch of DCs was used to detect maturation markers by flow cytometry and secreted cytokines by ELISA on day 4. The other batch of DCs was cocultured with naive T cells on day 4, and T-cell activation was evaluated on day 8. B, C MHC I (B) and CD86 (C) expression on DCs fed the indicated supernatants was detected by flow cytometry on day 4. n = 3. P values were determined by one-way ANOVA with Dunnett’s tests for multiple comparisons. D IL-12, TNF-α, and IL-1β levels in the culture medium of DCs fed with the indicated supernatants were determined by ELISA on day 4. n = 3. The P value was determined by a two-tailed Student’s t test. E Phase-contrast images of proliferating T-cell clones on day 8 are shown. The images are representative of three independent experiments. Scale bars, 50 μm. F IFN-γ, granzyme B, and perforin concentrations were determined by ELISA in the DC plus T-cell coculture system on day 8. n = 3. The P value was determined by a two-tailed Student’s t test. G-H, CD69, and CD44 expression on CD4⁺ T cells (G) and CD8⁺ T cells (H) was evaluated on T cells in the DC plus T-cell coculture system on day 8. n = 3. The P value was determined by a two-tailed Student’s t test. Data are shown as the mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001. See also in Supplementary Figs. S3 and S4.