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. 2021 Jul 29;218(9):e20200962. doi: 10.1084/jem.20200962

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© 2021 Fukuda et al.

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Figure 2. — Vaccination with AIM2-deficient DC improves the efficacy of ACT through activation of STING–type I IFN signaling. (A) IFN-β or CXCL10 in the supernatants of indicated BMDCs stimulated with 0, 0.1, or 1 µg/ml B16F10 DNA for 4 h (IFN-β) or 10 h (CXCL10; n = 3). (B) Immunoblotting for pTBK1, TBK1, pIRF3, IRF3, and vinculin in the lysates of indicated BMDCs stimulated with 0, 0.1, or 1 µg/ml B16F10 DNA for 4 h. (C–G) B16F10-bearing WT mice (B16F10 mice) were treated with ACT alone or ACT + 1.0 × 10⁶ WT, Aim2^−/−, or Aim2^−/−Sting^−/− DC-gp100. On day 20 after PMELs (1.0 × 10⁶ cells) transfer, tissues were harvested. (C) The therapy regimen scheme. (D) Tumor growth over time (left; n = 9). Sample photo of B16F10 tumor on day 20 after PMELs transfer (right). Scale bar, 10 mm. (E and F) Flow cytometry analysis of TILs (n = 9). (E) The numbers of PMELs, CD8⁺ T cells, and CD4⁺ T cells among 10⁴ live singlet cells, percentage of FoxP3⁺ cells in CD4⁺ T cells, and PMEL/T reg cell ratio. (F) Percentages of IFN-γ⁺ and TNF-α⁺ cells in PMELs. (G) IFN-β protein levels within the tumor, TdLN, and spleen (n = 7). (H and I) B16F10 DNA–stimulated WT or Aim2^−/− DC-gp100 was cocultured with CFSE-labeled PMELs for 72 h (n = 5). (H) Histograms of PMELs CFSE dilution. (I) Proliferation index of PMELs and amount of IFN-γ⁺ in the supernatants. (J and K) B16F10 mice were treated with ACT using 1.0 × 10⁶ CFSE-labeled PMELs + 1.0 × 10⁶ WT or Aim2^−/− DC-gp100. On day 3 after PMELs transfer, spleens were harvested. (J) The therapeutic regimen. (K) Histograms of PMELs CFSE dilution, proliferation index of PMELs, and numbers of PMELs among 10⁴ live singlet cells in the spleen (n = 6 or 7). Data are shown as mean ± SEM and are pooled from three (A and D–G) or two (I and K) experiments or are representative of at least two independent experiments (B, H, and K). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; two-way ANOVA with Tukey’s multiple-comparisons test (D), one-way ANOVA with Dunnett’s (A and I) or Tukey’s (E, F, and K) multiple-comparisons test, or Mann–Whitney test (G and I).