Extended Data Fig. 9: In vitro mechanism investigation of taltirelin sensitized ICB therapy.

a, Cell viability of B16-F10 cells after treatment with PBS, taltirelin (100 nM, 200 nM, 400 nM, 800 nM, 1600 nM) or TSH (10 nM, 20 nM, 40 nM, 80 nM) for 24 h. b, Flow cytometry gating strategy for the analysis of CD8⁺ T cells ex vivo. c,d, Representative flow cytometry images (c) and quantification (d) of the CFSE ^low cells of CD8⁺ T cells stimulated with PBS, taltirelin (1, 10, 100, 500, 1000 nM) or TSH (20 nM) in the presence or absence of 2.5 μg mL⁻¹ ConA for 5 days. e,f, Percentages of IFN-γ⁺ (e) and GzmB⁺ cells (f) in CD8⁺ T cells after treatment as indicated in c by flow cytometry. One-way ANOVA followed by Tukey’s HSD post hoc test. g, Percentage of PD-1⁺ cells in CD8⁺ OT-I T cells after treatment indicated in Fig. 6i by flow cytometry. h, Cell viability of B16-F10 cells after treatment with PBS or L-thyroxine (10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M) for 24 h. Data are presented as means ± s.d. i-k, CD86 (i), MHC-I SINFEKL (j) and MHC-II (k) MFI on BMDCs after treatment with PBS or L-thyroxine (10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M) in the presence or absence of 10 μg mL⁻¹ OVA for 24 h by flow cytometry. l, Percentages of IFN-γ⁺ in CD8⁺ T cells after treatment with PBS or L-thyroxine (10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M) in the presence or absence of 2.5 μg mL⁻¹ ConA for 5 days by flow cytometry. Data are presented as means ± s.d (a, d-l). n = 6 biologically independent samples (a,h). n = 3 biologically independent samples (d-g, i-l). One-way ANOVA followed by Bonferroni’s multiple comparisons post-test (a, g-l). One-way ANOVA followed by Tukey’s HSD post hoc test (d–f).