Figure 1.

Construction and functional validation of MART-1_27-35-specific CD4⁺ TCR-Ts. (A) IFN-γ ELISPOT results after co-culture of CD8⁺ T cells with MART-1_27-35 or DMSO-pulsed T2 cells. (B) FACS sorting of MART-1_27-35-specific T cells stained with tetramers. (C) Pie chart of TCR clonotype frequency from single-cell V(D)J sequencing. (D) Cytotoxicity of CD8⁺ TCR-Ts and CD4⁺ TCR-Ts expressing top4 TCR clonotypes to MART-1_27-35 or DMSO-pulsed T2 cells. (E) Cytokine production by CD8⁺ TCR-Ts and CD4⁺ TCR-Ts stimulated with MART-1_27-35 or DMSO-pulsed T2 cells. (F) Killing of A375_MART-1 melanoma cell line by CD4⁺ and CD8⁺ TCR-Ts. (G) Mean tumor growth curves of five groups after treatment. NOG mice were implanted with A375_MART-1 subcutaneously. Mice were divided into five groups after 7 days, and received different treatment through peritumoral injection at day 0 (start of treatment) and day 7: PBS (n = 5), CD4⁺ mock T (n = 3, 3.0×10⁷), CD4⁺ TCR-T (n=5, 3.0×10⁷), CD8⁺ mock T (n = 5, 3.0×10⁷) and CD8⁺ TCR-T (n = 5, 3.0×10⁷). Tumor volume was calculated every 2 days. (A-F), groups were compared with a two-sided, unpaired t-test. (G), groups were compared with the one-way ANOVA analysis. Error bars denote the SD. *:P < 0.05; **:P < 0.01; ***:P < 0.001; ****:P < 0.0001.