Figure 6. The superior effector function of CD8⁺ tumor-infiltrating T cells following treatment with dual-LNP. Mice were inoculated with YUMM1.7 tumors and treated with either PBS, dual-LNP, VISTA control dual-LNP (VISTA siRNA+non-stimulatory GPC), and CPG control dual-LNP (CPG+non targeting siRNA). (A–E) Tumors were harvested 12 days after the start of treatment with dual-LNP and PBS treated. Tumor-associated lymphocytes were stimulated ex vivo with antiCD3 and antiCD28 for 16 hours. The expression of effector molecules was examined by flow cytometry and shown as the percentages of CD8⁺ TILs. The percentages of IFN-γ⁺(A), TNF-α⁺(B), IFN-γ⁺ TNF-α⁺(C), and CD107a⁺ TNF-α⁺(D), CD8⁺ TILS in dual-LNP and PBS-treated tumors were shown. (E) Representative flow cytometry plots of IFN-γ, TNF-α, and CD107a staining. (F–J) Tumors were harvested and stimulated 14 days after the start of treatment with dual-LNP and control LNP (VISTA siRNA+non-stimulatory GPC). IFN-γ⁺(F), TNF-α⁺(G), IFN-γ⁺ TNF-α⁺(H), and CD107a⁺ TNF-α⁺(I), CD8⁺ TILS in dual CPG+VISTA siRNA LNP and PBS-treated tumors were shown. (J) Representative flow cytometry plots of IFN-γ, TNF-α, and CD107a staining. (K–O) Tumors were harvested and stimulated 14 days after the start of treatment with dual LNP and control LNP (CPG+non-targeting siRNA). IFN-γ⁺(K), TNF-α⁺(L), IFN-γ⁺ TNF-α⁺(M), and CD107a⁺ TNF-α⁺(N), CD8⁺ TILS in dual CPG+VISTA siRNA LNP and PBS-treated tumors were shown. (O) Representative flow cytometry plots of IFN-γ, TNF-α, and CD107a staining. All experiments were performed with at least six mice per group. Statistics were analyzed by Student’s t-test. LNP, lipid nanoparticle; VISTA, V-domain immunoglobulin suppressor of T cell activation; PBS, phosphate-buffered saline.