Figure 6. Depletion of NK cells reduces CD8⁺ T cell polyfunctionality.

(A–D) Bilateral-tumor-bearing mice received 3xTx on the primary tumor. During treatment, IgG (3xTx) or anti-NK1.1 antibody (3xTx-NK^dep) was injected intraperitoneally. On day 22, spleen-sorted CD8⁺ T cells were co-cultured with pre-plated M2w or M2h for 5 days. (A) Treatment regimen. (B) NK cell depletion efficacy (n = 6). (C) Analysis of TNF-α and (D) IFN-γ MFI in effector/memory CD8⁺ T cells (T_em; Selectin⁻CD44⁺) and naive CD8⁺ T cells (T_naive; Selectin⁺CD44⁻) (n = 8–9). (E–I) Bilateral-tumor-bearing mice received 3xTx with IgG (3xTx) or anti-NK1.1 antibody (3xTx-NKdep). On day 16 or day 22, the distant tumor was collected and analyzed. (E) Analysis of LAG-, IFN-γ-, and TNF-α-positive or (F) IFN-γ and TNF-α double-positive cells of tumor-infiltrating T_em on day 16 (n = 9–13). (G) Polyfunctional activation topology PCA plot. (H) Single-cell polyfunctional group analysis (left) and multiple protein detection (right). (I) Polyfunctional strength index (PSI) of CD8⁺ T cells. Data represent the mean value. Error bars indicate SD. ns, not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.