Fig. 3: imNA_{αPD1 & αPDL1} increases CD8⁺ T cell-mediated cytotoxicity in vitro.

a CD8⁺ T cell-mediated antitumor immunity was selected as a model to investigate the superiority of imNA; the following four groups were established: (1) IgG control; (2) Free_{αPD1 & αPDL1}; (3) NP_αPD1 & NP_αPDL1; and (4) imNA_{αPD1 & αPDL1}. b Extracellular fluorescence of B16-F10 cells after treatment with αFc-NP_IgG or imNA_{αPD1 & αPDL1} for different periods. NPs were labeled with FITC. a.u., arbitrary unit. Data were presented as mean ± s.d. n = 3 biologically independent samples. c Confocal images of B16-F10 cells showed cell associations with imNA_{αPD1 & αPDL1} after a 24 h incubation. The cell membrane was stained with PKH26 dye. Scale bar, 5 μm. d Extracellular fluorescence of CD8⁺ T cells after treatment with αFc-NP_IgG or imNA_{αPD1 & αPDL1} for different periods. Data were presented as mean ± s.d. n = 3 biologically independent samples. e Confocal images of CD8⁺ T cells showing cell association with imNA_{αPD1 & αPDL1} after a 24 h incubation. Scale bar, 5 μm. f Confocal images showing the conjugations of CD8⁺ T cells on B16-F10 tumor cells. CFSE-labeled CD8⁺ T cells and B16-F10-mCherry cells were co-incubated for 8 h in the presence of different treatments, and cell conjugations were detected after washing. Scale bar, 50 μm. g The conjugation rates were determined manually by observing the red/green fluorescence overlap in multiple nonoverlapping images. Data were presented as mean ± s.d. n = 12 nonoverlapping images. The concentrations of IFN-γ (h), Granzyme B (i), and Perforin (j) in the supernatant of coincubated cells were examined by ELISA. CD8⁺ T cells and B16-F10 cells (10:1) were coincubated and treated as indicated above for 24 h. Data are presented as means ± s.d. n = 3 biologically independent samples. k Viability of B16-F10 cells analyzed by a high content analysis (HCA) platform. l HCA images showing that imNA_{αPD1 & αPDL1}-facilitated cell interactions induced tumor cells apoptosis. Scale bar, 10 μm. Data are presented as means ± s.d. Statistical significance was calculated via one-way ANOVA with the Tukey post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are provided as a Source Data file.