Anti-PD-1 mAb activity promotes CD8 ⁺ T cell responses in the draining lymph node of EG7 tumor – bearing mice. (A–E) CD11c-eYFP mice were injected s.c. with EG7 tumor cells. After 10 days, mice were adoptively transferred with GFP-expressing OT-I CD8⁺ T cells and treated or not with anti-PD-1 mAb (250 µg, i.v.). Two-photon imaging of tumor-draining lymph nodes was performed on day 1. (A) Experimental setup. (B) Representative two-photon time-lapse images showing contacts between OT-I CD8⁺ T cells (pseudocolored in magenta) and CD11c reporter positive cells (pseudocolored in cyan) and OT-I CD8⁺ T cell tracks (during 10 min). Scale bar, 10 µm. (C) Quantification of the density of stable T cell–DC contacts (lasting >5 min). (D) Representative two-photon images of stable T cell–DC contacts, illustrating the blastic phenotype seen in anti-PD-1 mAb–treated mice. Scale bar, 10 µm. (E) Quantification of the size of OT-I CD8⁺ T cell stably interacting with DCs in the indicated groups. Results are compiled from 9–11 movies from two independent experiments. (F and G) C57BL/6 mice were injected s.c. with EG7 tumor cells. After 10 days, mice were adoptively transferred with CTV-labeled OT-I CD8⁺ T cells and treated or not with anti-PD-1 mAb (250 µg, i.v.). (F) OT-I CD8⁺ T cell size was estimated by flow cytometry on day 3 using the FSC-H parameter in each cell generation identified by CTV dilution. Representative of three independent experiments with two to three mice per group in each experiment. (G) MFI of PD-1 expression on OT-I CD8⁺ T cells was quantified on day 3 for each cell generation. Representative of three independent experiments with two to three mice per group in each experiment. Statistical analyses were performed using t test (E) or two-way ANOVA (C, F, and G). *, P < 0.05; **, P < 0.01; ***, P < 0.001.