Anti-PD-1 mAb primarily binds to a subset of CD4 ⁺ T cells in tumor-draining lymph nodes. (A–D) C57BL/6 mice were injected s.c. with MC38-OVA tumor cells. After 10 days, tumor-bearing mice were adoptively transferred with GFP-expressing OT-I CD8⁺ T cells and treated with AF594-labeled anti-PD-Ab or treated with an AF594-labeled IgG2a isotype. Draining lymph node and tumor were analyzed by flow cytometry 20 h later. (A) Representative histograms showing in vivo labeling by AF594-labeled anti-PD-1 (percentage shown in blue) or AF594-labeled control isotype (percentage shown in black) on different immune cell subsets in tumor-draining lymph nodes. Note only CD4⁺ T cells specifically bound anti-PD-1 mAb and that a small level (2–5%) of unspecific binding (isotype and anti-PD-1 mAb) is detected on B cells. NK, natural killer. (B) Quantification of cell labeling by the AF594-conjugated anti-PD-1 Ab in the indicated subset. (C) Representative histograms showing in vivo labeling of tumor-resident CD4⁺ and CD8⁺ T cells by AF594-conjugated anti-PD-1 Ab. (D) Quantification of AF594⁺ labeled anti-PD-1 cells in each cell subset. One experiment with five to seven mice per group. (E–G) C57BL/6 WT or PD-1^−/− mice were injected s.c. with MC38-OVA tumor cells. (E and F) PD-1 expression on CD4⁺ or CD8⁺ T cells, CD19⁺ B cells, and NK-1.1⁺ cells was quantified on day 13. Representative histograms (E) and quantification (F) of PD-1⁺–expressing cells in WT mice. Compiled from two independent experiments with a total of six mice per group. (G) MFI of PD-1 expressed at the cell surface on different PD-1⁺ CD4⁺ T cell populations, defined by the transcription factors RORγτ, T-bet, GATA3, FoxP3, and BCL6. One experiment with seven mice per group. Statistical analyses were performed using two-way ANOVA, *, P < 0.05; ***, P < 0.001.