PD-1 blockade exhibits antitumor effects on MHC-I⁻MHC-II⁺tumors via cytotoxic CD4⁺T cells. (A-C) CD44⁺CD62L⁻ effector/memory CD4⁺ T cells (A), TNF-α⁺IFN-γ⁺CD4⁺ T cells (B), and GrB⁺CD4⁺ T cells (C) in the TIL population of E.G7/B2MKO/CIITA tumors. Tumor cells (5.0 × 10⁶) were inoculated subcutaneously. Mice were grouped when the tumors reached ∼100 mm³ (day 0), and anti-PD-1 mAb was administered on days 0, 3, and 6. TILs were prepared from tumors 14 days after tumor cell inoculation and analyzed with flow cytometry (n = 6 per group). Representative staining (left) and summaries for the frequency of each cell population (right) are shown. (D) In vivo antitumor efficacy of anti-PD-1 mAb against E.G7/B2MKO/CIITA tumors in CD4⁺ or CD8⁺ T-cell–deleted mice. For CD4⁺ or CD8⁺ T-cell deletion, anti-CD4 mAb or anti-CD8β mAb, respectively, was administered intraperitoneally 1 day before tumor cell injection and every 7 days after tumor cell injection. All in vivo experiments were performed twice with similar results. *P < .05; **P < .01.