LAG-3 blockade exhibits antitumor effects on MHC-I⁻MHC-II⁺tumors mediated by CD4⁺T cells. (A) LAG-3 expression by tumor-infiltrating CD4⁺ T cells. Tumor cells (5.0 × 10⁶) were inoculated subcutaneously (n = 6 per group). TILs were prepared from tumors 14 days after tumor cell inoculation and analyzed with flow cytometry. Representative staining (left) and summary of the frequency of LAG-3⁺CD4⁺ T cells (right) are shown. (B) In vivo antitumor efficacies of anti-PD-1 mAb, anti-LAG-3 mAb, and a combination against E.G7/B2MKO/CIITA and MC-38/B2MKO/CIITA tumors. Tumor cells (E.G7, 5.0 × 10⁶, or MC-38, 1.0 × 10⁶) were inoculated subcutaneously. Mice were grouped when the tumors reached ∼100 mm³ (day 0), and ICIs were administered on days 0, 3, and 6 (n = 6 per group). Tumor growth was monitored every 3 days. (C) In vivo antitumor efficacy of anti-LAG-3 mAb against E.G7/B2MKO/CIITA tumors in CD4⁺ T-cell– or CD8⁺ T-cell–deleted mice. For CD4⁺ T-cell or CD8⁺ T-cell deletion, anti-CD4 mAb or anti-CD8β mAb, respectively, was administered intraperitoneally 1 day before tumor cell inoculation and every 7 days after. All in vivo experiments were performed twice with similar results. **P < .01.