CD4⁺ T cells are responsible for the inhibition of BL cells in vivo. (A) Determination of T cell subsets responsible for the observed antitumor immunity. Wild-type, CD4 KO, and CD8 KO mice were immunized with DCs/EBNA1-P_607–619 peptide, and 2 weeks later were challenged with 5 × 10⁵ B6-BL/EBNA1-GFP tumor cells. The tumor sizes were measured every 2 days after tumor challenge. Significant inhibition of tumor growth was observed in wild-type and CD8 KO mice immunized with DCs/EBNA1-P_572–584 peptide compared with other groups (P = 0.005), Similar results were obtained in 3 repeated experiments. (B) Antitumor immunity elicited in B6, MHC class I KO, but not in MHC class II KO mice. B6, class I, and class II KO mice were immunized with DCs/EBNA1-P_607–619 and were then challenged with B6-BL/EBNA1-GFP cells. DC/EBNA1-P572–584 served as a specificity control. Significant suppression of tumor growth was observed in B6 and MHC class I KO mice immunized with DC/EBNA1-P_607–619 peptide compared with other groups (P = 0.0065). (C) Depletion (depl.) of the subset of CD4⁺ T cells abolished their ability to suppress tumor growth. The immunized mice were treated with anti-CD4 (GK1.5) or anti-CD8 (2.43) mAb’s (200 μg in 500 μl volume) 1 day before tumor challenge and on days 1, 3, and 5 after challenge. Tumor growth was not inhibited in mice with depletion of CD4⁺ T cells, while depletion of CD8⁺ T cells did not affect antitumor immunity (P = 0.0127). B6 mice immunized with DC/EBNA1-P_607–619or DC/EBNA1-P_572–584 peptide served as positive and negative controls, respectively.