Spleen cells are involved in the CAT-13–induced antitumor protection. (A) Spleen cells from naive (□, n = 3) or CAT-13–treated challenged (day 73) mice (■, n = 3) were stimulated with mitomycin C-treated target EL4-huCD20 cells at a 50:1 responder/stimulator ratio for 4 days in the presence of IL-2. Stimulated cells were then mixed with ⁵¹Cr-labeled EL4-huCD20 cells at effector/target ratios of 50:1, 100:1, and 200:1. Effector values correspond to the number of CD3⁺ T cells. (B) A total of 5 × 10⁷ spleen cells from surviving CAT-13–treated mice, isolated 20 days after tumor challenge, were intravenously injected into naive recipient mice on day −1 (♦ n = 5). These mice were subsequently intravenously injected on day 0 with 5 × 10⁵ EL4-huCD20 cells, as were control naive mice (□, n = 5; log-rank analysis, *P = .03). (C) Spleen cells from untreated or surviving CAT-13–treated mice were analyzed by immunofluorescence 20 days after tumor injection (untreated animals) or tumor challenge (surviving CAT-13–treated animals). Spleen cells from naive mice were also tested. The CD4⁺/CD8⁺ T-cell ratio was calculated (top panel; Kruskal-Wallis analysis, †P = .01), and the number of CD4⁺ and CD8⁺ cells among 3 × 10⁵ CD3⁺ spleen cells was determined for each mouse (bottom panel; Kruskal-Wallis analysis, ††P = .008).