Hyperexpanded CD4⁺ and CD8⁺ T cells synergistically mediate tumor regression. (A) CD62L^low TDLN cells, or the purified CD4⁺ subset thereof, were activated as described in “Materials and methods” with anti-CD3 restimulations every 21 days as indicated. The total proliferation is plotted on a semi-log₁₀ scale. (B) Mice bearing 3-day subcutaneous tumors (n = 5/group) were treated with TBI, and then received intravenous transfer of HBSS, 4 × 10⁷ CD4⁺ T cells, 4 × 10⁷ CD8⁺ T cells, or the combination of 4 × 10⁷ CD4⁺ and CD8⁺ T cells activated for 85 days. Mice treated with the combination of CD4⁺ and CD8⁺ T cells had complete regression and superior therapeutic response than CD4⁺ or CD8⁺ T cells alone; error bars indicate SEM (P < .01 for each). (C) Mice bearing 3-day intracranial tumors (n = 5/group) were treated with TBI, and then received HBSS, CD4⁺, CD8⁺ T-cell subsets alone, or the combination of CD4⁺ and CD8⁺ T cells activated for 85 days. Survival for the CD4⁺ and CD8⁺ T-cell subsets was significantly better than HBSS control (P < .01 for each), but survival for the CD4⁺ combined with CD8⁺ T-cell group was significantly better than either the CD4⁺ or CD8⁺ T-cell subset (P < .01). (D) Mice bearing 10-day pulmonary metastases were treated with TBI then received the indicated number of CD4⁺ or CD8⁺ T cells alone or the combination of CD4⁺ and CD8⁺ T cells activated for 135 days. The number of metastases was counted on day 21. The combination of CD4⁺ and CD8⁺ T cells was superior to either subset alone (P < .01).