Antigen-specific cytokine secretion, proliferation, and cytotoxicity by transduced mouse CD8⁺ and CD4⁺ T cells. To evaluate cytokine secretion, unfractionated T cells (□), CD8⁺ T cells (dotted bars) and CD4⁺ T cells (diagonally striped bars) transduced with the scFv-CD28-ζ receptor or mock-transduced unfractionated T cells (▪), CD8⁺ T cells (cross-hatched bars), and CD4⁺ T cells (▤) were cocultured with media alone or irradiated MDA-MB-435-erbB2 cells or MDA-MB-435 parental cells or stimulated with plate-bound anti-CD3 and anti-CD28 mAbs in 12-well plates for 24 hours. Supernatants were harvested and assessed for IFN-γ (A), GM-CSF (B), IL-4 (C), and IL-2 (D) production by ELISA. Results are expressed as picogram per milliliter of cytokine secreted ± SE for duplicate samples of 3 representative experiments. The proliferative capacity of each transduced T-cell population was evaluated in a 72-hour [3H]-thymidine incorporation assay (E). Transduced unfractionated T cells (□), CD8⁺ T cells (dotted bars), and CD4⁺ T cells (diagonally striped bars) or mock-transduced unfractionated T cells (▪), CD8⁺ T cells (cross-hatched bars), and CD4⁺ T cells (▤) were incubated with media alone or stimulated with plate-bound anti-CD3 and anti-CD28 mAbs or anti-tag mAb for 3 days. Results are expressed as means ± SE of triplicate samples and are representative of 3 experiments. Specific cytolytic function of scFv-CD28-ζ–transduced mouse T cells was evaluated in a 6-hour 51Cr-release assay. Transduced unfractionated (▵) and CD8⁺ (○) T cells demonstrated greater lysis of MDA-MB-435-erbB2 target cells than CD4⁺-transduced (▪) T cells (F). No lysis of MDA-MB-435 parental target cells was observed by chimeric receptor-transduced T cells (G) and mock-transduced unfractionated T cells (▪), CD8⁺ T cells (▴), or CD4⁺ T cells (□) did not lyse either cell line. The spontaneous lysis was less than 10% in all assays. Results are expressed as percent specific 51Cr release ± SE (%) of triplicate samples representative of at least 2 experiments.