Adoptively transferred gene-modified CD4 and CD8 T cells retain enhanced reactivity to the antigens. Thy1.1-positive congenic B6 mice were subcutaneously injected with E.G7 cells (2 × 10⁶). Seven days later, Thy1.2-positive retrovirus-transduced OT-I (1 × 10⁷) and OT-II T cells (2 × 10⁶) were adoptively transferred to tumor-bearing mice. After 3 days, splenocytes were isolated for the analysis. (A) Splenocytes were stimulated with OVA_323-339 peptides (10 μg/mL) for 2 hours and further stimulated for additional 4 hours in the presence of GolgiStop solution. After fixation and permeabilization of the cells, CD4 and intracellular IL-2 or INF-γ were stained with antibodies. Representative flow cytometry profiles of GFP-positive cells from 4 separate experiments are shown. (B) Absolute numbers of retrovirus-transduced OT-II T cells (Thy1.2⁺CD4⁺) in the spleen were calculated from the flow cytometry analysis and live cell counting. Data were from 4 independent experiments using 3 mice per group. P values, Student t test. (C) CD8 T cells were purified using anti-CD8 microbeads. The defined number (2 × 10⁴) of OT-I T cells (Thy1.2⁺CD8⁺) was stimulated with OVA_257-264 in the presence of irradiated splenocytes for 48 hours. IFN-γ production was measured by ELISA.