Preferential expansion of TCR+CD3 double transduced T cells in vivo. Thy1.1⁺ CD8⁺ T cells from C57BL/6 mice were transduced with F5-TCR or F5-TCR+CD3-GFP. Thy1.2⁺ recipient C57BL/6 mice were sublethally irradiated (5.5 Gy) and injected subcutaneously with 1 × 10⁶ EL4.NP tumor cells on day 0. The next day, mice were intravenously injected with 3 × 10⁵ transduced Thy1.1⁺ T cells containing similar numbers of F5-TCR and F5-TCR+CD3-GFP T cells. (A) FACS analysis of transduced T cells before injection and recovered from mice 35 days after injection. Dot plots are gated on Thy1.1⁺ CD8⁺ T cells and the numbers indicate the percentage ofVβ11⁺ T cells expressing TCR-only or TCR+CD3-GFP. (B-C) Absolute numbers of transduced cells recovered from the spleen (B) and tumor draining lymph nodes (TDLN; C) are shown. Cells from spleen (D) and TDLN (E) were stained with antibodies for CD44 and CD62L to identify central and effector memory phenotype T cells. Graphs show the relative frequency of TCR-only and TCR+CD3-GFP T cells in the central and effector memory compartment. Data are representative of 5 mice/group. (F) Thy1.2⁺ irradiated C57BL/6 mice were injected subcutaneously with 1 × 10⁶ firefly luciferase-expressing EL4.NP tumor cells on day 0, followed by treatment with 3 × 10⁵ transduced Thy1.1⁺ T cells containing similar numbers of F5-TCR and F5-TCR+CD3-GFP cells. Mice were imaged before (day 8) and after tumor rejection (6 months). Tumor-free mice were rechallenged with 1 × 10⁶ irradiated EL4.NP tumor cells injected in the lower leg. After 5 days, mice were killed and T cells were collected from TDLN and contra-lateral control LNs (nonTDLN). FACS analysis was performed to determine the number of transferred Thy1.1⁺ T cells expressing TCR-only or TCR+CD3-GFP. *P < .05, **P < .01, ***P < .001.