Figure 2.

Analysis of cell surface CAR expression, in vivo transduction of EL-4 CAR by adenovirus, and CAR-mediated adenovirus transduction of other lymphocyte cell lines. (A) The expression of CAR was determined on the indicated cells by incubation of the cells with RmcB monoclonal against hCAR (or no primary antibody as a control, shown as a dotted line overlay) followed by a fluorescein isothiocyanate-linked goat α-mouse IgG1 antibody. EL-4 mCAR 7 is a single cell clone from the pooled EL-4 cells expressing mCAR. (B) EL-4 CAR 13 or EL-4 LXSN cells were injected i.p. into C57BL/6 mice (5 × 10⁶ in 0.5 ml of PBS per mouse). Seventy-two hours later, the mice were injected i.p. with 2 × 10⁷ focus-forming units of AdCMV-GFP in 0.4 ml of PBS. The mice were sacrificed 12 hr later. EL-4 cells were removed from the peritoneum by instilling then immediately withdrawing 5 ml of PBS injected into the peritoneum cavity with a 22-gauge needle. These cells were stained with Cy-chrome linked anti-CD3 (PharMingen 01088A), and then analyzed by flow cytometry. Cells gated for both size (as determined for uninjected EL-4 cells) and CD3 expression were analyzed for GFP expression as shown in B. The percentage of fluorescent cells (F bar) is indicated. (C) FL5.12, DPK.C7, and the hCAR expressing derivatives of either of these cell lines were transduced with either Ad-CON or AdCMV-GFP at the indicated MOIs and analyzed by flow cytometry, as described in Fig. 1. The percentage of fluorescent cells (C bar) is indicated.