Expression, cytotoxicity, and proliferation of scFv-CD28-ζ–transduced T cells from WT, IFN-γ^−/−, pfp^−/−, and pfp^−/−IFN-γ^−/−mice.

(A) Equivalent expression of the scFv-anti-erbB2-CD28-ζ chimeric receptor in primary mouse T lymphocytes from WT, IFN-γ^−/−, pfp^−/−, and pfp^−/−IFN-γ^−/− BALB/c mice as determined by flow cytometry. Cells were stained with an anti–tag mAb (solid-line histogram) or an IgG1 isotype control mAb (dashed-line histogram). (B) The cytolytic activity of the scFv-anti-erbB2-CD28-ζ–transduced T cells from WT and gene-targeted mice was evaluated in a 6-hour⁵¹Cr release assay. Lysis of MC-38-erbB2⁺ tumor cells (closed symbols) by transduced IFN-γ^−/− T cells (circles), but not pfp^−/− (triangles) or pfp^−/−INF-γ^−/− T cells (diamonds), was equivalent to that mediated by gene-modified WT T cells (squares). All transduced effector T-cell populations were unable to lyse the erbB2⁻MC-38 parental tumor cells (open symbols). Results from a representative experiment are expressed as percent specific⁵¹Cr release ± SE of triplicate samples. Spontaneous lysis was consistently less than 10%. (C) The proliferative capacity of scFv-anti-erbB2-CD28-ζ–transduced mouse T lymphocytes from WT (striped bars), pfp^−/− (dotted bars), IFN-γ^−/− (open bars), and pfp^−/−IFN-γ^−/− (closed bars) mice was determined by [³H]-thymidine incorporation at 72 hours. The ability of receptor-modified T cells from gene-targeted mice to proliferate in response to erbB2 antigen or antibody-mediated crosslinking of endogenous CD3 and CD28 receptors was comparable to that observed for transduced WT T cells. All transduced T-cell populations failed to proliferate in response to erbB2⁻MC-38 parental tumor cells. Incorporation of radioactivity by tumor cells was not detected. Results from a representative experiment are expressed as means ± SE of triplicate samples.