Antigen-specific costimulation of scFv-CD28-ζ–transduced T cells in vitro.

(A) Cytolytic function evaluated in a 6-hour ⁵¹Cr release assay. T cells transduced with the scFv-ζ (●) or scFv-CD28-ζ (○) chimeric receptors equivalently lysed the erbB2⁺tumors COLO 205, MDA-MB-435, or MC-38-erbB2, but not the erbB2⁻ tumors 24JK or MC-38. T cells transduced with the pLXSN retrovirus alone (▪) were unable to lyse the erbB2^−/+ tumors. The spontaneous lysis was less than 10% in all assays. Results are expressed as percent specific⁵¹Cr release ± SE of triplicate samples and are representative of at least 2 experiments. (B) Antigen-specific cytokine production. Mouse T cells transduced with pLXSN alone (dotted bars) or pLXSN encoding the scFv-anti-erbB2-ζ (■) or -CD28-ζ (▪) receptors and cultured with erbB2⁺ (Lovo, COLO 205, or MC-38-erbB2) or erbB2⁻ (24JK or MC-38) tumor cells or stimulated with soluble CD3 and CD28 mAbs for 24 hours. Harvested supernatants were analyzed for IFN-γ, GM-CSF (above) and TNFα, IL-2, IL-4, and IL-10 (data not shown) production by ELISA. T cells secreted minimal levels of IL-2 secretion and no TNF-α, IL-4, or IL-10. Results are expressed as pg/mL of cytokine secreted ± SE of duplicate samples and are representative of at least 5 experiments. (C) Proliferation was estimated by [³H]-thymidine incorporation at 72 hours. Mouse T cells transduced with pLXSN alone (▧) or pLXSN encoding the scFv-anti-erbB2-ζ (■) or scFv-anti-erbB2-CD28-ζ (▪) receptors were cultured in media alone, with irradiated MC-38-erbB2 or MC-38 tumor cells or stimulated with plate-bound CD3 and CD28 mAbs for 3 days. Incorporation of radioactivity by the tumor cells was not detected. Results are expressed as means ± SE of triplicate samples and are representative of at least 2 experiments. Cytokine induction and proliferation via scFv-anti-erbB2-CD28-ζ and -ζ receptors were statistically compared by Mann-Whitney test (*P < .01, **P < .05).