CD3 is rate limiting for expression and function of the WT1-TCR and the F5-TCR in T lymphoma cells. (A) Schematic diagram of the CD3 vector. The 4 CD3 chains (ζ-ϵ-δ-γ) are linked by the 2A sequences, followed by IRES and GFP. (B) T lymphoma cells (58^α−β−) were mock-transduced or transduced with WT1-TCR, WT1-TCR+control-GFP, or WT1-TCR+CD3-GFP, then stained with antibodies against CD3, Vβ2.1, or with HLA-A2/pWT126 tetramer. Cells (58^α−β−) transduced with F5-TCR, F5-TCR+control-GFP or F5-TCR+CD3-GFP, were stained with antibodies against Vβ11 or H2D^b/pNP tetramer. The numbers in the panels in TCR+CD3-GFP transduced cells indicate the fold increase in the level of CD3 or TCR expression (measured by MFI) in cells expressing TCR+CD3-GFP (Q2) compared with the cells expressing TCR only (Q1). No increase in CD3 or TCR expression was seen in cells transduced with TCR+control-GFP. The data are representative of 5 performed experiments. (C) Bulk transduced 58^α−β− cells were stimulated with RMA-S cells coated with increasing concentrations of pNP peptide or 10μM pSV9 control peptide. Supernatant was harvested and assessed for IL-2 production by ELISA. The graph demonstrates the mean IL-2 concentration ± SD of triplicate values. (D) Transduced 58^α−β− cells were purified by FACS to obtain cells expressing TCR-only or TCR+CD3. (E) FACS purified 58^α−β− cells were stimulated with RMA-S cells coated with increasing concentrations of pNP peptide or 10μM pSV9 control peptide. Supernatant was harvested and assessed for IL-2 production by ELISA. The graph demonstrates the mean IL-2 concentration ± SD of triplicate values.