Figure 2
Figure 2. CD3 is rate limiting for expression and function of the F5-TCR and the WT1-TCR in primary T cells. (A) C57BL/6 splenocytes were Vβ11 depleted before activation and transduced with the F5-TCR, F5-TCR+control-GFP, or F5-TCR+CD3-GFP. Cells were stained with antibodies against Vβ11 or H2Db/pNP tetramer. Alternatively, total splenic T cells were transduced with the WT1-TCR, WT1-TCR+control-GFP, or WT1-TCR+CD3-GFP and stained with antibodies against CD3, Vβ2.1, or with HLA-A2/pWT126 tetramer. The numbers in the panels indicate the fold increase in the level of CD3 or TCR expression (measured by MFI) in cells expressing TCR+CD3-GFP or TCR+control-GFP (Q2) compared with the cells expressing TCR only (Q1). The data are representative of 5 performed experiments. (B) Bulk transduced T cells (1 × 106) were stimulated with 5 × 105 T2 cells loaded with increasing concentrations of pWT126 or 10μM pMelanA control peptide. Supernatant was harvested and assessed for IFNγ production by ELISA. The graph demonstrates the mean IFNγ concentration ± SD of triplicate values. The data are from one of 3 performed experiments. (C) Intracellular cytokine production was assessed in CD8+ T cells expressing WT1-TCR+CD3-GFP or WT1-TCR-only. Transduced T cells were stimulated with increasing concentrations of pWT126 or 10μM pMelanA control peptide. The intracellular IFNγ staining was assessed in CD8+ T cells expressing TCR+CD3 or TCR-only (Vβ2.1+GFP+ cells and Vβ2.1+GFP− cells, respectively). The graph shows the percentage of Vβ2.1+GFP+ and Vβ2.1+GFP− T cells producing IFNγ. (D) CD8+ T cells from C57BL/6 mice were transduced with F5-TCR+CD3-GFP and FACS purified to obtain cells expressing TCR-only or TCR+CD3 (purity of 86%-96%, respectively). (E) F5-TCR or F5-TCR+CD3 T cells (1 × 105) were stimulated with 1 × 105 irradiated splenocytes coated with increasing concentrations of pNP or 10μM pWT126 control peptide, and peptide-specific proliferation was measured by thymidine incorporation. (F-G) 1 × 105 F5-TCR or TCR+CD3 T cells were stimulated with 1 × 105 irradiated splenocytes coated with increasing concentrations of pNP or 10μM pWT126 control peptide. Supernatant was harvested and assessed for (F) IFNγ and (G) IL-2 production by ELISA. The graph demonstrates the mean cytokine concentration ± SD of triplicate values. The data are from 2 performed experiments.

CD3 is rate limiting for expression and function of the F5-TCR and the WT1-TCR in primary T cells. (A) C57BL/6 splenocytes were Vβ11 depleted before activation and transduced with the F5-TCR, F5-TCR+control-GFP, or F5-TCR+CD3-GFP. Cells were stained with antibodies against Vβ11 or H2Db/pNP tetramer. Alternatively, total splenic T cells were transduced with the WT1-TCR, WT1-TCR+control-GFP, or WT1-TCR+CD3-GFP and stained with antibodies against CD3, Vβ2.1, or with HLA-A2/pWT126 tetramer. The numbers in the panels indicate the fold increase in the level of CD3 or TCR expression (measured by MFI) in cells expressing TCR+CD3-GFP or TCR+control-GFP (Q2) compared with the cells expressing TCR only (Q1). The data are representative of 5 performed experiments. (B) Bulk transduced T cells (1 × 106) were stimulated with 5 × 105 T2 cells loaded with increasing concentrations of pWT126 or 10μM pMelanA control peptide. Supernatant was harvested and assessed for IFNγ production by ELISA. The graph demonstrates the mean IFNγ concentration ± SD of triplicate values. The data are from one of 3 performed experiments. (C) Intracellular cytokine production was assessed in CD8+ T cells expressing WT1-TCR+CD3-GFP or WT1-TCR-only. Transduced T cells were stimulated with increasing concentrations of pWT126 or 10μM pMelanA control peptide. The intracellular IFNγ staining was assessed in CD8+ T cells expressing TCR+CD3 or TCR-only (Vβ2.1+GFP+ cells and Vβ2.1+GFP− cells, respectively). The graph shows the percentage of Vβ2.1+GFP+ and Vβ2.1+GFP− T cells producing IFNγ. (D) CD8+ T cells from C57BL/6 mice were transduced with F5-TCR+CD3-GFP and FACS purified to obtain cells expressing TCR-only or TCR+CD3 (purity of 86%-96%, respectively). (E) F5-TCR or F5-TCR+CD3 T cells (1 × 105) were stimulated with 1 × 105 irradiated splenocytes coated with increasing concentrations of pNP or 10μM pWT126 control peptide, and peptide-specific proliferation was measured by thymidine incorporation. (F-G) 1 × 105 F5-TCR or TCR+CD3 T cells were stimulated with 1 × 105 irradiated splenocytes coated with increasing concentrations of pNP or 10μM pWT126 control peptide. Supernatant was harvested and assessed for (F) IFNγ and (G) IL-2 production by ELISA. The graph demonstrates the mean cytokine concentration ± SD of triplicate values. The data are from 2 performed experiments.

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