Figure 5.
Figure 5. HLA-A24–restricted cytotoxicity of, and IFN-γ production by, TCR gene–transduced Tc1 and Th1 cells against freshly isolated leukemia cells. TCR gene–transduced CD8+ Tc1 and CD4+ Th1 cells were isolated by a FACSVantage instrument restimulated with WT1 peptide (CMTWNQMNL)–pulsed HLA-A24+ LCLs and expanded in the presence of IL-2. (A) After expansion, TCR gene–transduced Th1 and Tc1 cells were stained with FITC-labeled anti-CD4 mAb and PE-labeled anti-CD8 mAb, and the staining profile of Th1 and Tc1 cells was analyzed by a FACSCalibur instrument and Cell Quest software. Percentage of cells in each quadrant is indicated in flow cytometer plots. (B-C) Cytotoxic activity of TCR gene–transduced Tc1 (B) and Th1 cells (C) against freshly isolated leukemia cells was evaluated by 4-hour 51Cr release assay. (D-E) TCR gene–transduced Tc1 (D) and Th1 cells (E) were cocultured with leukemia cells. After 20 hours, culture supernatant was harvested, and IFN-γ levels in the supernatant were measured by ELISA. WT1 expression level of leukemia cells used in the experiments of Figure 5B-E was determined by quantitative real-time PCR to be HLA-A24+ AML M1 (3.5 × 10–1), AML M21 (2.7 × 10–1), AML M22 (8.6 × 10–2), AML M4 (8.8 × 10–1), ALL L2 (5.3 × 10–1), HLA-A24– AML M2 (1.5 × 100), AML M4 (2.5 × 10–1), and ALL L2 (5.8 × 10–1). Expression levels were expressed as relative values against K562 cells which strongly express WT1. Error bars indicate standard error (SE) in triplicate samples. (F-G) Cytotoxic activity of TCR gene–transduced Tc1 (F) and Th1 cells (G) against HLA-A24+ WT1+ freshly isolated leukemia cells and HLA-A24+ WT1– malignant lymphoma (ML) cells was evaluated by 4-hour 51Cr release assay. WT1 expression level of leukemia and lymphoma cells used in the experiments of Figure 5F-G was determined to be AML1 (1.5 × 100), AML2 (7.2 × 10–1), ML (DLBL) (7.3 × 10–5), ML (Burkitt)1 (3.5 × 10–4), and ML (Burkitt)2 (7.8 × 10–5). AML indicates acute myeloid leukemia; ALL, acute lymphoid leukemia; DLBL, diffuse large B cell lymphoma; and Burkitt, Burkitt lymphoma.

HLA-A24–restricted cytotoxicity of, and IFN-γ production by, TCR gene–transduced Tc1 and Th1 cells against freshly isolated leukemia cells. TCR gene–transduced CD8+ Tc1 and CD4+ Th1 cells were isolated by a FACSVantage instrument restimulated with WT1 peptide (CMTWNQMNL)–pulsed HLA-A24+ LCLs and expanded in the presence of IL-2. (A) After expansion, TCR gene–transduced Th1 and Tc1 cells were stained with FITC-labeled anti-CD4 mAb and PE-labeled anti-CD8 mAb, and the staining profile of Th1 and Tc1 cells was analyzed by a FACSCalibur instrument and Cell Quest software. Percentage of cells in each quadrant is indicated in flow cytometer plots. (B-C) Cytotoxic activity of TCR gene–transduced Tc1 (B) and Th1 cells (C) against freshly isolated leukemia cells was evaluated by 4-hour 51Cr release assay. (D-E) TCR gene–transduced Tc1 (D) and Th1 cells (E) were cocultured with leukemia cells. After 20 hours, culture supernatant was harvested, and IFN-γ levels in the supernatant were measured by ELISA. WT1 expression level of leukemia cells used in the experiments of Figure 5B-E was determined by quantitative real-time PCR to be HLA-A24+ AML M1 (3.5 × 10–1), AML M21 (2.7 × 10–1), AML M22 (8.6 × 10–2), AML M4 (8.8 × 10–1), ALL L2 (5.3 × 10–1), HLA-A24– AML M2 (1.5 × 100), AML M4 (2.5 × 10–1), and ALL L2 (5.8 × 10–1). Expression levels were expressed as relative values against K562 cells which strongly express WT1. Error bars indicate standard error (SE) in triplicate samples. (F-G) Cytotoxic activity of TCR gene–transduced Tc1 (F) and Th1 cells (G) against HLA-A24+ WT1+ freshly isolated leukemia cells and HLA-A24+ WT1– malignant lymphoma (ML) cells was evaluated by 4-hour 51Cr release assay. WT1 expression level of leukemia and lymphoma cells used in the experiments of Figure 5F-G was determined to be AML1 (1.5 × 100), AML2 (7.2 × 10–1), ML (DLBL) (7.3 × 10–5), ML (Burkitt)1 (3.5 × 10–4), and ML (Burkitt)2 (7.8 × 10–5). AML indicates acute myeloid leukemia; ALL, acute lymphoid leukemia; DLBL, diffuse large B cell lymphoma; and Burkitt, Burkitt lymphoma.

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