Figure 3.
Figure 3. DCs and CD40-B loaded with FluMP mRNA induce functional specific T cells in adult cancer patients. (A) CD40-B and DCs from the same prostate cancer patient as in Figure 1A were electroporated with FluMP or GFP mRNA and were cocultured with autologous PBLs for 7 days. T-cell cultures were analyzed with tetramers as in Figure 2. (B) Day 7 T-cell cultures stimulated with FluMP mRNA-loaded CD40-B or DCs were assayed by IFN-γ ELISPOT using either T2 cells (left and middle upper panels) pulsed with tax peptide or FluMP peptide (shown compared with T cells only) or mRNA-electroporated DCs (right upper panel) using GFP mRNA or FluMP mRNA (shown compared with T cells only). For T cells stimulated with FluMP mRNA–loaded CD40-B, the effect of mouse antihuman class I mAb was compared with mouse purified IgG (each at 25 μg/mL) or T cells only (lower panels). Targets were either T2 cells (left lower panel) pulsed with tax peptide or FluMP peptide or mRNA-electroporated DCs (right lower panel) using GFP mRNA or FluMP mRNA. Bars represent 1 standard deviation. P values shown are for comparisons between starred experiments. (C) CD40-B and DCs from a second prostate cancer patient were electroporated with FluMP or GFP mRNA, cocultured with autologous PBLs for 7 days, and analyzed with tetramers as in Figure 2. A range of T-cell/APC ratios for DCs (solid line) and CD40-B (dotted line) was examined. Tetramer results for cultures stimulated with FluMP APCs are shown. The negative control tax tetramer labeled less than 0.05% in each case (not shown). Labeling of GFP mRNA stimulated cultures with the FluMP tetramer was less than 0.1% in each case (not shown). (D) T-cell cultures were stimulated once with CD40-B loaded with FluMP mRNA (left panels), once with DCs loaded with FluMP mRNA (upper right panel), or once (lower left) or 3 times (lower right) with CD40-B loaded with GFP mRNA and were assayed for lysis of peptide-loaded T2 cells (• FluMP peptide; ○ tax peptide) or mRNA-loaded SW-480 carcinoma cells (lower left panel: ▴ or , FluMP mRNA–loaded targets; ▵ or ⋄, GFP mRNA–loaded targets; ▴ or ▵, FluMP mRNA–stimulated T cells; or ⋄, GFP mRNA–stimulated T cells). Results are representative of 1 to 3 experiments from 2 prostate cancer patients.

DCs and CD40-B loaded with FluMP mRNA induce functional specific T cells in adult cancer patients. (A) CD40-B and DCs from the same prostate cancer patient as in Figure 1A were electroporated with FluMP or GFP mRNA and were cocultured with autologous PBLs for 7 days. T-cell cultures were analyzed with tetramers as in Figure 2. (B) Day 7 T-cell cultures stimulated with FluMP mRNA-loaded CD40-B or DCs were assayed by IFN-γ ELISPOT using either T2 cells (left and middle upper panels) pulsed with tax peptide or FluMP peptide (shown compared with T cells only) or mRNA-electroporated DCs (right upper panel) using GFP mRNA or FluMP mRNA (shown compared with T cells only). For T cells stimulated with FluMP mRNA–loaded CD40-B, the effect of mouse antihuman class I mAb was compared with mouse purified IgG (each at 25 μg/mL) or T cells only (lower panels). Targets were either T2 cells (left lower panel) pulsed with tax peptide or FluMP peptide or mRNA-electroporated DCs (right lower panel) using GFP mRNA or FluMP mRNA. Bars represent 1 standard deviation. P values shown are for comparisons between starred experiments. (C) CD40-B and DCs from a second prostate cancer patient were electroporated with FluMP or GFP mRNA, cocultured with autologous PBLs for 7 days, and analyzed with tetramers as in Figure 2. A range of T-cell/APC ratios for DCs (solid line) and CD40-B (dotted line) was examined. Tetramer results for cultures stimulated with FluMP APCs are shown. The negative control tax tetramer labeled less than 0.05% in each case (not shown). Labeling of GFP mRNA stimulated cultures with the FluMP tetramer was less than 0.1% in each case (not shown). (D) T-cell cultures were stimulated once with CD40-B loaded with FluMP mRNA (left panels), once with DCs loaded with FluMP mRNA (upper right panel), or once (lower left) or 3 times (lower right) with CD40-B loaded with GFP mRNA and were assayed for lysis of peptide-loaded T2 cells (• FluMP peptide; ○ tax peptide) or mRNA-loaded SW-480 carcinoma cells (lower left panel: ▴ or , FluMP mRNA–loaded targets; ▵ or ⋄, GFP mRNA–loaded targets; ▴ or ▵, FluMP mRNA–stimulated T cells; or ⋄, GFP mRNA–stimulated T cells). Results are representative of 1 to 3 experiments from 2 prostate cancer patients.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal