Figure 2.
Figure 2. Appropriate cellular processing and expression of IL-15, IL-15Rα, and CD80 are observed after lentiviral transduction of 293T and 32Dp210 cell lines. (A) Levels of IL-15 secretion after lentiviral transduction of 293T cells. Murine IL-15 secretion was quantified by ELISAs of tissue culture supernatants 48 hours after transfection. On the left are designations of lentiviral vector constructs generated and used for transduction studies. Asterisks denote vectors that were used to generate the final 32Dp210 vaccines. Lanes 1 to 5 on the vertical axis depict IL-15 secretion detected as nanograms per milliliter as detected by ELISA for cells transduced with each of the designated constructs. Bars represent the mean cytokine concentration ± SEM. (B) Levels of IL-15 secretion after lentiviral transduction of 32Dp210 cells are comparable pre- and postirradiation. 32Dp210 cells transduced with lentiviral vectors containing mIL-15/IL-15Rα or CD80, or both IL-15/IL-15Rα and CD80, were expanded, and populations were selected that express similar levels of cell-surface IL-15Rα and/or CD80 by fluorescence-activated cell sorter. High-expressing populations were purified. Levels of IL-15 in culture supernatants (ng/mL) were measured before (red bars) and 48 hours after irradiation with 40 Gy (blue bars). 32Dp210, to the left of the uppermost 2 lanes, indicates IL-15 secretion in untransduced controls. As previously, designations to the left of the graph indicate the genes (IL-15, IL-15Rα, or CD80, or all 3 cassettes) contained in the lentiviral vector used to transduce the 32Dp210 cells analyzed. Levels of IL-15 detected in cell culture supernatants are indicated (ng/mL) on the horizontal axis. Bars represent the mean cytokine concentration ± SEM. (C) Transduced 32Dp210 cells exhibit high level cell-surface expression of IL-15Rα, CD80, and IL-15 after purification of transduced 32Dp210 populations. Lentivirally transduced, purified 32Dp210-derived cells were stained with anti-IL-15, anti-IL-15Rα, and anti-CD80 antibodies and subjected to flow cytometric analyses. 32Dp210 on the left indicates the untransduced parental cell line used as a control. The genes encoded by each lentiviral vector used to transduce the 32Dp210 cells are indicated to the left of the flow analysis, and the antibody used to stain cells is indicated above each flow plot. (D) Cell-surface expression of IL-15Rα and CD80 on 32Dp210 vaccines is stable 48 hours after irradiation with doses up to 50 Gy. Lentivirally transduced, purified 32Dp210 cells were stained with anti-IL-15Rα and anti-CD80 antibodies, and the effects of different doses of radiation on IL-15Rα and CD80 expression analyzed. The lentiviral vector used to transduce 32Dp210 cells is indicated above each plot, and the dose of irradiation indicated on the vertical axis. Antibodies used to analyze cell surface expression are indicated below the plots. Data showing cell surface IL-15Rα expression are shown in the left 2 graphs, and cell surface staining with anti-CD80 antibodies is presented in the right 2 graphs.

Appropriate cellular processing and expression of IL-15, IL-15Rα, and CD80 are observed after lentiviral transduction of 293T and 32Dp210 cell lines. (A) Levels of IL-15 secretion after lentiviral transduction of 293T cells. Murine IL-15 secretion was quantified by ELISAs of tissue culture supernatants 48 hours after transfection. On the left are designations of lentiviral vector constructs generated and used for transduction studies. Asterisks denote vectors that were used to generate the final 32Dp210 vaccines. Lanes 1 to 5 on the vertical axis depict IL-15 secretion detected as nanograms per milliliter as detected by ELISA for cells transduced with each of the designated constructs. Bars represent the mean cytokine concentration ± SEM. (B) Levels of IL-15 secretion after lentiviral transduction of 32Dp210 cells are comparable pre- and postirradiation. 32Dp210 cells transduced with lentiviral vectors containing mIL-15/IL-15Rα or CD80, or both IL-15/IL-15Rα and CD80, were expanded, and populations were selected that express similar levels of cell-surface IL-15Rα and/or CD80 by fluorescence-activated cell sorter. High-expressing populations were purified. Levels of IL-15 in culture supernatants (ng/mL) were measured before (red bars) and 48 hours after irradiation with 40 Gy (blue bars). 32Dp210, to the left of the uppermost 2 lanes, indicates IL-15 secretion in untransduced controls. As previously, designations to the left of the graph indicate the genes (IL-15, IL-15Rα, or CD80, or all 3 cassettes) contained in the lentiviral vector used to transduce the 32Dp210 cells analyzed. Levels of IL-15 detected in cell culture supernatants are indicated (ng/mL) on the horizontal axis. Bars represent the mean cytokine concentration ± SEM. (C) Transduced 32Dp210 cells exhibit high level cell-surface expression of IL-15Rα, CD80, and IL-15 after purification of transduced 32Dp210 populations. Lentivirally transduced, purified 32Dp210-derived cells were stained with anti-IL-15, anti-IL-15Rα, and anti-CD80 antibodies and subjected to flow cytometric analyses. 32Dp210 on the left indicates the untransduced parental cell line used as a control. The genes encoded by each lentiviral vector used to transduce the 32Dp210 cells are indicated to the left of the flow analysis, and the antibody used to stain cells is indicated above each flow plot. (D) Cell-surface expression of IL-15Rα and CD80 on 32Dp210 vaccines is stable 48 hours after irradiation with doses up to 50 Gy. Lentivirally transduced, purified 32Dp210 cells were stained with anti-IL-15Rα and anti-CD80 antibodies, and the effects of different doses of radiation on IL-15Rα and CD80 expression analyzed. The lentiviral vector used to transduce 32Dp210 cells is indicated above each plot, and the dose of irradiation indicated on the vertical axis. Antibodies used to analyze cell surface expression are indicated below the plots. Data showing cell surface IL-15Rα expression are shown in the left 2 graphs, and cell surface staining with anti-CD80 antibodies is presented in the right 2 graphs.

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