Figure 3.
Figure 3. Signaling DCs through CD40 increases transgene expression to protein levels similar to those induced by rAd-Luc. (A) Transgene expression in mature DCs, gene gun–transfected with pCMV-Luc or infected with rAd-CMV-Luc and cultured in the presence or in the absence of the agonist anti-CD40 mAb. Mature DCs infected with rAd expressed significantly higher levels of luciferase compared with mature DCs transfected with gene gun (P < .01). The enhancing effect of CD40 signaling was observed in mature DCs transfected with gene gun or rAd by the induction of similar levels of luciferase expression (P > .05). Controls included mature DC transfected and incubated in the presence of hamster antimouse IgM. (B) The efficiency of transfection after CD40 ligation was determined by the expression of cytoplasmic OVA in mature DC gene gun–transfected with pCMV-OVA-489 or infected with rAd-OVA-489. Numbers in graphs are percentages and MFI of OVA+ cells (parentheses). Negative controls included mature DCs transfected with pCMV-LacZ or infected with rAd LacZ and cultured with agonist anti-CD40 mAb and immunostained as DCs transfected with OVA-489. (C) Mature DCs transfected with gene gun showed numerous gold particles in the cytoplasm (arrows). (D-E) Expression of transmembrane OVA (D) or cytoplasmic EGFP (E) by mature DCs 24 hours after gene gun transfection. (F) As negative controls, mature DCs were transfected with pCMV-LacZ and labeled with anti-OVA mAb. (G-H) Enhanced expression of transmembrane OVA (G) or cytoplasmic EGFP (H) by mature DCs 24 hours after gene gun transfection plus agonist anti-CD40 mAb. Transgene expression is observed in DCs with gold particles in the cytoplasm (arrows). (C) Phase-contrast microscopy. (D-H) Fluorescence microscopy. Original magnification × 400. Bar = 10 μm. (I-J) Effect of CD40 signaling of mature DCs on gene expression is maintained for 72 hours and is attributed to the terminal differentiation of mature DCs. (I) Time-point curve showing that the high transgene expression observed in mature DCs transfected with gene gun and signaled through CD40 was sustained up to 72 hours. (J) DC terminal differentiation by CD40 signaling was assessed by the increase in the level of expression of the activation markers CD86 and CD54 in mature DCs, indicated by the significant increase of the MFI (numbers in graphs).

Signaling DCs through CD40 increases transgene expression to protein levels similar to those induced by rAd-Luc. (A) Transgene expression in mature DCs, gene gun–transfected with pCMV-Luc or infected with rAd-CMV-Luc and cultured in the presence or in the absence of the agonist anti-CD40 mAb. Mature DCs infected with rAd expressed significantly higher levels of luciferase compared with mature DCs transfected with gene gun (P < .01). The enhancing effect of CD40 signaling was observed in mature DCs transfected with gene gun or rAd by the induction of similar levels of luciferase expression (P > .05). Controls included mature DC transfected and incubated in the presence of hamster antimouse IgM. (B) The efficiency of transfection after CD40 ligation was determined by the expression of cytoplasmic OVA in mature DC gene gun–transfected with pCMV-OVA-489 or infected with rAd-OVA-489. Numbers in graphs are percentages and MFI of OVA+ cells (parentheses). Negative controls included mature DCs transfected with pCMV-LacZ or infected with rAd LacZ and cultured with agonist anti-CD40 mAb and immunostained as DCs transfected with OVA-489. (C) Mature DCs transfected with gene gun showed numerous gold particles in the cytoplasm (arrows). (D-E) Expression of transmembrane OVA (D) or cytoplasmic EGFP (E) by mature DCs 24 hours after gene gun transfection. (F) As negative controls, mature DCs were transfected with pCMV-LacZ and labeled with anti-OVA mAb. (G-H) Enhanced expression of transmembrane OVA (G) or cytoplasmic EGFP (H) by mature DCs 24 hours after gene gun transfection plus agonist anti-CD40 mAb. Transgene expression is observed in DCs with gold particles in the cytoplasm (arrows). (C) Phase-contrast microscopy. (D-H) Fluorescence microscopy. Original magnification × 400. Bar = 10 μm. (I-J) Effect of CD40 signaling of mature DCs on gene expression is maintained for 72 hours and is attributed to the terminal differentiation of mature DCs. (I) Time-point curve showing that the high transgene expression observed in mature DCs transfected with gene gun and signaled through CD40 was sustained up to 72 hours. (J) DC terminal differentiation by CD40 signaling was assessed by the increase in the level of expression of the activation markers CD86 and CD54 in mature DCs, indicated by the significant increase of the MFI (numbers in graphs).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal