American Society of Hematology

Figure 1.

$TAA/ecdCD40L protein produced by Ad-sig-TAA/ecdCD40-infected cells binds to DCs. (A) Proposed mechanism for induction of immune response by the Ad-sig-TAA/CD40L vector. Injecting Ad-sig-TAA/ecdCD40L induces in vivo activation and tumor-antigen loading of DCs, migration of the DCs to regional lymph nodes, and activation of CD8+ cytotoxic T cells, which are specific for cells carrying the tumor antigen. (B) In vitro expression of the E7/ecdCD40L transcription unit. Plasmid expression vectors encoding the nonsecretable E7/wtCD40 ligands (lane 1), the secretable ecd of the CD40 ligand (sig-ecdCD40L) alone (lane 2), and the secretable sig-E7/ecdCD40 ligand protein (lane 3) produced in a cell-free transcription/translation system are as predicted: lane 1, E7/wtCD40L is 39 kDa; lane 2, sig-ecdCD40L is 22 kDa; and lane 3, sig-E7/ecdCD40L is 32 kDa. Molecular weight markers are in the extreme right lane. (C) Western blot analysis of the expression of E7/ecdCD40L protein in 293 cells. Molecular weights of the TAA/ecdCD40L proteins produced from 293 cells infected by the Ad-sig-TAA/ecdCD40L vectors adenoviral vectors were as predicted: lane 1, lysates from cells infected with the Ad-sig-GFP/ecdCD40L vector; lane 2, lysates from cells infected with the Ad-sig-E7/ecdCD40L vector; lane 3, lysates from cells infected with the Ad-sig-ecdCD40L vector; and lane 4, lysates from the Ad-sig-ecdhMUC1/ecdCD40L vector. Molecular weight markers are in the extreme right lane. (D) Secretory form of TAA/ecdCD40L binds in vitro to DCs. Bone marrow-derived DCs were fractionated to 78% purity. (i-ii) FITC-labeled E7/ecdCD40L recombinant proteins released from Ad-sig-E7/ecdCD40L-infected 293 cells were incubated with bone marrow-derived DCs. Cells were portioned with light microscopy (left panels) to demonstrate the morphology of the DCs and then with fluorescence microscopy (right portion panels) to detect the binding of the fluoresceinated proteins. (i) DCs incubated with FITC-labeled proteins from the supernatant of cells infected with the Ad-sig-E7/ecdCD40L. (ii) DCs incubated with FITC-labeled proteins from the supernatants of cells infected with the Ad-sig-ecdCD40L vector. (iii-v) Proteins released from Ad-sig-ecdhMUC-1/ecdCD40L-infected 293 cells were fractionated on a Nickel column to purify the His-tagged ecdhMUC-1/ecdCD40L proteins. These proteins were fluorescein labeled, as outlined in “Materials and methods.” FITC-labeled ecdhMUC-1/ecdCD40L proteins and a PE-conjugated rat antimouse CD11C antibody were added to the purified DCs. (iii) Cells exposed to a laser excitatory for phycoerythrin. (iv) Cells exposed to a laser excitatory for FITC. (v) Overlay of the images from subpanels iii and iv. A Nikon Eclipse TE-2000-U microscope, which was equipped with a Perkin Elmer UltraView R55 spinning disk confocal attachment, was used at 20 × N.A. 0.5. Adobe Photoshop was the software used.$

TAA/ecdCD40L protein produced by Ad-sig-TAA/ecdCD40-infected cells binds to DCs. (A) Proposed mechanism for induction of immune response by the Ad-sig-TAA/CD40L vector. Injecting Ad-sig-TAA/ecdCD40L induces in vivo activation and tumor-antigen loading of DCs, migration of the DCs to regional lymph nodes, and activation of CD8⁺ cytotoxic T cells, which are specific for cells carrying the tumor antigen. (B) In vitro expression of the E7/ecdCD40L transcription unit. Plasmid expression vectors encoding the nonsecretable E7/wtCD40 ligands (lane 1), the secretable ecd of the CD40 ligand (sig-ecdCD40L) alone (lane 2), and the secretable sig-E7/ecdCD40 ligand protein (lane 3) produced in a cell-free transcription/translation system are as predicted: lane 1, E7/wtCD40L is 39 kDa; lane 2, sig-ecdCD40L is 22 kDa; and lane 3, sig-E7/ecdCD40L is 32 kDa. Molecular weight markers are in the extreme right lane. (C) Western blot analysis of the expression of E7/ecdCD40L protein in 293 cells. Molecular weights of the TAA/ecdCD40L proteins produced from 293 cells infected by the Ad-sig-TAA/ecdCD40L vectors adenoviral vectors were as predicted: lane 1, lysates from cells infected with the Ad-sig-GFP/ecdCD40L vector; lane 2, lysates from cells infected with the Ad-sig-E7/ecdCD40L vector; lane 3, lysates from cells infected with the Ad-sig-ecdCD40L vector; and lane 4, lysates from the Ad-sig-ecdhMUC1/ecdCD40L vector. Molecular weight markers are in the extreme right lane. (D) Secretory form of TAA/ecdCD40L binds in vitro to DCs. Bone marrow-derived DCs were fractionated to 78% purity. (i-ii) FITC-labeled E7/ecdCD40L recombinant proteins released from Ad-sig-E7/ecdCD40L-infected 293 cells were incubated with bone marrow-derived DCs. Cells were portioned with light microscopy (left panels) to demonstrate the morphology of the DCs and then with fluorescence microscopy (right portion panels) to detect the binding of the fluoresceinated proteins. (i) DCs incubated with FITC-labeled proteins from the supernatant of cells infected with the Ad-sig-E7/ecdCD40L. (ii) DCs incubated with FITC-labeled proteins from the supernatants of cells infected with the Ad-sig-ecdCD40L vector. (iii-v) Proteins released from Ad-sig-ecdhMUC-1/ecdCD40L-infected 293 cells were fractionated on a Nickel column to purify the His-tagged ecdhMUC-1/ecdCD40L proteins. These proteins were fluorescein labeled, as outlined in “Materials and methods.” FITC-labeled ecdhMUC-1/ecdCD40L proteins and a PE-conjugated rat antimouse CD11C antibody were added to the purified DCs. (iii) Cells exposed to a laser excitatory for phycoerythrin. (iv) Cells exposed to a laser excitatory for FITC. (v) Overlay of the images from subpanels iii and iv. A Nikon Eclipse TE-2000-U microscope, which was equipped with a Perkin Elmer UltraView R55 spinning disk confocal attachment, was used at 20 × N.A. 0.5. Adobe Photoshop was the software used.

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