American Society of Hematology

Figure 2.

$Figure 2. TAA/ecdCD40L protein from Ad-sig-TAA/ecdCD40L vector-infected cells binds to and activates DCs, which induce migration to regional lymphoid tissue. (A) Injection of the Ad-sig-E7/ecdCD40L vector generates the release of the E7/ecdCD40L protein around the vector injection site for up to 10 days. Skin section stained by anti-CD154 and DAPI 5 days (i) and 10 days (ii) after injection of the Ad-sig-E7/ecdCD40L vector. (B) Bone marrow-derived DCs release IL-12 and IFN-γ after exposure to the Ad-sig-E7/CD40L Vector. IL-12 (i) or IFN-γ (ii) released by vector-infected DCs into the supernatant medium was measured by ELISA in DCs stimulated for 24 hours (light gray bars) and 48 hours (dark gray bars) with the adenoviral vectors Ad-sig-E7/ecdCD40L, Ad-ecdCD40L, Ad-GFP, Ad-wtCD40L, and AD-E7/wtCD40L. (iii) Semiquantitative RT-PCR reaction was used to measure the levels of E7/CD40L RNA in 293 cells exposed to the Ad-sig-eE7/ecdCD40L vector or the Ad-E7/wtCD40L vector. 293 cells were infected with the vectors Ad-sig-ecdCD40L, Ad-E7/wtCD40L, and Ad-sig-E7/ecdCD40L at an MOI of 10. Then the RNA was isolated and PCR was carried out with primers specific for E7/CD40L mRNA. The cDNA generated was then fractionated on a molecular-weight gel. The electrophoretic species corresponding to the predicted molecular weight of the PCR product from the E7/CD40L template is indicated in the right-hand margin of the gel by the CD40L label. Electrophoretic mobility of a PCR cDNA product using the same RNA but primers specific for GAPDH (loading control) is indicated in the right-hand margin by glyceraldehyde phosphate dehydrogenase (GAPDH). (C) Up-regulation of CCR-7 mRNA in DCs exposed to the Ad-sig-E7/ecdCD40L vector. Lane 1: the Ad-sig-ecdCD40L vector. Lane 2: the Ad-wtCD40L vector. Lane 3: the Ad-sig-E7/ecdCD40L vector. Lane 4: the Ad-E7 vector. Lane 5: the Ad-E7-wtCD40L vector. Lane 6: uninfected cells (control). (D) In vivo study of migration of DCs to regional lymph nodes after loading of DCs with CFDA SE dye and infection with the Ad-sig-E7/ecdCD40L vector. Bone marrow-derived DCs were loaded in vitro with the CFDA SE supravital dye, exposed in vitro to the following vectors at an MOI of 200. (i) Ad-sig-E7/ecdCD40L. (ii) Ad-ecdCD40L. (iii) Ad-E7/wtCD40L. (iv) Ad-wtCD40L. DCs were then injected subcutaneously into the hind flanks of the test mice. Two days later, regional lymph nodes were dissected and frozen sections were studied under a fluorescence microscope. Color micrographs were obtained.$

TAA/ecdCD40L protein from Ad-sig-TAA/ecdCD40L vector-infected cells binds to and activates DCs, which induce migration to regional lymphoid tissue. (A) Injection of the Ad-sig-E7/ecdCD40L vector generates the release of the E7/ecdCD40L protein around the vector injection site for up to 10 days. Skin section stained by anti-CD154 and DAPI 5 days (i) and 10 days (ii) after injection of the Ad-sig-E7/ecdCD40L vector. (B) Bone marrow-derived DCs release IL-12 and IFN-γ after exposure to the Ad-sig-E7/CD40L Vector. IL-12 (i) or IFN-γ (ii) released by vector-infected DCs into the supernatant medium was measured by ELISA in DCs stimulated for 24 hours (light gray bars) and 48 hours (dark gray bars) with the adenoviral vectors Ad-sig-E7/ecdCD40L, Ad-ecdCD40L, Ad-GFP, Ad-wtCD40L, and AD-E7/wtCD40L. (iii) Semiquantitative RT-PCR reaction was used to measure the levels of E7/CD40L RNA in 293 cells exposed to the Ad-sig-eE7/ecdCD40L vector or the Ad-E7/wtCD40L vector. 293 cells were infected with the vectors Ad-sig-ecdCD40L, Ad-E7/wtCD40L, and Ad-sig-E7/ecdCD40L at an MOI of 10. Then the RNA was isolated and PCR was carried out with primers specific for E7/CD40L mRNA. The cDNA generated was then fractionated on a molecular-weight gel. The electrophoretic species corresponding to the predicted molecular weight of the PCR product from the E7/CD40L template is indicated in the right-hand margin of the gel by the CD40L label. Electrophoretic mobility of a PCR cDNA product using the same RNA but primers specific for GAPDH (loading control) is indicated in the right-hand margin by glyceraldehyde phosphate dehydrogenase (GAPDH). (C) Up-regulation of CCR-7 mRNA in DCs exposed to the Ad-sig-E7/ecdCD40L vector. Lane 1: the Ad-sig-ecdCD40L vector. Lane 2: the Ad-wtCD40L vector. Lane 3: the Ad-sig-E7/ecdCD40L vector. Lane 4: the Ad-E7 vector. Lane 5: the Ad-E7-wtCD40L vector. Lane 6: uninfected cells (control). (D) In vivo study of migration of DCs to regional lymph nodes after loading of DCs with CFDA SE dye and infection with the Ad-sig-E7/ecdCD40L vector. Bone marrow-derived DCs were loaded in vitro with the CFDA SE supravital dye, exposed in vitro to the following vectors at an MOI of 200. (i) Ad-sig-E7/ecdCD40L. (ii) Ad-ecdCD40L. (iii) Ad-E7/wtCD40L. (iv) Ad-wtCD40L. DCs were then injected subcutaneously into the hind flanks of the test mice. Two days later, regional lymph nodes were dissected and frozen sections were studied under a fluorescence microscope. Color micrographs were obtained.

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