1. Volume 13, Issue 2, Pages 301-309 (February 2006)
Potent Vaccine Therapy with Dendritic Cells Genetically Modified by the Gene- Silencing-Resistant Retroviral Vector GCDNsap 
Tsukasa Nabekura, Makoto Otsu, Toshiro Nagasawa, Hiromitsu Nakauchi, Masafumi Onodera 
Molecular Therapy 
Volume 13, Issue 2, Pages (February 2006)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Structure of the retroviral vector GCDN/OVA and characteristics of the OVA-transduced DCs. (A) The retroviral vector GCDNsapI/E contains the PCMV LTR with intact splice donor and splice acceptor sequences and the EGFP cDNA downstream of the internal ribosome entry site. The OVA cDNA was inserted between the BamHI and the XhoI sites to generate GCDN/OVA. Sequences present in the vector are labeled as follows: MoMLV, Moloney murine leukemia virus LTR; PCMV, PCC4 cell-passaged myeloproliferative sarcoma virus LTR; ψ+, packaging signal; S.D., splice donor; S.A., splice acceptor; IRES, internal ribosome entry site. (B) Morphological appearance of OVA-transduced DCs. The DCs were cytospun followed by May–Gruenwald–Giemsa staining. The scale bar represents 20 μm. (C) Surface markers and transduction efficiency of the transduced DCs. Transduction efficiency of DCs was determined by EGFP expression. Representative data are shown. (D) lacZ activation of B3Z by OVA-transduced DCs. Target cells (▵, EL4; ▴, E.G7-OVA; □, DCs; ▪, OVA peptide-pulsed DCs; ♦, OVA-transduced DCs) were incubated with 1 × 105 B3Z (effector cells) at various cell ratios as indicated. Absorption of each well at OD570 is indicated. *P < 0.01, compared with values for EL4 and DCs. n = 5 to 7.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Active immunotherapy with OVA-transduced DCs against the OVA-expressing tumor E.G7-OVA. (A) Mice left untreated (○) or inoculated with 5 × 105 OVA peptide-pulsed DCs (▪) or OVA-transduced DCs (♦) on Days −14 and −7 were implanted with 5 × 106 E.G7-OVA on Day 0. The tumor size was measured over time and represented as the tumor index. Results from four mice per group are presented. *P < 0.05 compared with the untreated group. (B) Mice left untreated (○) or inoculated with 5 × 105 OVA-transduced DCs (♦) on Days −14 and −7 were implanted with 5 × 106 EL4 on Day 0.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Induction of cellular immunity against E.G7-OVA. (A) The number of splenic CD8+ T cells recognizing the OVA peptide (SIINFEKL) in mice inoculated with OVA peptide-pulsed DCs (□) or OVA-transduced DCs (♦) was measured by FACS with anti-CD8α antibody and OVA peptide tetramer. The y axis indicates the percentage of the peptide-specific CD8+ T cells in mice inoculated with the number of DCs indicated on the x axis. (B) The prevalence of total CTLs against EL4 or E.G7-OVA. Target cells (2 × 104 EL4/EGFP or E.G7-OVA/EGFP) were incubated with 5 × 104 splenic CD8+ T cells from untreated mice (solid bars), mice treated with the peptide-pulsed DCs (dotted bars), or mice treated with the transduced DCs (hatched bars). Representative data from one of three independent experiments are shown.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Involvement of OVA-specific CD4+ T cells in the anti-tumor effects. (A) IgG subclasses against OVA in mice with the indicated immunization were measured by enzyme-linked immunosorbent assay (OD450). Sera of mice immunized with OVA mixed with Freund's adjuvant (OVA + FA) were used as a control. (B) OVA-specific Th1 was detected by Th1 intracellular cytokine staining. Splenic CD4+ T cells from mice immunized with or without OVA-transduced DCs were cocultured with the DCs, and intracellular Th1 cytokines were stained with anti-IL-2 or anti-interferon-γ antibody. The patterns are shown for CD4+ gated cells. (C) Anti-tumor effects in MHC class II-deficient mice immunized with (⋄, n = 6) or without (○, n = 8) 1 × 105 OVA-transduced DCs (Days −14 and −7) were compared with those in wild-type mice immunized with (♦, n = 8) or without (●, n = 8) the DCs. The tumor size of the mice was measured after implantation of 5 × 106 E.G7-OVA. *P < 0.05 compared with the nonimmunized MHC class II-deficient mice group. **P < 0.01 compared with the other groups. In the immunized wild-type mice group, three mice completely rejected E.G7-OVA.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Eradication of the preexisting tumor by transduced DC vaccine. Mice implanted with 2 × 106 E.G7-OVA on Day −3 or −2 were left untreated (•, n = 2) or inoculated with 5 × 105 transduced DCs (♦, n = 4). Transduced DCs were injected peritumorally on Days 0, 2, and 4. Mice inoculated with transduced DCs were again implanted with 2 × 106 E.G7-OVA on Day 79 (arrow).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions