In Vivo Generation of Dendritic Cells by Intramuscular Codelivery of FLT3 Ligand and GM-CSF Plasmids  Yoav Peretz, Zheng Frank Zhou, Fawaz Halwani, Gérald J. Prud'homme  Molecular Therapy  Volume 6, Issue 3, Pages 407-414 (September 2002) DOI: 10.1006/mthe.2002.0677 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 Serum concentrations of FLT3-L and GM-CSF. Mice were coinjected intramuscularly with VR-FLT3-L and VR-GM-CSF and serum was recovered between 0 and 14 days. (A) GM-CSF level and (B) FLT3-L level. Each point represents the mean ± SD (n = 3). a,bP < 0.05 versus VR-Blank. Molecular Therapy 2002 6, 407-414DOI: (10.1006/mthe.2002.0677) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Increase in total splenocytes and splenic DC populations following intramuscular plasmid injection. (A) Percent increase in splenocyte numbers versus untreated mice on day 7. The data from each group of mice inoculated with plasmids as labeled, represent the mean ± SD (n = 3) of one representative experiment. aP < 0.05 versus VR-Blank. bP < 0.01 versus either VR-Blank or VR-FLT3-L. (B) Representative flow cytometric histograms of CD11c-, MHC II-, CD8α-, CD11b-, and F4/80-stained splenocytes from mice injected with plasmids as denoted on the histograms. All horizontal axes represent CD11c staining. Percent values from the total splenocyte population are also listed in the relevant quadrants. (C) Increase in numbers of CD11c+/MHC II+ DCs per spleen expressed as a percent or total cell number. aP < 0.005 versus either no injection or VR-Blank. bP < 0.001 versus either no injection, VR-Blank, or VR-FLT3-L. cP < 0.001 versus either no injection, VR-Blank, VR-FLT3-L, or VR-GM-CSF. Molecular Therapy 2002 6, 407-414DOI: (10.1006/mthe.2002.0677) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 Total number of CD11c+/MHC II+ DCs over time. A single intramuscular injection of VR-Blank or a single coinjection of VR-FLT3-L with VR-GM-CSF was performed. Each point represents the mean number of cells per spleen ± SD (n = 3). a,bP < 0.05 versus VR-Blank. Molecular Therapy 2002 6, 407-414DOI: (10.1006/mthe.2002.0677) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 Augmentation of myeloid-type and lymphoid-type DCs. Mice were injected once intramuscularly with either VR-Blank, VR-FLT3-L, or VR-GM-CSF, or were coinjected once with VR-FLT3-L and VR-GM-CSF. The data from each group represent the mean ± SD (n = 3) for one representative experiment. (A) Lymphoid-type (CD11c+/CD8α+) DCs, total number and percent. aP < 0.01 versus either no injection, VR-Blank, VR-FLT3-L or VR-GM-CSF. bP < 0.001 versus either no injection, VR-Blank, or VR-GM-CSF. (B) Myeloid-type (CD11c+/CD11b+) DCs, total number and percent; or DCs expressing F4.80, total number and percent. aP < 0.001 versus either no injection, VR-Blank, VR-FLT3-L, or VR-GM-CSF. bP < 0.001 versus either no injection, VR-Blank, or VR-FLT3-L. cP < 0.001 versus either no injection, VR-Blank, VR-FLT3-L, or VR-GM-CSF. dP < 0.001 versus either no injection, VR-Blank, or VR-FLT3-L. Molecular Therapy 2002 6, 407-414DOI: (10.1006/mthe.2002.0677) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Maturity markers of CD11c+ DCs. Representative flow cytometry histograms of the expression levels of (A) MHC II, (B) B7.1, (C) B7.2, and (D) CD40 on gated CD11c+ splenocytes of mice treated with FLT3-L and GM-CSF cDNA versus untreated (control) mice. Molecular Therapy 2002 6, 407-414DOI: (10.1006/mthe.2002.0677) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 6 Stimulation of MLC by DCs. CD11c+ splenic DCs from C57BL/6 treated mice were positively selected and irradiated. Varying numbers of DCs were cultured for 3 days with 1 × 105 allogeneic CD4+ T cells (BALB/cJ). DCs were obtained from untreated mice, or 7 days after inoculation with VR-Blank, VR-FLT3-L, VR-GM-CSF, or VR-FLT3-L and VR-GM-CSF. T-cell proliferation was monitored by [3H]thymidine incorporation. Each point represents the mean cpm ± SEM of triplicate cultures. aP< 0.005 versus VR-FLT3-L and VR-GM-CSF. Molecular Therapy 2002 6, 407-414DOI: (10.1006/mthe.2002.0677) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 7 Concentration of IL-12 (p70) in MLC culture supernatants. CD11c+ splenic DCs from C57BL/6-treated mice were positively selected and irradiated. DCs (104) isolated from mice treated with VR-FLT3-L or VR-GM-CSF were cultured for 24, 48, and 72 hours with 105 allogeneic CD4+ T cells (BALB/cJ). Culture supernatants were harvested and assayed by ELISA to determine the IL-12 concentration. DCs alone did not produce IL-12 (data not shown). Results represent mean levels ± SEM of triplicate cultures. Molecular Therapy 2002 6, 407-414DOI: (10.1006/mthe.2002.0677) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 8 Presentation of soluble antigen (OVA) by DCs. CD11c+ splenic DCs from BALB/cJ treated mice were positively selected and irradiated. Varying numbers were cultured for 5 days with OVA and 1 × 105 syngeneic CD4+ T cells from OVA-TCR transgenic DO11.10 mice. DCs were obtained from untreated mice, or 7 days after injection of VR-Blank or VR-FLT3-L and VR-GM-CSF. T cell proliferation was monitored by [3H]thymidine incorporation. Each point represents the mean CPM ± SEM of triplicate cultures. aP < 0.05 versus untreated mice. Background counts were < 300 cpm in cultures maintained without OVA (data not shown). Molecular Therapy 2002 6, 407-414DOI: (10.1006/mthe.2002.0677) Copyright © 2002 American Society for Gene Therapy Terms and Conditions