DNA vaccines targeting tumor antigens to B7 molecules on antigen-presenting cells induce protective antitumor immunity and delay onset of HER-2/Neu-driven mammary carcinoma. to express indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme that Indisulam (E7070) induces and activates Treg cells (16). Since CTLA-4-Ig has the potency to induce Treg cells, we investigated whether the OVA-CTLA-4 DNA vaccine Indisulam (E7070) encoding fusion protein OVA-CTLA-4 in the present study binds to the B7 ligand on DCs and induces the generation of Treg cells. To establish whether Treg cells play a role in allergen-CTLA-4-encoding DNA vaccination, we also measured the percentages of Treg cells in the spleens of vaccinated mice. MATERIALS AND METHODS Animals. Male BALB/c mice (6 to 10 weeks old) and weighing 18 to 22 g were purchased Indisulam (E7070) from the Animal Centre at Jinling Hospital. Animal care and experimental procedures were performed in accordance with the animal ethics regulations of the Institutional Animal Care and Use Committee. Construction of the OVA-CTLA-4-pcDNA3.1 immunization vector. OVA and a DNA fragment encoding the extracellular domain name of mouse CTLA-4 were amplified by PCR. The two fragments were inserted into a pcDNA3.1(+) vector to generate the recombinant plasmids OVA-pcDNA3.1 and OVA-CTLA-4-pcDNA3.1. The resulting plasmids were then sent to ShengXing Corp. (Nanjing, China) for DNA sequencing. Sequencing confirmed that the two plasmids had been constructed successfully. The large-scale purification of plasmids was conducted by using a Maxiprep GFII Endo-Free kit (Qbiogene, Inc., Montreal, Quebec, Canada) according to the manufacturer’s instructions. Expression and purification of ectopic protein. COS-7 cells were seeded in 100-mm culture flasks (2 106 cells/flask) in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (Gibco, Grand Island, NY). The expression plasmids OVA-pcDNA3.1 and OVA-CTLA-4-pcDNA3.1 were transfected into COS-7 cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After 72 h, the supernatants were collected, and ectopic protein OVA and OVA-CTLA-4 were purified from the culture supernatants by using anti-OVA antibodies (Zhongshan Co., Beijing, China) coupled to CNBr-Sepharose 4B affinity chromatography (Amersham Biosciences, Piscataway, NJ). The purified protein OVA and OVA-CTLA-4 were confirmed by Western blotting with anti-OVA antibodies. Detection of OVA-CTLA-4 binding to B7 ligands on DCs for 15 min). Anti-CD80 or anti-CD86 antibody was added to the diluted samples, and after overnight incubation at 4C protein A-coupled agarose beads (15 l) were added, followed by further incubation for at least 8 h at 4C. After three washes with Tris buffer, the beads were suspended in SDS-PAGE loading buffer (0.125 M Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 0.1 M dithiothreitol, 0.01% bromphenol blue) and boiled for 5 min, a process which also cleaves the cross-linker DSP. After centrifugation at 14,000 for 5 Indisulam (E7070) min, the supernatants were analyzed by Western blotting with anti-OVA antibody. Induction of Foxp3+ Treg cells by OVA-CTLA-4 = 6 mice per group) as follows: (i) mice treated and sensitized and challenged with phosphate-buffered saline (PBS) (control); (ii) mice treated with PBS and sensitized and challenged with OVA (model); (iii) mice treated with the pcDNA3.1 plasmid (100 g/mouse) and sensitized and challenged with OVA (pcDNA3.1); (iv) mice treated with OVA-pcDNA3.1 (100 g/mouse) and sensitized and challenged with OVA (OVA-pcDNA3.1); (v) mice treated with a low dose of OVA-CTLA-4-pcDNA3.1 (50 g/mouse) and sensitized and challenged with OVA [OVA-CTLA-4-pcDNA3.1(L)]; and (vi) mice treated with a high dose of OVA-CTLA-4-pcDNA3.1 (100 g/mouse) and sensitized and challenged with OVA [OVA-CTLA-4-pcDNA3.1(H)]. The Col1a1 establishment of the mouse allergic asthma model and the vaccination protocols have been described previously (14). In brief, mice were sensitized to OVA (grade V; Sigma-Aldrich, St. Louis, MO) by intraperitoneal injection of 100 l of alum-precipitated antigen comprising 10 g of OVA assimilated onto 4 mg of aluminum potassium sulfate (Pierce Biotechnology, Rockford, IL) on day 0, followed by inhalation of 1% OVA (grade II; Sigma-Aldrich) diluted in PBS for 30 min on days 8 and 9. The mice were then vaccinated with PBS, pcDNA3.1, OVA-pcDNA3.1, or OVA-CTLA-4-pcDNA3.1 on days 10 and 25. On day 39 the mice were exposed to aerosol challenge with 1% OVA (grade II) diluted in PBS for 30 min (Fig. 1). At 24 h after the final challenge, blood, bronchoalveolar lavage fluid (BALF), lungs, and spleens were harvested for further analysis. Open in a separate window Fig. 1. Immunization protocol. Histological analysis of lung tissue. At 24 h after the final allergen challenge, the excised lungs were harvested and.