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Blood, Vol. 113, Issue 18, 4262-4272, April 30, 2009
Antigen mRNA-transfected, allogeneic fibroblasts loaded with NKT-cell ligand confer antitumor immunity
Blood Fujii et al.
113: 4262
Supplemental materials for: Fujii et al
Reagents
The following monoclonal antibodies (mAbs) were purchased from BD PharMingen (San Diego, CA): anti-mouse CD1d (1B1), CD8α (53-6.7), CD11c (HL3), CD19 (1D3), CD40 (3/23), CD70 (FR70), CD80 (16-10A1), CD86 (B7-2), CD119 (GR20), NK1.1(PK136), TCRβ (H57-597), Vα2 TCR (B20.1), H-2Kb (AF16-88.5), I-Ab(KH74), IFN-γ (XMG1.2), IL-4 (11B11), IL-12p40/p70 (C15.6) and mouse IgG1 (A85-1). Biotinylated mAbs were detected with streptavidin-APC. For flow cytometry of OVA257–264 peptide (SIINFEKL)-specific CD8+ T cells, we used Kb/OVA257–264-tetramer-PE (Beckman Coulter Inc., Fullerton, CA). For analysis, FACSCalibur™ instrument and CELLQuest™ (BD Biosciences) or FlowJo (Tree Star, San Carlos, CA) software was used. Cloning of pSP64 poly(A) vector constructs EGFP, OVA and TRP-2 gene
Genes encoding enhanced GFP (EGFP), chicken OVA and TRP-2 were cloned into the vector downstream of the SP64 promoter using HindIII-BamHI sites. All the cDNAs were obtained by PCR. The amplification products were sequenced before insertion into pSP64 vector. To amplify EGFP coding region, the forward primer 5′-CCCCCAAGCTTTCGCCACCATGGTGAGCAAGGGCG-3′ and reverse primer 5′-CCCCCGGATCCTTACTTGTACAGCTCGTCC-3′ were used. For amplification of OVA cDNA we used the pAc-neo-OVA plasmid as a template. The following primers were used to amplify full length chicken OVA gene (NM_205152). The forward primer 1: 5′-CCCCCAAGCTTTCGCCACCCTCGAAAGACAACTCAGAGTTC-3′, the reverse primer 2: 5′-CCCCCGGATCCTCTAGATTAAGCTCCAGC-3′. To amplify full-length cDNA of TRP-2 coding region, B16 melanoma cell line was used as the template. The following primer pairs were used: The forward primer 5′- CCCCCAAGCTTTCGCCACCATGGGCCTTGTGGGATG-3′ and reverse primer 5′- CCCCCGGATCCCTAGGCTTCCTCCGTGTATC-3′. Cytokine assays
ELISPOT assays for IFN-γ secreting iNKT cells were performed on 96-well filtration plates (Millipore, Bedford, MA) coated with rat anti-mouse IFN-γ capture antibody at 10 µg/mL (BD Pharmingen) by culturing with or without α-GalCer for 16 hours as previously described.1 Then, biotinylated anti-mouse IFN-γ detection antibody was added at 2 µg/mL (BD PhaMingen) for 2 hours and spots were developed with an avdin-peroxidase complex (Vectastain Elite Kit, Vector Labs, Burlingame, CA) and stable DAB substrate (Research Genetics, Huntsville, AL). The number of ligand-dependent IFN-γ spots was analyzed with the series 3B ImmunoSpot image Analyzer (Cellular Technology, Cleveland, OH). For intracellular cytokine staining of NK cells by FACS, the cells from spleen were cultured with brefeldin A (BD Bioscience) for 2 hours. Then, these cells were preincubated with 2.4G2 culture medium to block FcγR, washed, stained with anti-NK1.1-APC and anti–CD3-FITC mAb for NK cells for cell surface markers. After the cell surface was labeled with mAbs, cells were permeabilized in Cytofix-Cytoperm Plus (BD Bioscience) and stained with anti–IFN-γ–PE.
To identify T cell specific for OVA antigen as assays, spleen cells were isolated 7 days after immunization. CD8+T cells from spleen cells were purified by positive selection, and were incubated with OVA-peptide-pulsed CD11c+ spleen DCs for 36 hours which had been isolated from C57BL/6 mice. ELISPOT assays for OVA antigen specific IFN-γ secreting cells were performed as described above. Assays for antigen presentation in vivo
We prepared OVA mRNA-NIH3T3 transfectants loaded with or without α-GalCer 2 days before transfection with OVA mRNA. To analyze OVA presentation, mice were adoptively transferred with 2 × 106 OT-I cells, and were immunized with 5 × 105 CD1dhi-NIH3T3/Gal-ova or nothing on the following day. Spleen cells were analyzed 3 days later to measure expanded OT-I cells. CD1dhi-B16/Gal-ova as modified CD1dhi-tumor/Gal was administered to mice as a positive control and OT-I transferred mice were used as a negative control. In some experiments to assess host DC presentation of cell-associated antigens to T cells, CFSE-labeled, OT-I cells were transferred to C57BL/5 mice or CD11c-DTR mice that had been treated with diphtheria toxin (DT) (Sigma-Aldrich, St. Louis, MO) as recipients.2 Subsequently, these mice were immunized with CD1dhi-NIH3T3/Gal-ova. OT-1 cell proliferation was monitored by dilution of CSFE 3 days later. REFERENCES 1. Fujii S, Shimizu K, Kronenberg M, Steinman RM. Prolonged interferon-γ producing NKT response induced with α-galactosylceramide-loaded dendritic cells. Nat Immunol. 2002;3:867–874.
2. Jung S, Unutmaz D, Wong P, et al. In vivo depletion of CD11c+ dendritic cells abrogation priming of CD8+ T cells by exogenous cell-associated antigens. Immunity. 2002;17:211–220.
Files in this Data Supplement:
- Figure S1. Strategy of mRNA expression from antigen cDNA carrying pSP64 poly(A) vector (JPG, 54.3 KB)
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As described in materials and methods, each full length cDNA was subcloned using the pSP64 poly(A) vector. The vector carrying each cDNA was amplified and then linearized. After making Capped-mRNA, Ribo m7G cap analog (Ambion, Austin, TX) was incorporated during the RiboMax transcription reaction for amplifying mRNA with RiboMax Large scale RNA production systems –SP6 (Promega). For the transfection of mRNAs, in vitro transcribed RNAs were transfected into different cell lines with TransMessenger transfection kit (Qiagen) following by the manufacture’s protocols.
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