Despite significant attractiveness of anti-sense oligonucleotide/RNAi technology, its clinical application has
Despite significant attractiveness of anti-sense oligonucleotide/RNAi technology, its clinical application has been precluded by a lack of methods for targeted delivery and transduction of primary immune cells in vivo. Midland Certified Reagent Company (Midland, TX); Ambion in vivo siRNA to Roscovitine mouse IL10 (s68180), FoxP3 (A, s73597 and B, s73595) and control siRNA (in vivo ready) were from Ambion Products (Austin, TX). The following antibodies were used: anti-mouse CD4-FITC, anti-mouse CD25-PE (Biolegend) and anti-mouse Foxp3-APC (eBioscience); rabbit anti-mouse CCR4 Ab (Capralogics, Biolegend); anti-human CCL17 Ab (Abcam, ab9816), Fc blocker (anti-CD16/32; BD Biosciences). Chemoarp production TARC-arp and RANTES-arp (collectively named chemoarp) encode mature sequences of human chemokines CCL17 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002987″,”term_id”:”22538801″,”term_text”:”NM_002987″NM_002987) and CCL5 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002985″,”term_id”:”22538813″,”term_text”:”NM_002985″NM_002985) fused in frame with hypothetical single DNA/RNA-binding domain (RBD) of HBcAg of HBV  (Arya et al., Patent is pending). TARC-FN was created from TARC-arp by replacing RBD with irrelevant peptide of the same size. Coding sequences of chemoarps were cloned using XhoI and NotI restriction sites under signal sequence of yeast -factor into pPIC9 plasmid (Invitrogen). All Rabbit Polyclonal to NDUFS5 constructs were verified by DNA sequencing (Keck DNA Sequencing Lab, New Haven, CT). Chemoarps were produced using methanol-inducible Pichia expression kit (Invitrogen) in GS115 following manufacturers instructions. Briefly, after one week of methanol induction, culture chemoarp-containing supernatants were collected by centrifugation at 3000C5000g and filtered through 0.25 filter. Then, chemoarp was purified using SP-Sepharose? Fast Flow and Heparin-HP trap column chromatography (GE Healthcare) with Fast performance liquid chromatography (FPLC) (Bio-Rad BioLogic Duoflow). Chemoarp was eluted using NaCl gradient elution in 20 mM Na-phosphate buffer, pH8.0. Chemoarp-containing fractions were combined and dialysed against PBS in dialysis chambers with 3000 cutoff (Pierce, Thermo Fisher Scientific Inc.). Purity of proteins was (>95%), as verified by Coomassie Blue staining and western blotting with respective antibodies. Cells and mice The 4T1 mouse mammary carcinoma cells (CRL-2539), human acute T-lymphoblastic leukemia cell lines CCRF-CEM (CEM, CCL-19) were from the American Type Culture Collection, Rockville, MD); 4T1.2 is a single cell subclone of 4T1 cells and a gift from Dr. Robin L. Anderson (Peter McCallum Cancer Center, Australia). Cells were cultured in RPMI 1640 (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum, HEPES-Sodium Pyruvate, non-essential amino acids, 2-Mercaptoethanol, L-glutamine, and Penicillin-Streptomycin (complete RPMI). Mouse CD3+ T cells were isolated from na?ve mouse spleen using T cell enrichment columns (R&D Systems, Minneapolis, MN). To generate non-Tregs (purity >99.5 %), CD4+ T cells were isolated by CD4 negative selection kit (Miltenyi Biotec Inc) and separated from CD25+ cells using CD25 Microbead kit (Miltenyi Biotec Inc). The CD25+CD4+ cells were used as Tregs. The lung mononuclear cells were isolated using Ficoll density separation after digesting lungs with collagenase/DNase/elastase mixture (Sigma). In vitro siRNA manipulations siRNA binding was evaluated by incubating 2 pmol siRNA with serial dilutions of TARC-arp in PBS on ice for 15 min. Upon binding with TARC-arp, siRNA losses ability to be separated by electrophoresis in 2% ethidium bromide stained agarose gel in TAE buffer (Invitrogen). To evaluate siRNA uptake, 4T1 cells (20,000 cells/well) were treated with 20 pmol of Invitrogens BLOCK-iT? Alexa Fluor? Red Fluorescent Oligo (Invitrogen) complexed with TARC-arp for 18 hours at 37C. After washing 3 times in PBS to remove free siRNA, fluorescence was visualized using a Zeiss Axiovert 200 microscope (Carl Zeiss, Heidelberg, Germany). Images were processed using NIH ImageJ software. Lipofectamine-2000 (Invitrogen) C mediated siRNA transfection was used as the positive control. Viability of 4T1.2 cells was tested using WST-1 assay (Roche) following manufacturers instructions. In brief, titrated amounts of TARC-arp (5C25 g/ml, with or without siRNA) were in vitro cultured with 20,000 cells in triplicate 96-well plates in cRPMI for 24, 48 and 72h. To inhibit Roscovitine gene expression, indicated amounts of siRNA and TARC-arp were mixed in PBS and pre-incubated on ice for 15C30 min prior adding onto cells. For example, 6 g/ml TARC-arp complexed with 18 g/ml siRNA was incubated with freshly isolate 50,000C100,000 CD4+ T cells/50 l/well in serum-free RPMI for 1h at Roscovitine 37C. Then, after.