(a) Representative frequency\histogram flow\cytometry plots showing effect of 5 g/ml UPM on CD1c+ mDC expression of CD83, CCR7, CD40 and HLA\ABC after 20 hr of culture. been investigated previously, but little work has focused on CD8 JAK2-IN-4 T\lymphocyte responses despite their importance in anti\viral immunity. To address this, we examined the effects of UPM Rabbit Polyclonal to EDG4 on DC\stimulated naive CD8 T\cell responses. Expression of the maturation/activation markers CD83, CCR7, CD40 and MHC class I on human myeloid DCs (mDCs) was characterized by flow cytometry after stimulation with UPM in the presence/absence of granulocyteCmacrophage colony\stimulating factor (GM\CSF). The capacity of these mDCs to stimulate naive CD8 T\lymphocyte responses in allogeneic co\culture was then assessed by measuring T\cell cytokine secretion using cytometric bead array, and proliferation and frequency of interferon\(IFN\(IFN\and tumour necrosis factor\(TNF\housekeeping gene was conducted in triplicates by real\time quantitative PCR using Taqman Universal PCR MasterMix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and an Applied Biosystems Viia 7 real\time thermal cycler. Results were analysed using viia 7 software (Applied Biosystems). Taqman primers were purchased from Applied Biosystems. Allogeneic co\cultures After 20 hr of culture, naive CFSE\labelled CD8 T lymphocytes from a different donor were then added at a 1 : 5 ratio of DCs to naive CD8 T cells to produce an allogeneic mixed lymphocyte reaction. On day 5 of co\culture, supernatant was removed and stored at ?20 for subsequent analysis of cytokine protein concentrations, and an aliquot of T cells was removed for flow cytometric analysis of cell proliferation. The DC : T\cell co\cultures were then expanded in fresh media containing 5 U/ml recombinant human interleukin\2 (IL\2) (Eurocetus, Harefield, UK) before performing intracellular cytokine staining 2 days later. Cells were JAK2-IN-4 stimulated for 2 hr with 5 ng/ml PMA and 500 ng/ml Ionomycin (Sigma\Aldrich, St Louis, MO) and then 2 m Monensin was added for a further 2 hr to block cytokine secretion. Cells were incubated with 2 m of 7\aminoactinomycin D (7\AAD; Sigma) to assess their viability before being fixed and permeabilized using BD PermFix (BD Bioscience). The cells were then stained with allophycocyanin\labelled anti\IFN\(BioLegend) for 40 min at room temperature before wash in Cytofix/Cytoperm buffer and then were analysed on an Attune Acoustic Focusing Cytometer (Applied Biosystems). In some experiments cells were additionally stained with phyoerythrin\labelled anti\Granzyme A (BioLegend) and Alexa Fluor 647\labelled anti\Granzyme B (BioLegend) but without CFSE labelling. Flow cytometry data were analysed using flowjo (FlowJo LLC; version 10, San Diego, CA). Cytometric bead array The concentration of cytokines in cell culture supernatants was assessed by Cytometric Bead Array multiple cytokine assay (BD Biosciences). Human exposure studies Sixteen healthy non\smoking volunteers, free from respiratory infection or pre\existing allergic disease were recruited and exposed on two separate occasions, once to filtered air and once to diesel engine exhaust, with exposures separated by at least 3 weeks to limit carry\over effects. Each exposure lasted for 1 hr, during which the subjects alternated between 15 min of rest and exercise (20 l/min/m2 body surface). Diesel exhaust was generated by an idling Volvo diesel engine Volvo (TD45, 45 L, 4 Cylinders, 1991, 680 rpm). JAK2-IN-4 This exposure duplicated an earlier protocol used to investigate the pro\inflammatory nature of diesel exhaust.29 The protocol was approved by the local Ethical Review Board at Ume? University, and performed in accordance with the JAK2-IN-4 Declaration of Helsinki with written informed consent of all participating volunteers. Further information is given in the Supplementary material (Appendix S1). Bronchoscopy was performed 6 hr after the diesel and filtered air exposures using a flexible video bronchoscope (Olympus BF IT160, Japan) with proximal and lower airway samples obtained by bronchial wash (BW, 2 20 ml) and bronchoalveolar lavage (bronchoalveolar (BAL), 3 60 ml) respectively, using sterile saline. Granzyme A was determined in cell\free BW and BAL fluid samples using a commercial ELISA kit (BioVendor, Brno, Czech Republic). Messenger RNA was generated from BAL leucocytes using the Qiagen RNeasy Mini Kit and the Superscript III First\Strand Synthesis System for quantitative PCR kit from Invitrogen Technologies (Paisley, UK), following the manufacturer’s instructions. Glyceraldehyde 3\phosphate dehydrogenase (GAPDH) was selected as a reference gene for this data set. BAL cell cytospins were fixed in 2% paraformaldehyde and after washing, were used for immunocytochemistry analysis.
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