PMC:2193592 / 3231-9765 JSONTXT

{"project":"2_test","denotations":[{"id":"11828008-8145033-59569240","span":{"begin":1581,"end":1582},"obj":"8145033"},{"id":"11828008-3944470-59569241","span":{"begin":4840,"end":4842},"obj":"3944470"}],"text":"Materials and Methods\n\nAntibodies and Blocking Reagents.\nAntibodies used for flow cytometry were: anti-CD86-FITC, -CD83-PE, -HLA-DR-PerCP, -CD69-FITC or -PE, -CD25-FITC, -CD3-FITC or -PE, -CD19-FITC, -CD14-FITC, -CD1a-FITC, -CD80-FITC or -PE, and -CD56-FITC or -PE (Becton Dickinson). Staining of cells was performed according to standard protocols and flow cytometric analysis was performed using a FACSCalibur™ cytometer. Antibodies used for magnetic activated cell sorting (MACS®) and subset purifications were: anti-CD3, anti-CD14, and anti-CD19 (Becton Dickinson). Blocking/neutralizing reagents used in different experiments were: anti–IFN-α (mAb MMHA-1 and sheep polyclonal anti–human; Ancell); anti–IFN-β (sheep polyclonal anti–human; Biosource International); r-hu-fas-ligand/Fc chimera (CD178; R\u0026D Systems); hu-CTLA-4 muIg fusion protein (CD152; Ancell); anti–LFA-1 (CD11a, mAb G43–25B; Becton Dickinson); anti–TNF-α (mAb B154.2); anti–IL-12 p40 (mAb C8.6); and anti–IFN-γ (mAb B133.3) (all provided by G. Trinchieri, Schering Plough Corp., Lyon, France); humanized anti-CD40 (mAb 5H7; supplied by K. Chu, Chiron Corp., Emeryville, CA). All neutralizing antibodies and reagents were used at, at least, two different concentrations which were equal to and exceeded the manufacturers' instructions or their previously published levels for neutralization.\n\nCell Preparations and Cultures.\nPBMCs were prepared from healthy blood donors (buffy coats or lab volunteers) by separation on a Ficoll-Hypaque gradient. iDCs were generated as described by Sallusto and Lanzavecchia (8). Briefly, highly purified (\u003e98%) monocyte populations were obtained from PBMCs by MACS® (Miltenyi Biotec), using a one step positive selection of CD14+ cells with anti-CD14–coated magnetic microbeads (Miltenyi Biotec). The monocytes were then cultured for 5–6 d in RPMI 1640 medium (Life Technologies) supplemented with 10% FCS (Hyclone); IL-4 (10% of supernatant from an IL-4–secreting cell line, provided by A. Lanzavecchia, Institute for Research in Biomedicine, Bellinzona, Switzerland) and 50 ng/ml of GM-CSF (Leucomax®). After culture the iDC populations were routinely checked for CD1a and CD80 expression and their lack of CD14 expression. Purified (85–95% CD56+/CD3−), resting NK cells were obtained from PBMCs by MACS® (Miltenyi Biotec) using a two step protocol: PBMCs were first incubated with purified anti-CD3, anti-CD14, and anti-CD19 mAb's (Becton Dickinson) for 30 min followed by negative selection with goat anti–mouse microbeads (Miltenyi Biotec). After which the NK cells (which contained \u003c1% contamination with CD3+, CD19+, and CD14+ cells but between 5–15% lineage negative cells) were frozen in 90% FCS; 10% DMSO for 5–6 d. Prior to their use in experiments with cultured, autologous iDCs, they were gently thawed, washed, and resuspended in RPMI 1640/10% FCS medium (RPMIc). Purified (99% CD56+/CD3−), activated NK cells were obtained using the method of Perussia et al. with slight modification (9). Briefly, PBMCs were cultured with the irradiated (5,000 rads) EBV-transformed B cell line, RPMI 8866, in 24-well plates at a ratio of 5:1 (PBMC:8866). For experiments with buffy coats the PBMCs were cultured for 5–6 d, whereas for PBMCs from lab volunteers who donated blood twice (first for NK cell cultures and then for iDC cultures), they were cultured for 8 d under the published conditions (9). After culture, purified NK cells were obtained by negative selection by MACS® (Miltenyi Biotec) as described above for resting NK cells.\n\nNK–iDC Cultures.\nThe specific details of the different experiments are described in the figure legends. In general, activated/cultured or resting NK cells were added to autologous iDCs at the ratios (NK/DC) indicated and the cells were cocultured in RPMIc medium for 24–48 h in the presence or absence of the indicated doses of LPS (Sigma-Aldrich). Prior to coculture, the iDCs were removed from their GM-CSF/IL-4 containing medium by washing with RPMI 1640 medium. Control experiments comparing NK activation or iDC maturation in the presence or absence of the DC culture medium (GM-CSF/IL-4) showed no differences.\n\nMeasurement of Cytokine Production.\nCytokine production was quantified in culture supernatants by specific sandwich ELISA's for TNF-α (capture/secondary; mAb's B154.9/B154.7), IL-12 p40 (mAb's C11.79/C8.6), and IL-10 (mAb's JES3–907/JES3–12G8). The antibodies for TNF-α and IL-12 p40 ELISA were provided by G. Trinchieri and those for IL-10 were obtained from BD PharMingen. Recombinant standards for all assays were obtained from R\u0026D Systems.\n\nCell-mediated Cytotoxicity Assays.\nNK cell killing of iDCs or K562 was determined using standard 4-h chromium release assays and a flow cytometric assay for NK cell killing developed by McGinnes et al. with slight modifications (10). Briefly, iDCs or K562 cells were loaded with 5 μM CFDA SE for 5 or 15 min, respectively, and incubated in 96-well plates (U-bottom; Costar) with activated/cultured NK cells at the indicated NK/target ratios. After the indicated times (4 or 24 h) each sample was resuspended in a final volume of 600 μl of PBS, to which 50 μl of Flow Check fluorospheres (Beckman Coulter) were added. Using the cytometer, a fixed number (700) of fluorescent beads was acquired and the percentage of killing was calculated using the following equation: (the number of CFDA SE+ cells acquired in the control without NK cells) − (the number of CFDA SE+ cells in the sample)/(the sample of CFDA SE+ cells in the control) × 100. Comparison of 4-h chromium release assays with the 4-h flow cytometric procedure revealed minor differences with the flow cytometric technique being more sensitive (unpublished data).\n\nNK Cell Conjugate Formation Assays.\nNK–iDC or NK-K562 conjugates were detected by flow cytometry using NK cells labeled with CFDA SE (Molecular Probes) and iDCs or K562 labeled with SNARF-1 (Molecular Probes). After dye loading (NK cells and K562, 15 min at 37°C with 5 μM of CFDA SE or 2 μM SNARF-1, respectively, and iDCs, 5 min at 37°C with 2 μM of SNARF-1) the cells were washed and incubated alone or together (NK–K562 or NK–iDC) at the indicated ratios for 30 min at 37°C. Prior to incubation the cells were centrifuged at 200 g for 3 min at room temperature. The percentage of conjugates formed was calculated on a FACSCalibur™ cytometer (Becton Dickinson) by gating on the FL3+ cells (SNARF-1/iDCs or K562) and determining the percent of this population also positive for FL1 (CFDA SE/NK).\n"}