Interleukin 6 (il 6)

Human monocytes from nine human leukocyte antigen (HLA) -A2, and two non-HLA-A2 healthy donors were obtained from the NIH Blood Bank. The monocytes were cryopreserved in liquid N2 until use. To generate dendritic cells, the cryopreserved monocytes were thawed, put in tissue culture plate for 4 hrs at 37°C. The non-adherent cells were removed by washing with phosphate buffered saline (PBS). The adherent cells were cultured in complete RPMI 1640 medium (Mediatech, Inc. Manassas, VA) supplemented with human GM-CSF (1000 U/ml, BD Biosciences, San Jose, CA) for two days to generate pre-immature dendritic cells (piDC) or with human GM-CSF (1000 U/ml) and IL-4 (50 ng/ml, BD Biosciences) for 4 days to generate immature dendritic cells (iDC), or with human GM-CSF (1000 U/ml) and IL-4 (50 ng/ml) for 4 days and additional 2 days with TNFα (20 ng/ml, BD Biosciences) and CD40 ligand (200 ng/ml, InvivoGen, San Diego, CA) to generate mature dendritic cells (mDC) (Table 1). Two donors (D1 and D2) were also matured by culturing 2 days with a cytokine cocktail, which contained IL-1β (10 ng/ml, BD Biosciences), IL6 (10 ng/ml, InvivoGen), TNFα (10 ng/ml) and PGE₂ (10⁻⁷ M, Sigma-Aldrich, St. Louis, MO).

Phosphorylation of STAT1, 3 and 5 was determined as previously described [76 (link), 77 (link)]. Total CD4⁺ T cells were thawed, rested overnight in RF10, plated at 300,000 cells per condition in RF10 in a U bottom 96 well plate and rested for 90 min followed by a 15 min, or otherwise indicated, stimulation with r-hIFN (1–10,000 U/mL as indicated) at 37°C. As positive control for STAT phosphorylation, cells were incubated for 15 min with 20 U/mL r-hIL-2, 50 ng/mL IL-6 (InvivoGen) and 10,000 U/mL IFNα. Cells were fixed with formaldehyde (1.6% final concertation), permeabilized with ice-cold methanol and incubated with m-a-hpSTAT1-AF647 (clone 4a pY701), m-a-hSTAT3-PerCP-Cy5.5 (clone 4/P-STAT3, pY705) and m-a-hSTAT5-PE-Cy7 (clone 47, pY694). Expression was quantified using a BD LSR II (BD Biosciences) and results were analyzed using Weasel software. BD CompBeads and fluorescent-minus-one (FMO) controls were used for instrument and gating setup.

The SCNP assay was performed as described previously [7 (link)]. Modulators and concentrations were as follows: 1000 IU/ml IFN-α (PBL); 50 ng/ml IL-4, 5 μg/ml anti-IgD (BD Biosciences); 50 ng/ml IL-2, 50 ng/ml IL-6, 50 ng/ml IL-27, 5 μg/ml R848 (Invivogen); 40 nM PMA (Sigma Aldrich), 3 μg/ml anti-CD3 (eBioscience), 10 μg/ml anti-mouse (Santa Cruz Biotechnology), 10 μg/ml anti-IgM (Southern Biotech). For TCR stimulation, cells were exposed to anti-CD3 for 12 min, with anti-mouse added for the last 2 min; the anti-IgM modulation time was 10 min; all other modulation times were 15 min. Staining was performed using Ab cocktails with each cocktail consisting of 5 Abs to detect phenotypic markers and 2 Abs to detect intracellular protein readouts. Abs used include anti-CD3, -CD4, -CD45RA, -CD20, -p-NF-κB, -c-poly(ADP-ribose) polymerase, -p-Stat1, -p-Stat3, -p-Stat5, -p-Stat6, -p-Erk, -p-ZAP70/Syk (BD Biosciences); -p-Akt, -p-S6 (CST); and -CD14 (Beckman Coulter). Flow cytometry data was acquired on FACS Canto II Flow Cytometers (BD Biosciences). All flow cytometry data were analyzed with WinList (Verity House Software). PBMC subpopulations were delineated according to the immunophenotypic gating scheme described previously [7 (link)].