N downstream of IDO, was measured by spectrophotometry. In the first step, 50 of 30 trichloroacetic acid (TCA) was added to 100 DC supernatant, and the samples were vortexed and centrifuged at 8,000 for 5 minutes. An equal volume of Ehrlich reagent (100 mg P-dimethylbenzaldehyde in 2 ml glacial acetic acid) was added to the supernatant from the TCA precipitation, and the optical density at 492 nm was measured. L-kynurenine production was quantified by reference to a standard curve of defined kynurenine concentrations. Flow cytometry The following fluorochrome-conjugated antibodies were used: FITC-anti-CD80, FITC-antiCD86, PE-anti-CD83, PE-anti-TNF, FITC-anti-IFN, APC-anti-Foxp3, PE-anti-B7-H1, PE-anti-ICOSL (all from eBioscience), APC-anti–catenin, FITC-anti-CCR5, APC-antiCCR7, FITC-anti-CCR4, PE-anti-CXCR4 (all from R D Systems), AF647-anti-p38phos, PE-anti-ERK 1/2phos, PE-anti-NF-kB p65phos (all from BD Biosciences), FITC-anti-PD-1 (Bio-Legend), and APC-anti-CTLA-4 (BD PharMingen). Foxp3 and CTLA-4 were stained following fixation and permeabilization (with buffers from eBioscience, according to the manufacturer’s protocol). NF-kB p65phos, ERK 1/2phos, and p38phos were stained following fixation (BD Cytofix, Cat. No. 554655) and permeabilization (BD Phosflow permeabilization buffer III, Cat. No. 558050).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCancer Immunol Immunother. Author manuscript; available in PMC 2014 May 01.Cannon et al.PageIntracellular cytokine expression was assayed following antigen stimulation of CD4+ T cells. Briefly, LCL were loaded overnight with peptide antigen (50 /ml in serum-free DC medium), irradiated (7,500 cGy), and washed once with PBS prior to coculture with responder CD4+ T cells. LCL (2.5 105/well) and T cells (5 105 to 1 106/ well) were placed in 24-well Costar plates and incubated overnight in the presence of Brefeldin A (eBioscience). Intracellular expression of TNF and IFN was detected following fixation and permeabilization (fixation/permeabilization buffer concentrate, Cat.Pacritinib No.Polatuzumab 00-5123-43, diluent, Cat.PMID:27217159 No. 00-5223-56, and permeabilization buffer, Cat. No. 00-8333-56, all from eBioscience). All samples were analyzed with a FACSCalibur flow cytometer, using CellQuest software (BD Biosciences). Appropriate isotype controls were used throughout. Multiplex analyses of DC cytokine expression and signaling pathways DC cytokine expression was determined with a Milliplex MAP human cytokine/chemokine multiplex assay (Millipore, Cat. No. MPXHCYTO-60 K) for the following analytes: IFN, IL L-1, IL-6, IL-10, 12(p70), IL-15, TNF, and MDC (CCL22). Samples were read with a Luminex 100/200 analysis system (Luminex Corp.). DC signaling pathways were analyzed with an 8-plex multi-pathway signaling kit (Milliplex # 48-680, Millipore) for the determination of ERK 1/2, JNK, p38 MAPK, STAT3, STAT5A/B, p70 S6 kinase, CREB, and IB phosphoproteins. Samples were read with a Luminex 100/200 analysis system, with unstimulated and activated HeLa cell lysates as internal controls. Cytotoxicity assays Cytotoxicity was tested in a standard chromium release assay. Control or peptide antigenloaded (50 /ml) LCL were incubated in serum-free DC medium overnight at 37 . Peptide pulsed targets were then labeled with 50 i Na2[51Cr]O4 for an additional hour and washed twice with PBS and once with DC medium before use. Target cells were plated at 1 104/well in 96-well round-bottomed plates with effe.