Complete data set for each microarray experiment was lodged in the Gene Expression Omnibus public repository at NCBI (www.ncbi.nlm.nih.gov/geo/) (accession number GSE6863). Validation of a subset of randomly selected genes was carried out by qRT-PCR. HRE consensus Atezolizumab chemical structure elements consisting of a 4nt core (CGTG) flanked by degenerated sequences ((T|G|C)(A|G)(CGTG)(C|G|A)(G|C|T)(G|T|C)(C|T|G)) were mapped in the promoter regions of genes represented in the chip, as detailed . Real-time PCR (qRT-PCR) was performed on a 7500 Real Time PCR System (Applied), using SYBR Green PCR Master Mix and sense/antisense oligonucleotide primers designed using Primer-3
software from sequences in the GenBank and obtained from TIBMolbiol (Genova) or from Quiagen (RSP18), as detailed . Expression data were normalized on the values obtained in parallel for three reference genes check details (ARPC1B, RPS18, RPS19), selected among those not affected by hypoxia in the Affymetrix analysis, using the Bestkeeper software, and relative expression values were calculated using Q-gene software, as detailed [23, 24]. Twelve-well flat-bottom tissue culture plates (Corning Life Sciences) precoated with 10 μg/mL of agonist anti-TREM-1 mAb (R&D Systems, containing less than 0.1 EU per 1 μg of the antibody by the LAL method), control HLA-I (Serotec), irrelevant
isotype-matched Ab, or left uncoated were incubated overnight at 37°C before seeding 8 × 105 H-iDCs/well/mL of fresh RPMI 1640 without cytokines. Plates were briefly spun at 130 g to engage TREM-1. After 24 h stimulation under hypoxic conditions, supernatants were harvested by centrifugation and tested for cytokine/chemokine content by ELISA and H-iDCs were used to stimulate allogeneic T cells. T cells were purified by negative selection from peripheral blood mononuclear cells using a PanT kit (Miltenyi Biotec). Total of 1 × 106/mL T cells were cultured with allogeneic H-iDCs previously triggered with anti-TREM-1 mAb or control HLA-I at a 20:1 T:DC ratio. After 4 days, supernatants were collected
to measure released cytokines by ELISA. To assess proliferation, T cells were pulsed with 1 μCi of 3H-thymidine (Perkin Elmer) for a further 16 h culture, and 3H-thymidine incorporation was quantified Fludarabine in vivo using a TopCount microplates scintillation counter (Canberra Packard). All tests were performed in triplicate. Data are expressed as cpm ×10−3. Conditioned medium (CM) from monocyte-derived iDCs was replaced on day 3 of generation with fresh medium supplemented with cytokines for 24 h, both under normoxic and hypoxic conditions. On day 4, CM were collected, and tested for soluble (s)TREM-1 content by ELISA (R&D Systems). Secreted TNF-α, IL-12, CXCL8, IL-1β, CCL-5, CCL-17, and OPN were measured in the supernatants from iDCs triggered with anti-TREM-1 mAb or control mAbs, whereas IFN-γ, IL-17, IL-4, and IL-10 were quantified in the supernatants of T:DCs cocultures by specific ELISA (R&D Systems).