Il 6 pe

CFSE staining was done using 5 µM CFSE (Invitrogen, Cat# C34554) as described by Parish et al. [42] (link) and stimulated with cytokines for 6 days prior to flow cytometric analysis. Antibodies used: CD8-APC (BD, Cat# 345775), CD3-APC (BD, Cat# 555335), CD4-APC (Biolegend, Cat# 300514), CD56-APC (eBioscience, Cat#17-0569-42), CD16-PE (Biolegend, Cat#302007), HLA-DR-PE (Biolegend, Cat# 307605), NKG2D-APC (RnD Systems, Cat# FAB139A). For intracellular staining, IL-6-PE (eBioscience, Cat#12-7069) and IFN-γ-FITC (BD, Cat# 554700) were used. Isotype controls were IgG1 APC (BD, cat # 555751), rat IgG1-PE (Invitrogen, Cat # R104) and IgG1-FITC (BD, Cat #555748). PBMCs were incubated for 6 h +/− LPS (Sigma-Aldrich, L2654, 1 µg/mL) and cells were stained using the BD Cytofix/Cytoperm Kit (BD, Cat# 554714), Golgistop (BD, Cat# 554724) or Golgiplug (BD, Cat# 555029) according to the manufacturer’s protocol. Samples were run on an Accuri C6 Flow cytometer and data were analyzed using FCS Express v. 3.

For intracellular staining, monocytes (1x10⁶ cells/well/mL) were cultured overnight, followed by Brefeldin A (1 μg/mL, 4h) and surface stained with CD14-FITC [Biolegend, San Diego, USA. They were then stained for IL-6-PE, IL-1β-PE (eBioscience, San Diego, USA)], IL-8-APC, IL-12p40-PE, Latency associated peptide (LAP)-TGF-β1-APC (Biolegend, San Diego, USA) along with isotype controls and acquired in a flow cytometer.
5000 monocytes were acquired and data analyzed by Cell-Quest pro software. The frequency of cells with a particular phenotype was expressed as % of monocytes and was calculated by dividing percentages of the upper right quadrant (CD14⁺ marker⁺) by the sum of the upper and lower right quadrant (CD14⁺ marker^-).

Zymosan A from Saccharomyces cerevisiae was purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-mouse mAbs, such as CD11b-FITC, F4/80-PECy7, CD11c-PerCP-Cy5.5, CD19-BV650, MHC II (I-A/I-E)-PECy5, Siglec-F-PE, LY6G-APC, LY6C-APC-CY7, IL-6-PE and MCP-1-APC were purchased from eBioscience (San Diego, CA, USA). Recombinant mouse B7-H1/Fc chimera (1019-B7) was purchased from R&D Systems (Minneapolis, MN, USA). Human IgG for control experiments was purchased from Sigma–Aldrich. Anti-mouse PD-1 (clone J43) were purchased from eBioscience. LY294002, PD98059, SP600125 and SB203580 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA). The following mAbs were used for western blotting and immunoprecipitation: anti mPD-1 (RPMI-30; eBioscience), anti-phosphotyrosine Ab (Cell Signaling Technology, Beverly, MA, USA), anti-SHP-1 (sc-287), anti-SHP-2 (sc-280), p-STAT1, STAT1, p-STAT6 and STAT6 (Cell Signaling Technology), anti-mouse IgG-HRP, anti-rabbit IgG-HRP Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA).