Cd40 pe

Astaxanthin (mol wt 596.84), LPS derived from Escherichia coli 026: B6, FITC-Dextran (mol wt 40,000) and Cobalt protoporphyrin (CoPP, a HO-1 inducer) were from Sigma-Aldrich. Alexa Fluor 647-Dextran (mol wt 10,000) was from Thermo Fisher. Carboxyfluorescein succinimidylester (CFSE) and RPMI 1640 medium were from Invitrogen. Fetal bovine serum (FBS) was from Hyclone. Recombinant CCL19, GM-CSF, and IL-4 were from Peprotech. CCK-8 kit was from Beyotime. CD4⁺ T cell isolation kit was from Miltenyi Biotech. Fluorescent-labeled anti-mouse mAbs, PerCP-Cy5.5 CD69, FITC-MHCII, PE-CD40, PE-CD80, FITC-CD86, PE-CCR7 or respective isotype controls, were from BD PharMingen. Alexa Fluor 647 HO-1 or respective isotype was from Abcam. PE-Nrf2 or respective isotype was from Cell Signaling Technology. Tin protoporphyrin IX (SnPP, a HO-1 inhibitor) was from MedChemExpress.

Cells were first stained with LiveDead aqua or blue (Life Technologies). Following FcR-Block (2.4G2), cells were stained using the following monoclonal antibodies (all used at 1:200 dilution): CD3-APCe780, CD4-APC or A700, CD8α-PE/Cy7, CD11c-APC or BV421, MHC-II-PerCP/Cy5.5 or eFlour450, CD11b-BV711 or PE, CD19-e780, CD80-PerCP/Cy5.5, CD40-PE, CD44-BV570, CD69-FITC CD86-AF488, Foxp3-e450, F4/80-PE/Cy7, Gr1-FITC, HuCD2-PE, IgM-APCe780, SiglecF-PE and TCRβ-APCe780 (BD Biosciences, BioLegend or eBioscience). Foxp3 staining was performed with the eBioscience FoxP3 staining kit. Samples were acquired using FACS LSR II or FACS Canto II using BD FACSDiva software and analysed with FlowJo v.9 software (Tree Star). Cytokines were measured in culture supernatants by ELISA using paired monoclonal antibody, and recombinant cytokine standards, or Duosets (eBioscience, BD Biosciences, BioLegend, R&D Systems and Peprotech).

To characterize monocyte subsets, freshly purified PBMCs were analyzed by six-color flow cytometry on FACS LSRII apparatus. The gating strategy was based on a previous report [15 (link)]. Monocytes were subdivided into three major subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺ and non-classical CD14⁺CD16⁺⁺ monocytes. The following anti-human mAbs were used: CD45-Amcyan (BD Biosciences), HLA-DR-PerCP (BD Biosciences), CD19-ECD (Beckman Coulter), CD14-QDot655 (Invitrogen), CD16-APC-H7 (Beckman Coulter, Villepinte, France). The Live/Dead blue Dye (Invitrogen) was used to exclude dead cells.
Samples of the purified monocytes used to generate MD-DCs and of the resulting MD-DCs were routinely stained with the following anti-human mAbs: CD14-FITC, CD11c-APC, CD40-PE, HLA-I-FITC, HLA-DR-PerCP, CD80-PE, CD83-APC and CD86-FITC (all from BD Bioscience) and analyzed by flow cytometry on FACS canto II apparatus (BD Biosciences).

Bone marrow-derived dendritic cells (BMDCs) were generated from BALB/CByJ mice as described previously [20 (link)]. At day seven post-isolation, BMDCs were either exposed to 10 µM OR141-killed mesothelioma cells (as described above) at a ratio of 1:1 or LPS (from E. Coli, 0.5 μg/mL). DC maturation was analyzed with antibodies against CD11c-BV421 (BD Biosciences, San Jose, CA, USA, 565452), MHCII (I-A/I-E)-APC (BD Pharm, San Jose, CA, USA, 565367), CD40-PE (BD Pharm, 553791), CD80-PE (eBioscience, San Diego, CA, USA, 12-0801), CD86-PE (eBioscience, 12-0862) or CCR7-PE (BioLegend, San Diego, CA, USA, 120105). Live–dead exclusion was achieved by staining with FVD eFluor780 (eBioscience, 65-0865-14). Flow cytometry analysis was performed on FACS Canto II and data were analyzed using FlowJo software. For mouse vaccination, 2 × 10⁶ DC (in 100 μL PBS) were injected i.p. three times at one-week intervals; in parallel, another group of mice was also injected i.p. with 100 µg anti-CTLA4 (CD152) antibody (Bio X Cell, West Lebanon, NH, USA).

Immunostaining was performed as described previously.^{41 (link)} Briefly, after 10 min of incubation at 4 °C with Fc receptor blocker (BD Biosciences, San Joses, CA, USA), the cells were stained with the corresponding antibodies. The antibodies used for DC phenotyping were CD11c-PE, CD14-FITC, CD40-PE, CD54-FITC, CD80-PE, CD86-FITC, H-2K[d]-PE, and I-A[d]-FITC; all these antibodies were purchased from BD Biosciences. For intracellular cytokine staining, the cells were incubated with brefeldin A (BD Biosciences) for 1 h. The cells were stained with surface markers, such as CD4-FITC and CD25-APC (BD Biosciences). Then, the cells were permeabilized using Cytofix/Cytoperm reagents (BD Biosciences) and stained for intracellular markers as follows: IFN-γ-PE, IL-4-PE (BD Biosciences), IL-17A-PE, and Foxp3-PE (eBioscience, San Diego, CA, USA). Stained cells were acquired using a BD FACSCanto II flow cytometer (BD Biosciences) and analyzed using the FlowJo software (v.10, Ashland, OR, USA).