Anti cd40 apc

The analysis of immune cell populations in the spleen was performed 12 days after tumor inoculation as described previously [32 (link)]. The following staining and antibodies were used: anti-CD45.2-APC eFluor 780, anti-CD3-BV421, anti-CD4-Brilliant Violet 605 (clone RM4-5, Biologend, San Diego, CA, USA), anti-CD8α-APC-R700 (clone 53-6.7, BD Bioscience, Franklin Lakes, NJ, USA), anti-CD19-BV655, anti-CD40-APC, and anti-CD86-FTIC.

Dendritic cells were harvested, and 1 × 10⁶ cells were washed with cold 1× DPBS and then incubated with anti-CD40-APC (BD Biosciences; San Jose, CA, USA), anti-CD80-PE (eBioscience; San Diego, CA, USA), anti-CD83-PE-Cy5 (BD Biosciences; San Jose, CA, USA), anti-CD86-PE (eBioscience; San Diego, CA, USA), anti-HLA-DR-PE (eBioscience; San Diego, CA, USA), and anti-CCR7-PE-Cy7 (BD Biosciences; San Jose, CA, USA) labeled antibodies (BD Biosciences; San Jose, CA, USA) for 30 min on ice. After washes, cells were fixed with 1% paraformaldehyde (Fisher Scientific; Waltham, MA, USA) and data were collected on an LSRII flow cytometer (BD Biosciences; San Jose, CA, USA). Expression of cell surface markers was analyzed on gated GFP positive cells using FlowJo 6.3.4 software (FlowJo; Ashland, OR, USA).

Antibodies were purchased as followed: Anti-CD4-V450, anti-CD8α-APCcy7, anti-B220-V450, anti-CD138-Pe-cy7, anti-CD86-Pe-cy5, anti-FAS-PE, anti-GL-7-APC, anti-CD40-APC, anti-MHCII-FITC, anti-IL-4-PE, anti-IL-5-PE, anti-IgM-Pe-cy7, anti-IgG1-APC, anti-IFN-r-Percp5.5, anti-TNF-α-PE, and anti-IL-17-Pe-cy7 were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-PDL-1-biotin (eBioscience, San Diego, CA), anti-XBP-1s (Cell Signaling Technology, Danvers, MA), and anti-Rabbit IgG-FITC (Thermo Fisher Scientific, Waltham, MA) antibodies were purchased from commercial sources. Recombinant mouse IL-4 (PeproTech, Rocky Hill, NJ) and LPS (Sigma-Aldrich, St. Louis, MO) were purchased from the commercial companies. Goat F(ab’)2 Anti-Mouse IgM (1022-01, SouthernBiotech, Birmingham, AL) and anti-mouse CD40 (BE0016-2, BioXCell, Lebanon, NH) were commercially purchased from companies.

Recipient splenocytes and thymocytes were isolated and stained for surface receptors and intracellular cytokines using standard flow cytometric protocols as previously described [22 (link), 24 (link), 26 (link)]. The following Abs were used for cell-surface staining: anti-CD4–FITC, or–V450, anti-CD8α–FITC, or–allophycocyanin-cy7, anti-B220–v450 (RA3-6B2), anti-CD80–FITC, anti- CD86–FITC, anti-CD40 APC, Biotin-anti-CD29 (β1 integrin), anti-CD229.1–Biotin or PE, anti- CD5.1-FITC, purchased from BD Biosciences. Intracellular staining was carried out using anti–IFN-γ–PE or Per-cp 5.5 (XMG1.2; BD Biosciences), anti–IL-17–allophycocyanin (17B7; eBioscience), anti–IL-4–PE (11B11; BD Pharmingen), anti–IL-5–PE (TRFK5; BD Pharmingen), anti-TNFα–PE, or PE-Cy7 (MP6-XT22; BD Pharmingen), anti-Foxp3–PE (FJK-16s; eBioscience), and the appropriate isotype controls. Cell isolates were analyzed using Diva software, LSR II (BD Biosciences,San Jose, CA), FACS Verse (BD Biosciences, San Jose, CA), and FlowJo (TreeStar, Ashland, OR).

Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll density gradient and stored with fetal bovine serum (FBS) and 10% dimethylsulfoxide (DMSO) in liquid nitrogen until the time of the assay. Cells were stained with a viability marker (LIVE/DEAD Fixable Violet Dead Cell Stain Kit 405nm, Life Technologies) and different fluorochrome-conjugated antibodies for 35 min at RT to assess the expression of surface marker expression. Anti-CD3, anti-CD19, anti-CD20, and anti-CD56 conjugated with V450 were used as a dump channel. Anti-CD14-BV650 and anti-CD16-PeCF594 (BD biosciences, USA) were used to classify different subsets of monocytes. Anti-TLR2-FITC, anti-HLA-DR-BV570, anti-CD40-APC (all BD Biosciences, USA), and anti-TLR4-BV786 (Biolegend, USA) were determined as activation markers. Anti-CX3CR1-PerCPCy5,5, anti-CCR2-BV605, anti-CD49d- BV711, anti-CD142-PE (all Biolegend, USA), anti-CCR5- APC-Cy7, and anti-CD11b-AF700 (all from BD Biosciences, USA) were used as maturation and homing markers. The cellular markers were analyzed by multiparametric flow cytometry using the Fortessa LSR II cytometer (BD Biosciences, Madrid, Spain). A minimum of 1x10⁶ events were acquired per sample. Data were analyzed using Flowjo 9.3.2 software.

Cell viability was determined using Fixable Viability Dye eFluor™ 780 (Invitrogen # 65-0865-14) according to manufacturer's instructions. Briefly, cells were incubated with Fixable Viability Dye eFluor™ 780 (1:2,000) at 4°C for 20 min. Subsequently, cells were washed and analyzed by flow cytometry. The following primary monoclonal antibodies (mAbs) were used to determine the maturation state of the DCs: anti–CD80- APC, anti–CD40-APC (all BD Bioscience, San Jose, CA). Measurements were performed on FACSVerse flowcytometers (BD).