Anti cd16 32 mab

Cells were incubated with anti-CD16/32 MAbs (eBioscience) in goat serum (Sigma) to block the nonspecific uptake of antibodies and then subsequently labeled with the following MAbs conjugated to fluorescent labels: anti-CD4, anti-CD3, anti-CD8, anti-B220, anti-CD25, anti-CD103, anti-PD1, anti-PDL-1, anti-PDL-2 PE, anti-Fas, anti-FasL, anti-CD69, anti-CD44, anti-CD62L, anti-major histocompatibility complex class II (MHC-II) (IA-IE), anti-Nrp1, anti-CD11b (all from eBioscience), anti-CD49b (BioLegend), and anti-LAG3 (CD223) (BD Bioscience). Intracellular staining was performed according to the manufacturer's protocol for anti-FoxP3, anti-Helios, and anti-cytotoxic-T lymphocyte-associated antigen 4 (CTLA-4) (eBioscience). All flow cytometry data were acquired by using the Cyan ADP analyzer or the BD LSR Fortessa analyzer. Flow data were analyzed by using FlowJo software v7.6.5 (Tree Star, Inc.) or Summit v4.3 (DakoCytomation).

Splenocytes from naïve B6 mice and cervical lymph node cells from tongue tumor-bearing mice were cultured with Th17 promoting agents for 3 days. Cells were pre-treated with cordycepin or other ingredients for 3 hours, following co-cultured with Th17 promoting agents for 3 days. The Th17 promoting agents were composed of anti-CD3 mAbs (1 μg/ml, clone 145–2C11), anti-CD28 mAbs (1 μg/ml, clone 37.51), anti-IFN-γ mAbs (1 μg/ml, clone XMG1.2), anti-IL-4 mAbs (1 μg/ml, clone 11B11), rIL-6 (50 ng/ml, Biolegend), rIL-23 p19 (5 ng/ml, Biolegend), and rTGF-β (1 ng/ml, Biolegend). After 3 days, cells and supernatants were collected. Cells were re-stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 8 hours with monensin and brefeldin A. Subsequently, cells were harvested, permeabilized with Cytofix/Cytoperm solution (BD Biosciences, CA, USA), blocked with anti-CD16/32 mAbs (eBioscience, CA, USA), and stained with anti-CD3 (clone 145–2C11), anti-CD4 (clone RM4-5), and anti-IL-17A (clone TC11-18H10) mAbs for detection of IL-17A-expressing cells by flow cytometry. Levels of IL-17A and LDH (Takara Bio Inc., Kyoto, Japan) in the culture supernatants were analyzed with ELISA kits.

In vivo cell proliferation was determined by bromodeoxyuridine (BrdU) incorporation. Mice received 1 mg BrdU (Sigma) intraperitoneally (i.p.) daily for the final 4 days before harvest of the sdLN. The sdLN cells were then processed to a single-cell suspension and blocked with anti-CD16/32 MAbs (eBioscience) in goat serum (Sigma) and later surface stained for anti-CD3 eF450 and anti-CD4 phycoerythrin (PE)-Cy7 (both from eBioscience) in phosphate-buffered saline (PBS) supplemented with 1% FCS. Subsequently, cells were washed in PBS–1% FCS and incubated in 1× Fixation/Permeabilization buffer (eBioscience) for at least 1 h at 4°C. Cells were then washed in PBS–1% FCS and incubated at 37°C in 100 μg DNase (Sigma) for 1 h. Afterwards, cells were washed in PBS–1% FCS and stained for 45 min at room temperature with anti-BrdU allophycocyanin or rat IgG1 allophycocyanin (eBioscience) in 1× permeabilization buffer, according to the manufacturer's protocol.