Saturating anti cd16 cd32

HaP-T1 tumor-bearing hamsters were intratumorally injected with PBS, αPD-1 (10 mg/kg), oAd/IL12/GM-RLX (7×10⁷ VP), or oAd/IL12/GM-RLX (7×10⁷ VP) plus αPD-1 (10 mg/kg). At 12 days after the initial treatment, lymphocytes were isolated from draining lymph node (DLN) or tumor tissue as previously reported.19 (link) Before staining, cells were treated with saturating anti-CD16/CD32 (Biolegend) in staining buffer (2% FBS and 0.02% sodium azide in PBS). Then, cells were stained with hamster anti-mouse CD3e monoclonal Ab (BD Bioscience, San Jose, California, USA), mouse anti-rat CD4 monoclonal Ab (ebioscience), or mouse anti-rat CD8 monoclonal Ab (ebioscience) for the assessment of the CD4 or CD8 and interferon (IFN)-γ coexpressing T-cell population. After staining of surface markers (CD3, CD4, and CD8), cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD pharMingen, San Jose, California, USA) and then stained with a rabbit anti-hamster IFN-γ Ab (Abclon, Seoul, Korea). Samples were analyzed using a FACScan flow cytometer with CellQuest software (Beckton-Dickinson) as previously reported.19 (link)

Spleen and draining lymph nodes (DLNs) were harvested at 12 days following first viral inoculation of the B16-F10 tumor-bearing mice. Before staining, cells were treated with saturating anti-CD16/CD32 (Biolegend, San Diego, CA) in staining buffer (2% FBS and 0.02% sodium azide in PBS). Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (eBioscience), percp-CY5.5-conjugated anti-CD4 (BD Biosciences Pharmingen), PE-CY7-conjugated anti-CCR4 (Biolegend), and/or PE-conjugated anti-CCR7 (Biolegend). To determine the percentage of CCR4- or CCR7-expressing CD4⁺ T cell population, splenocytes or lymphocytes were gated by plotting forward vs. side scatter followed by gating on CD4⁺ cells. Gated cells were then analyzed for CCR4⁺ or CCR7⁺ cells. Samples were analyzed using BD Biosciences BD-LSR II Analytic Flow Cytometer and FACSDiva software (BD Biosciences Pharmingen).