Flow Cytometry Analysis of T-Cell Responses

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)