Dexamethasone suppresses tumor-specific T cell proliferation and differentiation but not activity in antigen-experienced mice.

Healthy mice were administered activated pmel-1 T cells labeled with the congenic marker Thy1.1 on day -21 and vaccinated for the first time with a gp100 vaccine on day -19. From day -2 until day 6, the mice were administered 3 mg/kg dexamethasone daily. On day 0, they were administered the vaccine a second time. Spleens were harvested for flow cytometry on day 7. The cells were stimulated with 1 µg/ml hgp100 peptide in the presence of 10 µg/ml brefeldin A at 37°C for 4 h. The cells were stained with Zombie Yellow, FITC-conjugated anti-Thy1.1, APC/Cyanine7-conjugated anti-CD8, Pacific Blue-conjugated anti-CD3, PE/Dazzle594-conjugated anti-B220, anti-F4/80, anti-NK1.1 and anti-Ly6G, followed by fixation, permeabilization and staining with PE/Cyanine7-conjugated anti-IFNγ, PE-conjugated anti-TNFα and APC-conjugated anti-Granzyme B (BioLegend) using Intracellular Staining Fixation Buffer and Intracellular Staining Permeabilization Wash Buffer (10X) (BioLegend) according to the manufacturer's protocols.