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 stained with Zombie Yellow to eliminate dead cells. The cells were then pretreated with Fc Block, stained with FITC-conjugated anti-Thy1.1, PerCP/Cyanine5.5-conjugated anti-CD4, APC-conjugated anti-CD3, APC/Cyanine7-conjugated anti-CD8, PE/Dazzle594-conjugated anti-B220, anti-F4/80, anti-NK1.1 and anti-Ly6G, followed by fixation, permeabilization and staining with PE-conjugated anti-Foxp3 and PE/Cyanine7-conjugated anti-Ki67 (Thermo Fisher Scientific) using Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's protocols.健康小鼠于第-21天接受经同系标记物（congenic marker）Thy1.1标记的活化pmel-1 T细胞输注，并于第-19天首次接种gp100疫苗。自第-2天至第6天，小鼠每日给予3 mg/kg地塞米松。第0天进行第二次疫苗接种。第7天收获脾脏，用于流式细胞术（flow cytometry）检测。 细胞经Zombie Yellow染色以剔除死细胞。随后用Fc封闭剂（Fc Block）预处理细胞，依次用FITC标记的抗Thy1.1、PerCP/Cyanine5.5标记的抗CD4、APC标记的抗CD3、APC/Cyanine7标记的抗CD8、PE/Dazzle594标记的抗B220、抗F4/80、抗NK1.1及抗Ly6G抗体进行染色；之后依照制造商说明书的操作流程，使用Thermo Fisher Scientific提供的Foxp3/转录因子染色缓冲液套装（Foxp3/Transcription Factor Staining Buffer Set）对细胞进行固定与透化处理，再采用PE标记的抗Foxp3及PE/Cyanine7标记的抗Ki67抗体（Thermo Fisher Scientific）完成染色。