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.健康小鼠于第-21天给予经同系异体标记物（congenic marker）Thy1.1标记的活化pmel-1 T细胞，并于第-19天首次接种gp100疫苗。第-2天至第6天，每日给予小鼠3 mg/kg地塞米松。第0天，小鼠接受第二次疫苗接种。第7天采集脾脏用于流式细胞术检测。 将细胞于37℃条件下，在10 µg/ml布雷菲德菌素A（brefeldin A）存在时，以1 µg/ml人gp100肽段（hgp100 peptide）刺激4小时。随后使用僵尸黄（Zombie Yellow）、异硫氰酸荧光素（FITC）标记的抗Thy1.1抗体、别藻蓝蛋白/花青素7（APC/Cyanine7）标记的抗CD8抗体、太平洋蓝（Pacific Blue）标记的抗CD3抗体、藻红蛋白/Dazzle594（PE/Dazzle594）标记的抗B220抗体，以及抗F4/80、抗NK1.1、抗Ly6G抗体完成细胞表面染色；之后依照制造商的操作规程，使用BioLegend公司的细胞内染色固定液（Intracellular Staining Fixation Buffer）与10×细胞内染色通透洗涤缓冲液（Intracellular Staining Permeabilization Wash Buffer (10X)）对细胞进行固定与通透处理，再采用BioLegend公司的藻红蛋白/花青素7（PE/Cyanine7）标记的抗干扰素γ（IFNγ）抗体、藻红蛋白（PE）标记的抗肿瘤坏死因子α（TNFα）抗体、别藻蓝蛋白（APC）标记的抗颗粒酶B（Granzyme B）抗体完成胞内染色。