Saito T, Dworacki G, Gooding W, Lotze M T, Whiteside T L
University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pennsylvania 15213, USA.
Clin Cancer Res. 2000 Apr;6(4):1351-64.
Peripheral blood mononuclear cells (PBMCs) obtained from patients with advanced melanoma but not from healthy individuals were found to undergo spontaneous ex vivo apoptosis upon incubation in medium. PBMCs were evaluated for evidence of apoptosis using Annexin V binding, caspase-3 activation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). PBMCs of patients with melanoma contained a significantly higher proportion (P = 0.0027) of spontaneously apoptotic cells than PBMCs of controls after 24-h incubation in medium alone. The relative proportion of activated Fas+ and tumor necrosis factor receptor 1-positive (TNFR1+) PBMCs was significantly higher in patients with melanoma than that observed in controls. To demonstrate that the TNF family of receptors and ligands was involved in this type of apoptosis, PBMCs were incubated in the presence of agonistic anti-Fas antibody (CH-11) or TNF-alpha. The proportion of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive PBMCs undergoing spontaneous apoptosis was found to be comparable with that induced by CH-11 antibody or TNF-alpha. Three-color flow cytometry revealed that CD3+ Fas+ T cells were especially sensitive to apoptosis and were preprogrammed in vivo to die. Apoptosis occurred in all subsets of PBMCs but was significantly higher (P = 0.01) in the CD3+ CD8+ T-cell subset in patients relative to controls. In two patients with melanoma, who responded clinically to dendritic cell-based peptide vaccines, the proportion of apoptotic T cells was decreased by half after therapy. In patients who were treated previously with vaccination-based therapies, levels of T-cell apoptosis were lower than in the other melanoma patients. The observed accelerated death of T cells, which are activated and susceptible to apoptosis in patients with melanoma, may contribute to a rapid turnover of immune cells, resulting in a decreased immunocompetence.
从晚期黑色素瘤患者而非健康个体获取的外周血单个核细胞（PBMCs）在培养基中孵育时会发生自发的体外凋亡。使用膜联蛋白V结合、半胱天冬酶-3激活和DNA片段化（末端脱氧核苷酸转移酶介导的dUTP缺口末端标记）评估PBMCs的凋亡证据。在仅培养基中孵育24小时后，黑色素瘤患者的PBMCs中自发凋亡细胞的比例显著高于对照组（P = 0.0027）。黑色素瘤患者中活化的Fas +和肿瘤坏死因子受体1阳性（TNFR1 +）PBMCs的相对比例显著高于对照组。为了证明TNF受体和配体家族参与了这种类型的凋亡，将PBMCs在激动性抗Fas抗体（CH-11）或TNF-α存在下孵育。发现经历自发凋亡的末端脱氧核苷酸转移酶介导的dUTP缺口末端标记阳性PBMCs的比例与CH-11抗体或TNF-α诱导的比例相当。三色流式细胞术显示CD3 + Fas + T细胞对凋亡特别敏感，并且在体内预先编程死亡。凋亡发生在PBMCs的所有亚群中，但患者的CD3 + CD8 + T细胞亚群中的凋亡显著更高（P = 0.01），相对于对照组。在两名对基于树突状细胞的肽疫苗有临床反应的黑色素瘤患者中，治疗后凋亡T细胞的比例降低了一半。在先前接受基于疫苗治疗的患者中，T细胞凋亡水平低于其他黑色素瘤患者。在黑色素瘤患者中观察到的活化且易发生凋亡的T细胞加速死亡可能导致免疫细胞的快速更新，从而导致免疫能力下降。
