背景与目的 本研究旨在探索细胞毒性T淋巴细胞相关抗原(cytotoxic T lymphocyte associated anti-gen-4,CTLA-4)、程序坏死因子(programmed death 1,PD-1)和程序坏死因子配体(programmed death ligand 1,PD-L1)在小细胞肺癌(small cell lung cancer,SCLC)患者外周血中的分布情况,探索其免疫作用机制并评估其作为生物标志物的临床价值.方法 招募290例SCLC患者及60例健康志愿者,收集治疗前和治疗2nd周期末SCLC患者EDTA抗凝血2 mL.应用流式细胞仪检测CTLA-4、PD-1和PD-L1在外周血CD3、CD4、CD8及CD25的分布,分析其与临床病理特征的相关性;采用细胞免疫化学法和流式细胞法检测PD-L1在SCLC细胞系H446中的表达.结果 SCLC患者外周血中CTLA-4+细胞和PD-1+细胞水平分别为(1.56±1.24)%和(8.07±3.97)%、CTLA-4在CD3细胞和CD4细胞中的表达水平无明显差异,分别为(4.87±5.18)%和(3.85±2.60)%,均低于PD-1在CD3+或CD4+细胞中的表达(26.63±9.04)%和(20.79±9.41)%,与健康对照组相比,SCLC中CD4+CD25+CTLA-4+细胞水平明显升高(1.91±1.27)%vs(7.09±5.09)%,P<0.001;PD-1+(CD8+)细胞表达水平明显降低,分别为(22.56±4.21)%vs(11.47±5.85)%,P<0.001.CD4+CD25+CTLA-4+细胞或CD8+PD-1+细胞水平与患者的年龄、性别、吸烟状况、临床分期以及是否转移等因素无关(P>0.05).化疗两周期末CD4+CD25+CTLA-4+和CD8+PD-1+细胞的水平对比化疗前明显下降,分别为(5.11±2.60)%vs(6.94±4.91)%;(8.74±3.39)%vs(11.48±5.91)%,P值均<0.000,1,但与无疾病进展生存和总生存无显著相关性.PD-L1高表达于SCLC细胞系H446中并定位在细胞膜和细胞浆,但在外周血中未见表达.结论 本研究首次证实SCLC外周血中CTLA4高表达于调节性T细胞中,而PD-1低表达于效应性T细胞,该结果为揭示SCLC免疫监测点免疫逃逸机制提供了理论依据,可能作为一种新的无创性且可实时监测的生物标志物.%Background and objective The aim of this study is to explore cytotoxic T lymphocyte associated anti-gen 4 (CTLA-4), programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) distribution and clinical value in liq-uid biopsy (such as blood) of small cell lung cancer (SCLC) patients. Methods A total of 60 healthy and 290 chemotherapy-naive patients with SCLC were recruited. Venous blood samples were collected prior to chemotherapy (baseline) and after the second cycle of chemotherapy (2nd cycle), and flow cytometry was used to analyze the level of CTLA-4, PD-1 or PD-L1 with or without CD3, CD4, CD8 or CD25. Immunocytochemical method was used to detect PD-L1 expression in SCLC cell line H446. Results Cells of CTLA-4+ and PD-1+ in SCLC peripheral blood were (1.56±1.24)% and (8.07±3.97)%;there is no significant difference between CD3+CTLA-4+ and CD4+CTLA-4+, (4.87±5.18)% and (3.85±2.60)%, but show lower expression than CD3+PD-1+ and CD4+PD-1+ (26.63±9.04)% and (20.79±9.41)%, respectively. However, the level of CD4+CD25+CTLA-4+cells were remarkably higher in SCLC than that in control, (7.09±5.09)% vs (1.91±1.27)%, P<0.001 and CD8+PD-1+cells were less in SCLC than that in control, (11.47±5.85)% vs (22.56±4.21)%, P<0.001, both of which were not associated with age, sex, smoke or disease stage. Level of CD4+CD25+CTLA-4+cells and CD8+PD-1+cells was dropped (5.11± 2.60)% vs (6.94±4.91)% and (8.74±3.39)% vs (11.48±5.91)% after 2nd cycle compare to that at baseline (P<0.000,1). Neither the level of CD4+CD25+CTLA-4+ nor CD8+PD-1+ cells before or after treatment was related to progression-free disease or overall survival of patients. Although PD-L1 was highly expressed in H446 cell cytoplasm and membrane, it was rarely found in peripheral blood. Conclusion The data we presented here showed that CTLA-4 was highly expressed in regulatory T cells and PD-1 decreased in CD8+ T cells in peripheral blood of SCLC patients, suggesting their unique mechanisms involved in im-mune regulation. CD4+CD25+CTLA-4+level changed after treatment implies its potential role in predicting treatment e?cacy.
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