干扰素α免疫治疗后患者间STAT1磷酸化的多参数流式细胞术分析。

Multiparametric flow cytometric analysis of inter-patient variation in STAT1 phosphorylation following interferon Alfa immunotherapy.

作者信息

Lesinski Gregory B, Kondadasula Sri Vidya, Crespin Tim, Shen Lei, Kendra Kari, Walker Michael, Carson William E

机构信息

Department of Human Cancer Genetics, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, OH 43210, USA.

出版信息

J Natl Cancer Inst. 2004 Sep 1;96(17):1331-42. doi: 10.1093/jnci/djh252.

DOI:10.1093/jnci/djh252

PMID:15339971

Abstract

BACKGROUND

Regulation of gene expression by signal transducer and activator of transcription 1 (STAT1) within host tissues mediates the antitumor effects of interferon alfa (IFN alpha). We used a novel flow cytometric assay to examine phosphorylation-mediated activation of STAT1 within immune effector cell subsets following in vitro or in vivo IFN alpha treatments.

METHODS

Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 17) or melanoma patients (n = 19) were treated in vitro with interferon alfa-2b (IFN alpha-2b) or phosphate-buffered saline (PBS) and subjected to multiparametric flow cytometry to measure the levels of phosphorylated STAT1 (P-STAT1) within immune cell subsets. We similarly analyzed PBMCs isolated from melanoma patients before and 1 hour after immunotherapy with IFN alpha-2b. All statistical tests were two-sided.

RESULTS

P-STAT1 levels in all major immune cell subsets increased within 15 minutes of in vitro IFN alpha-2b treatment of PBMCs; the increase was most pronounced in T lymphocytes and monocytes. Relatively low doses of IFN alpha-2b (i.e., 10(2)-10(3) IU/mL) induced maximal STAT1 activation in vitro. Compared with melanoma patients, healthy donors had higher basal levels of P-STAT1 (specific fluorescence [Fsp]; i.e., Fsp(PBS), the level of P-STAT1 in PBS-treated cells) in total PBMCs, natural killer (NK) cells, and T cells (mean Fsp(PBS) in total PBMCs: 5.5 in healthy donors versus 1.6 in patients, difference = 3.9, 95% confidence interval [CI] = 1.4 to 6.5, P =.004; mean Fsp(PBS) in NK cells: 4.6 in healthy donors versus 0.9 in patients, difference = 3.7, 95% CI = 1.7 to 5.7, P =.001; mean Fsp(PBS) in T cells: 6.8 in healthy donors versus 0.9 in patients, difference = 5.9, 95% CI = 2.5 to 9.3, P =.002). P-STAT1 was detected in the NK and T cells of two patients who received IFN alpha-2b immunotherapy (20 MU/m2 [MU = million units], administered by intravenous injection). P-STAT1 levels in the PBMCs of a patient treated sequentially with 5 MU/m2 and 10 MU/m2 IFN alpha-2b (administered by subcutaneous injection) also increased in response to treatments with IFN alpha-2b but did not increase further with the increased dosage of IFN alpha-2b.

CONCLUSION

This flow cytometry method can be used to monitor STAT1 activation within subsets of immune cells from patients undergoing IFN alpha immunotherapy.

摘要

背景

信号转导子和转录激活子1（STAT1）在宿主组织内对基因表达的调控介导了干扰素α（IFNα）的抗肿瘤作用。我们使用一种新型流式细胞术检测方法，来检查体外或体内IFNα治疗后免疫效应细胞亚群内STAT1的磷酸化介导激活情况。

方法

从健康供者（n = 17）或黑色素瘤患者（n = 19）中分离出的外周血单个核细胞（PBMC），在体外分别用干扰素α-2b（IFNα-2b）或磷酸盐缓冲盐水（PBS）处理，然后进行多参数流式细胞术检测，以测量免疫细胞亚群内磷酸化STAT1（P-STAT1）的水平。我们同样分析了黑色素瘤患者在接受IFNα-2b免疫治疗前和治疗后1小时分离出的PBMC。所有统计检验均为双侧检验。

结果

在体外对PBMC进行IFNα-2b处理后的15分钟内，所有主要免疫细胞亚群中的P-STAT1水平均升高；在T淋巴细胞和单核细胞中升高最为明显。相对低剂量的IFNα-2b（即10² - 10³ IU/mL）在体外诱导了最大程度的STAT1激活。与黑色素瘤患者相比，健康供者在总PBMC、自然杀伤（NK）细胞和T细胞中的P-STAT1基础水平更高（特异性荧光[Fsp]；即Fsp（PBS），PBS处理细胞中P-STAT1的水平）（总PBMC中Fsp（PBS）的平均值：健康供者为5.5，患者为1.6，差异 = 3.9，95%置信区间[CI] = 1.4至6.5，P = 0.004；NK细胞中Fsp（PBS）的平均值：健康供者为4.6，患者为0.9，差异 = 3.7，95% CI = 1.7至5.7，P = 0.001；T细胞中Fsp（PBS）的平均值：健康供者为6.8，患者为0.9，差异 = 5.9，95% CI = 2.5至9.3，P = 0.002）。在两名接受IFNα-2b免疫治疗（20 MU/m²[MU = 百万单位]，静脉注射）的患者的NK细胞和T细胞中检测到了P-STAT1。一名先后接受5 MU/m²和10 MU/m² IFNα-2b（皮下注射）治疗的患者，其PBMC中的P-STAT1水平也因IFNα-2b治疗而升高，但并未随着IFNα-2b剂量的增加而进一步升高。