Varker Kimberly A, Kondadasula Sri Vidya, Go Michael R, Lesinski Gregory B, Ghosh-Berkebile Rupa, Lehman Amy, Monk J Paul, Olencki Thomas, Kendra Kari, Carson William E
Department of Surgery, Ohio State University Comprehensive Cancer Center, Columbus, OH 43210, USA.
Clin Cancer Res. 2006 Oct 1;12(19):5850-8. doi: 10.1158/1078-0432.CCR-06-1159.
Treatment with interleukin (IL)-2 (Proleukin) yields a 10% to 20% response rate in patients with metastatic melanoma or metastatic renal cell carcinoma. IL-2 is known to activate distinct signals within lymphocytes, including the Janus-activated kinase-signal transducer and activator of transcription (STAT) pathway. We examined the phosphorylation of STAT5 (P-STAT5) in IL-2-stimulated immune cells of normal subjects and in patients receiving IL-2 therapy using a novel flow cytometric assay to characterize the pattern and level of activation within immune subsets.
Normal peripheral blood mononuclear cells (PBMC) were treated in vitro with IL-2 and analyzed for P-STAT5 using an intracellular flow cytometric assay. PBMC were simultaneously evaluated for the induction of STAT5-regulated genes at the transcript level. PBMC were also obtained from patients immediately before and 1 hour after treatment with high-dose IL-2 and analyzed for the presence of P-STAT5 within immune cell subsets by dual-variable intracellular flow cytometry.
In vitro IL-2 treatment produced a rapid and dose-dependent increase in P-STAT5 within normal PBMC that correlated with the induction of transcript for the IL-2-responsive genes CIS, Pim-1, and SOCS1 (correlation coefficients 0.8628, 0.6667, and 0.7828, respectively). Dose-dependent induction of P-STAT5 was detected in PBMC for up to 18 hours following in vitro pulse stimulation with IL-2. P-STAT5 was detected within a subset of normal donor CD4(+) T cells (52.2 +/- 15.0%), CD8(+) T cells (57.6 +/- 25.8%), and CD56(+) natural killer (NK) cells (54.2 +/- 27.2%), but not CD14(+) monocytes or CD21(+) B cells, following in vitro IL-2 treatment. The generation of P-STAT5 within immune cell subsets after the therapeutic administration of IL-2 varied significantly between individuals. NK cells were noticeably absent in the posttreatment sample, a finding that was consistent for all patients examined. Surprisingly, activated STAT5 persisted within CD4(+) and CD8(+) T lymphocytes, as well as CD56(+) NK cells, for up to 3 weeks post-IL-2 treatment in three patients who exhibited a clinical response to therapy and in a fourth who exhibited a significant inflammatory response after 11 doses of therapy (first cycle).
The flow cytometric assay described herein is a highly efficient and reliable method by which to assess the cellular response to IL-2 within PBMC and specific immune effector subsets, both in vitro and in the clinical setting. Assessment of P-STAT5 in patient PBMC in response to therapeutic IL-2 administration reveals disparate responses between immune cell subsets as well as interpatient variation. Persistent activation of STAT5 within NK and T cells was an unexpected observation and requires further investigation.
白细胞介素（IL）-2（普罗力）治疗转移性黑色素瘤或转移性肾细胞癌患者的有效率为10%至20%。已知IL-2可激活淋巴细胞内不同信号，包括Janus激活激酶-信号转导子和转录激活子（STAT）途径。我们使用一种新型流式细胞术检测方法，检测正常受试者经IL-2刺激的免疫细胞以及接受IL-2治疗患者的免疫细胞中STAT5（P-STAT5）的磷酸化情况，以表征免疫亚群内激活的模式和水平。
正常外周血单个核细胞（PBMC）在体外经IL-2处理，并使用细胞内流式细胞术检测方法分析P-STAT5。同时在转录水平评估PBMC中STAT5调节基因的诱导情况。还在高剂量IL-2治疗前及治疗后1小时从患者获取PBMC，并通过双变量细胞内流式细胞术分析免疫细胞亚群中P-STAT5的存在情况。
体外IL-2处理使正常PBMC中P-STAT5迅速且呈剂量依赖性增加，这与IL-2反应性基因CIS、Pim-1和SOCS1转录本的诱导相关（相关系数分别为0.8628、0.6667和0.7828）。体外经IL-2脉冲刺激后，PBMC中P-STAT5呈剂量依赖性诱导，持续长达18小时。体外经IL-2处理后，在正常供体的一部分CD4(+) T细胞（52.2±15.0%）、CD8(+) T细胞（57.6±25.8%）和CD56(+)自然杀伤（NK）细胞（54.2±27.2%）中检测到P-STAT5，但在CD14(+)单核细胞或CD21(+) B细胞中未检测到。IL-2治疗后免疫细胞亚群中P-STAT5的产生在个体间差异显著。治疗后样本中明显未检测到NK细胞，这一发现对所有检测患者均一致。令人惊讶的是，在三名对治疗有临床反应的患者以及第四名在接受11剂治疗（第一周期）后出现显著炎症反应的患者中，IL-2治疗后长达3周，活化的STAT5持续存在于CD4(+)和CD8(+) T淋巴细胞以及CD56(+) NK细胞中。
本文所述的流式细胞术检测方法是一种高效且可靠的方法，可用于评估体外和临床环境中PBMC及特定免疫效应亚群对IL-2的细胞反应。评估患者PBMC中对治疗性IL-2给药的P-STAT5反应，揭示了免疫细胞亚群之间以及患者间的不同反应。NK细胞和T细胞中STAT5的持续激活是一个意外发现，需要进一步研究。
