Phenotyping of immune cell subsets in clinical trials is limited to well-defined phenotypes, due to technological limitations of reporting flow cytometry multi-dimensional phenotyping data. We developed a multi-dimensional phenotyping analysis tool and applied it to detect nitric oxide (NO) levels in peripheral blood immune cells before and after adjuvant ipilimumab co-administration with a peptide vaccine in melanoma patients. We analyzed inhibitory and stimulatory markers for immune cell phenotypes that were felt to be important in the NO analysis. The pipeline allows visualization of immune cell phenotypes without knowledge of clustering techniques and to categorize cells by association with relapse-free survival (RFS). Using this analysis, we uncovered the potential for a dichotomous role of NO as a pro- and anti-melanoma factor. NO was found in subsets of immune-suppressor cells associated with shorter-term (≤ 1 year) RFS, whereas NO was also present in immune-stimulatory effector cells obtained from patients with significant longer-term (> 1 year) RFS. These studies provide insights into the cell-specific immunomodulatory role of NO. The methods presented herein can be applied to monitor the pro- and anti-tumor effects of a variety of immune-based therapeutics in cancer patients.

Clinical Trial Registration Number: NCT00084656 (https://clinicaltrials.gov/ct2/show/NCT00084656).

由于报告流式细胞术多维表型数据的技术局限性，临床试验中免疫细胞亚型的表型仅限于定义明确的表型。我们开发了多维表型分析工具，并将其用于在黑色素瘤患者佐剂ipilimumab与肽疫苗共同施用之前和之后检测外周血免疫细胞中的一氧化氮（NO）水平。我们分析了免疫细胞表型的抑制性标记和刺激性标记，这些标记在NO分析中被认为是重要的。该管道可在不了解聚类技术的情况下可视化免疫细胞表型，并通过与无复发生存期（RFS）关联对细胞进行分类。使用该分析，我们发现了NO作为促黑素瘤和抗黑素瘤因子的二分作用的潜力。在短期（≤1年）RFS相关的免疫抑制细胞亚群中未发现NO，而长期（> 1年）RFS明显升高的患者的免疫刺激效应细胞中也未见NO。这些研究提供了NO的细胞特异性免疫调节作用的见解。本文提出的方法可以应用于监测癌症患者中各种基于免疫的治疗剂的促肿瘤和抗肿瘤作用。

临床试验注册号： NCT00084656（https://clinicaltrials.gov/ct2/show/NCT00084656）。