设计、优化和标准化评估黑色素瘤患者t细胞免疫表型的高维光谱流式细胞术工作流程

IF 4.6 2区 医学 Q2 IMMUNOLOGY Clinical & Translational Immunology Pub Date : 2023-09-07 DOI:10.1002/cti2.1466
Jack M Edwards, Miles C Andrews, Hayley Burridge, Robin Smith, Carole Owens, Mark Edinger, Katherine Pilkington, Juliette Desfrancois, Mark Shackleton, Sashendra Senthi, Menno C van Zelm
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引用次数: 0

摘要

尽管免疫检查点阻断疗法取得了成功,但大多数转移性黑色素瘤患者对治疗没有反应或出现严重的毒性。在治疗前或早期评估生物标志物和免疫表型将有助于了解有利的反应并改善治疗结果。我们提出了一种高维的血液t细胞分析方法,使用三种多参数细胞计数面板:(1)用于绝对细胞计数的TruCount面板,(2)评估t细胞标记的27色光谱面板,(3)评估细胞内细胞因子表达的20色光谱面板。在11批染色前,冷冻保存患者和健康对照的治疗前血液单个核细胞。使用单供体对照跟踪批处理效果,并评估归一化的适用性。采用人工门控和高维策略对数据进行分析。结果批与批之间的差异是最小的,正如批控制样本的维数降低所证明的那样,标准化并没有改善人工或高维分析。工作流程的应用证明了面板的容量,并显示患者的淋巴细胞比对照组少(P = 0.0027),这是由于初始CD4+ (P = 0.015)和CD8+ (P = 0.011) T细胞和滤泡辅助T细胞(P = 0.00076)较低。患者表现出CD4+和CD8+记忆亚群中Ki67和il -2表达细胞比例升高的趋势,TCRγδ+ T细胞中CD57和EOMES表达增加。我们优化的高参数光谱细胞术方法提供了深入的血液T细胞图谱,并发现了基线时患者免疫表型的差异。我们工作流程的稳健性,正如最小批量效应所证明的那样,使得这种方法非常适合免疫治疗效果的纵向评估。
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Design, optimisation and standardisation of a high-dimensional spectral flow cytometry workflow assessing T-cell immunophenotype in patients with melanoma

Objectives

Despite the success of immune checkpoint blockade, most metastatic melanoma patients fail to respond to therapy or experience severe toxicity. Assessment of biomarkers and immunophenotypes before or early into treatment will help to understand favourable responses and improve therapeutic outcomes.

Methods

We present a high-dimensional approach for blood T-cell profiling using three multi-parameter cytometry panels: (1) a TruCount panel for absolute cell counts, (2) a 27-colour spectral panel assessing T-cell markers and (3) a 20-colour spectral panel evaluating intracellular cytokine expression. Pre-treatment blood mononuclear cells from patients and healthy controls were cryopreserved before staining across 11 batches. Batch effects were tracked using a single-donor control and the suitability of normalisation was assessed. The data were analysed using manual gating and high-dimensional strategies.

Results

Batch-to-batch variation was minimal, as demonstrated by the dimensionality reduction of batch-control samples, and normalisation did not improve manual or high-dimensional analysis. Application of the workflow demonstrated the capacity of the panels and showed that patients had fewer lymphocytes than controls (P = 0.0027), due to lower naive CD4+ (P = 0.015) and CD8+ (P = 0.011) T cells and follicular helper T cells (P = 0.00076). Patients showed trends for higher proportions of Ki67 and IL-2-expressing cells within CD4+ and CD8+ memory subsets, and increased CD57 and EOMES expression within TCRγδ+ T cells.

Conclusion

Our optimised high-parameter spectral cytometry approach provided in-depth profiling of blood T cells and found differences in patient immunophenotype at baseline. The robustness of our workflow, as demonstrated by minimal batch effects, makes this approach highly suitable for the longitudinal evaluation of immunotherapy effects.

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来源期刊
Clinical & Translational Immunology
Clinical & Translational Immunology Medicine-Immunology and Allergy
CiteScore
12.00
自引率
1.70%
发文量
77
审稿时长
13 weeks
期刊介绍: Clinical & Translational Immunology is an open access, fully peer-reviewed journal devoted to publishing cutting-edge advances in biomedical research for scientists and physicians. The Journal covers fields including cancer biology, cardiovascular research, gene therapy, immunology, vaccine development and disease pathogenesis and therapy at the earliest phases of investigation.
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