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
{"title":"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","authors":"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","doi":"10.1002/cti2.1466","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> Objectives</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Methods</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>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 (<i>P</i> = 0.0027), due to lower naive CD4<sup>+</sup> (<i>P</i> = 0.015) and CD8<sup>+</sup> (<i>P</i> = 0.011) T cells and follicular helper T cells (<i>P</i> = 0.00076). Patients showed trends for higher proportions of Ki67 and IL-2-expressing cells within CD4<sup>+</sup> and CD8<sup>+</sup> memory subsets, and increased CD57 and EOMES expression within TCRγδ<sup>+</sup> T cells.</p>\n </section>\n \n <section>\n \n <h3> Conclusion</h3>\n \n <p>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.</p>\n </section>\n </div>","PeriodicalId":152,"journal":{"name":"Clinical & Translational Immunology","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2023-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cti2.1466","citationCount":"0","resultStr":"{\"title\":\"Design, optimisation and standardisation of a high-dimensional spectral flow cytometry workflow assessing T-cell immunophenotype in patients with melanoma\",\"authors\":\"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\",\"doi\":\"10.1002/cti2.1466\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n \\n <section>\\n \\n <h3> Objectives</h3>\\n \\n <p>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.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Methods</h3>\\n \\n <p>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.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Results</h3>\\n \\n <p>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 (<i>P</i> = 0.0027), due to lower naive CD4<sup>+</sup> (<i>P</i> = 0.015) and CD8<sup>+</sup> (<i>P</i> = 0.011) T cells and follicular helper T cells (<i>P</i> = 0.00076). Patients showed trends for higher proportions of Ki67 and IL-2-expressing cells within CD4<sup>+</sup> and CD8<sup>+</sup> memory subsets, and increased CD57 and EOMES expression within TCRγδ<sup>+</sup> T cells.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Conclusion</h3>\\n \\n <p>Our optimised high-parameter spectral cytometry approach provided in-depth profiling of blood T cells and found differences in patient immunophenotype at baseline. 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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.
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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