Institute of Laboratory Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Philipps University Marburg, German Center for Lung Research (DZL), Marburg, Germany.
Centre d'Immunophénomique Centre d'Immunophénomique (CIPHE), Aix Marseille Université, INSERM, CNRS Philipps-Universität Marburg (UMR), Marseille, France.
Front Cell Infect Microbiol. 2024 Jan 15;13:1336489. doi: 10.3389/fcimb.2023.1336489. eCollection 2023.
Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2-vaccinated and SARS-CoV-2-infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.
了解抗原特异性 T 细胞反应，例如病毒感染或过敏原暴露后的反应，对于疫苗和治疗药物的开发具有重要意义。我们旨在通过改进流式细胞术和质谱流式细胞术的染色程序来优化抗原刺激后 T 细胞的免疫表型分析。我们的方法可用于来自小鼠和人类的原代细胞，使用双条形码系统对个体样本和条件进行低频 T 细胞反应的检测。首先，在 T 细胞刺激试验之前，使用抗 CD45 抗体进行活细胞条形码标记。其次，为了区分刺激条件并防止细胞丢失，将样品条形码与商业条形码解决方案相结合。这种双重条形码方法可节省细胞，因此特别适用于细胞数量较少的样本。为了进一步减少细胞丢失并提高多重样品的去条形码效率，我们将我们的双重条形码方法与新的无离心层流（Curiox™）洗涤系统相结合。最后，为了证明我们建立的方案的优势，我们通过高参数 CyTOF 分析检测了 SARS-CoV-2 疫苗接种和 SARS-CoV-2 感染患者的病毒特异性 T 细胞反应，并与健康未暴露个体进行了比较。我们可以揭示出抗原特异性刺激后 CD4、CD8 和 gd-T 细胞之间表型的异质性。我们的方案允许从同一供体样本中检测针对病毒衍生肽、过敏原或其他抗原的微小 T 细胞群体的抗原特异性反应，以研究定性和定量差异。
