Pejoski David, Tchitchek Nicolas, Rodriguez Pozo André, Elhmouzi-Younes Jamila, Yousfi-Bogniaho Rahima, Rogez-Kreuz Christine, Clayette Pascal, Dereuddre-Bosquet Nathalie, Lévy Yves, Cosma Antonio, Le Grand Roger, Beignon Anne-Sophie
University of Paris South, U1184, 92265 Fontenay-aux-Roses, France; French Atomic Energy and Alternative Energies Commission, Organization for the Direction of Fundamental Research/Institute of Emerging Diseases and Innovative Therapies, U1184, Immunology of Viral Infections and Autoimmune Diseases, Infectious Disease Models and Innovative Therapies Infrastructure, 92265 Fontenay-aux-Roses, France; INSERM, U1184, 94276 Le Kremlin-Bicêtre, France; Vaccine Research Institute, Henri Mondor Hospital, 94010 Créteil, France;
ImmunoPharmacology and Biosafety Laboratory, BERTIN Pharma, French Atomic Energy and Alternative Energies Commission, 92265 Fontenay-aux-Roses, France; and.
J Immunol. 2016 Jun 1;196(11):4814-31. doi: 10.4049/jimmunol.1502005. Epub 2016 May 2.
Broadening our understanding of the abundance and phenotype of B cell subsets that are induced or perturbed by exogenous Ags will improve the vaccine evaluation process. Mass cytometry (CyTOF) is being used to increase the number of markers that can be investigated in single cells, and therefore characterize cell phenotype at an unprecedented level. We designed a panel of CyTOF Abs to compare the B cell response in cynomolgus macaques at baseline, and 8 and 28 d after the second homologous immunization with modified vaccinia virus Ankara. The spanning-tree progression analysis of density-normalized events (SPADE) algorithm was used to identify clusters of CD20(+) B cells. Our data revealed the phenotypic complexity and diversity of circulating B cells at steady-state and significant vaccine-induced changes in the proportions of some B cell clusters. All SPADE clusters, including those altered quantitatively by vaccination, were characterized phenotypically and compared using double hierarchical clustering. Vaccine-altered clusters composed of previously described subsets including CD27(hi)CD21(lo) activated memory and CD27(+)CD21(+) resting memory B cells, and subphenotypes with novel patterns of marker coexpression. The expansion, followed by the contraction, of a single memory B cell SPADE cluster was positively correlated with serum anti-vaccine Ab titers. Similar results were generated by a different algorithm, automatic classification of cellular expression by nonlinear stochastic embedding. In conclusion, we present an in-depth characterization of B cell subphenotypes and proportions, before and after vaccination, using a two-step clustering analysis of CyTOF data, which is suitable for longitudinal studies and B cell subsets and biomarkers discovery.
拓宽我们对外源性抗原诱导或扰动的B细胞亚群丰度和表型的理解，将改善疫苗评估过程。质谱流式细胞术（CyTOF）正被用于增加可在单细胞中研究的标志物数量，从而以前所未有的水平表征细胞表型。我们设计了一组CyTOF抗体，以比较食蟹猴在基线时以及第二次用安卡拉痘苗病毒进行同源免疫后8天和28天的B细胞反应。使用密度归一化事件的生成树进展分析（SPADE）算法来识别CD20(+) B细胞簇。我们的数据揭示了稳态下循环B细胞的表型复杂性和多样性，以及疫苗诱导的一些B细胞簇比例的显著变化。对所有SPADE簇，包括那些因接种疫苗而在数量上改变的簇，进行表型特征分析，并使用双重层次聚类进行比较。疫苗改变的簇由先前描述的亚群组成，包括CD27(hi)CD21(lo)活化记忆B细胞和CD27(+)CD21(+)静止记忆B细胞，以及具有新型标志物共表达模式的亚表型。单个记忆B细胞SPADE簇的先扩增后收缩与血清抗疫苗抗体滴度呈正相关。通过一种不同的算法——非线性随机嵌入的细胞表达自动分类，也产生了类似的结果。总之，我们使用CyTOF数据的两步聚类分析，对疫苗接种前后的B细胞亚表型和比例进行了深入表征，该分析适用于纵向研究以及B细胞亚群和生物标志物的发现。
