Mapping the Diversity of Follicular Helper T Cells in Human Blood and Tonsils Using High-Dimensional Mass Cytometry Analysis

• High-dimensional analysis of relationships between T cell phenotype and function
• Identification of IFN-γ+ TH1-, IL-17+ TH17-, and Foxp3+ Treg-like TFH cells
• Functional diversity of TFH cells concentrated at extrafollicular stages
• Memory TFH cell accumulation in tonsils during childhood

SummarySingle-cell analysis technologies such as mass cytometry allow for measurements of cellular heterogeneity with unprecedented dimensionality. Here, we applied dimensionality reduction and automated clustering methods on human T helper (TH) cells derived from peripheral blood and tonsils, which showed differential cell composition and extensive TH cell heterogeneity. Notably, this analysis revealed numerous subtypes of follicular helper T (TFH) cells that followed a continuum spanning both blood and tonsils. Furthermore, we identified tonsillar CXCR5loPD-1loCCR7lo TFH cells expressing interferon-γ (IFN-γ), interleukin-17 (IL-17), or Foxp3, indicating that TFH cells exhibit diverse functional capacities within extrafollicular stages. Regression analysis demonstrated that CXCR5loPD-1− and CXCR5loPD-1lo cells accumulate during childhood in secondary lymphoid organs, supporting previous findings that these subsets represent memory TFH cells. This study provides an in-depth comparison of human blood and tonsillar TFH cells and outlines a general approach for subset discovery and hypothesizing of cellular progressions.

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