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

Single-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 (T_H) cells derived from peripheral blood and tonsils, which showed differential cell composition and extensive T_H cell heterogeneity. Notably, this analysis revealed numerous subtypes of follicular helper T (T_FH) cells that followed a continuum spanning both blood and tonsils. Furthermore, we identified tonsillar CXCR5^loPD-1^loCCR7^lo T_FH cells expressing interferon-γ (IFN-γ), interleukin-17 (IL-17), or Foxp3, indicating that T_FH cells exhibit diverse functional capacities within extrafollicular stages. Regression analysis demonstrated that CXCR5^loPD-1^- and CXCR5^loPD-1^lo cells accumulate during childhood in secondary lymphoid organs, supporting previous findings that these subsets represent memory T_FH cells. This study provides an in-depth comparison of human blood and tonsillar T_FH cells and outlines a general approach for subset discovery and hypothesizing of cellular progressions. In this study, Wong et al. use mass cytometry to comprehensively probe the phenotypic and functional profiles of human blood- and tonsil-derived CD4⁺ T cells. This analysis uncovers multiple subtypes of T_FH cells spanning blood and tonsils. Regression analysis reveals that during childhood, memory T_FH cells accumulate with age.