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(lo)PD-1(lo)CCR7(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(lo)PD-1(-) and CXCR5(lo)PD-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.