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 TH 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) TFH cells expressing interferon-gamma (IFN-gamma), 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.