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 (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.
