Animal models have highlighted the importance of innate lymphoid cells (ILCs) in multiple immune responses. However, technical limitations have hampered adequate characterization of ILCs in humans. Here, we used mass cytometry including a broad range of surface markers and transcription factors to accurately identify and profile ILCs across healthy and inflamed tissue types. High dimensional analysis allowed for clear phenotypic delineation of ILC2 and ILC3 subsets. We were not able to detect ILC1 cells in any of the tissues assessed, however, we identified intra-epithelial (ie)ILC1-like cells that represent a broader category of NK cells in mucosal and non-mucosal pathological tissues. In addition, we have revealed the expression of phenotypic molecules that have not been previously described for ILCs. Our analysis shows that human ILCs are highly heterogeneous cell types between individuals and tissues. It also provides a global, comprehensive, and detailed description of ILC heterogeneity in humans across patients and tissues.

Depending on the tissue microenvironment, T cells can differentiate into highly diverse subsets expressing unique trafficking receptors and cytokines. Studies of human lymphocytes have primarily focused on a limited number of parameters in blood, representing an incomplete view of the human immune system. Here, we have utilized mass cytometry to simultaneously analyze T cell trafficking and functional markers across eight different human tissues, including blood, lymphoid, and non-lymphoid tissues. These data have revealed that combinatorial expression of trafficking receptors and cytokines better defines tissue specificity. Notably, we identified numerous T helper cell subsets with overlapping cytokine expression, but only specific cytokine combinations are secreted regardless of tissue type. This indicates that T cell lineages defined in mouse models cannot be clearly distinguished in humans. Overall, our data uncover a plethora of tissue immune signatures and provide a systemic map of how T cell phenotypes are altered throughout the human body.

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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.

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