Fig. 3: Flow cytometric ILCs identification and quantification in human tonsils (n = 5 independent biological samples performed in a single batch).

A Example of the gating strategy used for identification of ILCs in tonsil, with ILCs defined as lineage negative (Lin⁻) (CD141⁻ CD14⁻ CD11c⁻ CD20⁻ FcεRIα⁻ CD3⁻ CD34⁻ CD123⁻ CD1a⁻) and CD45⁺ CD127(IL-7Rα)⁺ CD56⁻. Following viability assessment, unwanted lineage markers were excluded, and ILCs pool population was defined through the expression of IL-7Rα and the absence of expression of CD56 NK cell marker. B ILCs identification according to Crinier A. et al.²⁴, Yudanin et al.²², Bjorklünd et al.⁴³ based on the relative co-expression of CD117 and CD294 (CRTH2) markers. ILC1 were defined as CD117⁻ CRTH2⁻, ILC2 were defined as CD117^± CRTH2⁺, ILC3 were defined as CD117⁺ CRTH2⁻. Co-expression of T-Bet/EOMES, GATA3/ICOS, and AHR/RORγT assessment in each determined ILCs subset. C ILCs identification according to mIF gating strategy. ILCs pool was defined as LIN⁻ CD45⁺ CD3⁻ CD56⁻ CD127⁺ and the populations were defined as T-Bet⁺ EOMES^± for ILC1, GATA3⁺ ICOS⁺ for ILC2 and RORγT⁺ AHR⁺ for ILC3. Violin plot represents the quantification of ILC1, ILC2, and ILC3 subsets according to the mIF gating strategy in tonsil. T-Bet and EOMES expression (“ILC1 profile”) were assessed in NK cells populations either positive or negative for IL-7Rα expression. D FC and mIF quantification obtained values (% relative to IL-7Rα⁺ population) were compared through normalized means of cell percentages observed. Source data are provided as a Source Data file.