Figure 1.

Innate lymphoid cells frequency and subtypes in AML patients at diagnosis. Total circulating ILCs were identified as Lineage [(FITC-conjugated anti-CD3, anti-CD4, anti-CD14, anti-CD16, anti-CD19 (Beckman Coulter), anti-CD8, anti-CD15 (AbD Serotech), anti-CD20, anti-CD33, anti-CD34, anti-CD203c and anti-FceRI (BioLegend)] negative CD127⁺ (PerCP-Cy5.5- or Brilliant Violet 421-conjugated anti-CD127 (BioLegend)] lymphocytes. Since a non-negligible proportion of ILC3 can express CD56,¹⁰ we excluded this marker from the lineage cocktail. (A) Representative example of the gating strategy to determine, by multiparameter flow cytometry, in healthy donors (HD) and AML patients, the percentage (B) and the absolute number (C) of total circulating ILCs. Histograms represent the mean ± SEM of total Lin⁻CD127⁺ ILCs in PB of healthy subjects and AML patients. (D) Correlation between the number of circulating ILCs and the percentage of circulating blasts. (E) Representative example of the gating strategy to determine ILC1, ILC2 and ILC3 according to CRTH2 versus cKit expression and subsequent evaluation of T-bet expression (PE CF594-conjugated) in ILC1, GATA-3 (PE-Cy7-conjugated) in ILC2 and Rorγt (PE-conjugated) in ILC3 (all from BD Pharmingen) of one representative healthy donor. In clear gray the histogram of the isotype control. ILC subsets were identified as previously described,⁷ by using PE CF594- or Brilliant Violet 421-conjugated anti-CRTH2 (BD Pharmingen); PerCP-Cy5.5- or PE-Cy7-conjugated anti-NKp46; PE⁻ or APC-conjugated anti-cKit; APC-eFluor780-conjugated anti-CD56 (eBioscience). (F and G) Histograms represent the mean ± SEM of the percentage of ILC subsets within total Lin⁻CD127⁺ ILCs of healthy donors and AML patients at diagnosis (F), or of BM and PB of AML paired samples at diagnosis (G), setting total ILCs as the 100%. **P<0.01; ***P<0.001.