Figure 1: ILC composition of mucosal LNs differs from peripheral LNs.

(a) Gating strategy for detection of ILC populations by flow cytometry with T-bet⁺ ILC1, GATA-3⁺ ILC2 and RORγ⁺ ILC3. T-bet isotype control is shown in grey. Plots are representative of 10 mice from three independent experiments. (b) Enumeration of ILC1, ILC2 and ILC3 cells per individual inguinal (i; n=8), brachial (b; n=12 except for ILC1 where=8) or mesenteric (m; n=14 except for ILC1 where=10) LNs of WT mice. Data pooled from a minimum of three independent experiments. (c) Pie charts showing the median percentage of total Lin⁻IL-7Rα⁺ cells that are ILC1, ILC2 and ILC3 populations in different LNs, including also popliteal (p; n=3) and mediastinal (md; n=3) LNs. Data pooled from a minimum of three independent experiments; white segment shows % cells outside transcription factor gates. (d) Percentage of ILCs expressing RORγ in different LNs (n=3, 8, 12, 14, 3 mice per group), pooled from a minimum of three independent experiments. (e) Percentage of RORγ⁺ ILC3s expressing CCR6, CD4, MHCII and NKp46 in mLN. (f) Expression of MHCII and NKp46 by RORγ⁺ ILC3s within the mLN. Plots are representative of 10 mice from three independent experiments. (g) Expression of IL-22 by CD4⁺ RORγ⁺ ILC3s. Plots are representative of eight mice from three independent experiments. (h) Percentage of CD4⁺ and CD4⁻ RORγ⁺ ILC3s in mLN and iLN expressing IL-22 after culture with IL-23 (n=8, 9). Data pooled from three independent experiments. (i) Number of 2W1S:I-A^b+ CD4 T cells per peripheral LN pool from H2-Ab1^fl/fl and MHCII^ΔILC3 mice (n=4, 4) 6 days post i.p. immunization with 2W1S peptide. Data pooled from one experiment. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 (Mann–Whitney non-parametric, two-tailed test). Bars represent the median values in all graphs.