Extended Data Fig. 9: ILC2s are uniquely activated by the microbiota in the middle colon.

a, Number of DEGs between SPF and GF mice in immune cell populations from the single-cell data (stars mark the section without comparison due to low number of cells, N < 30). b, Expression of genes from single-cell RNA-seq data of ILC2s from different regions of colon (A-D; proximal-distal). Data summarize pooling of SPF, GF, and FMT treated mice. c, Representative flow cytometric gating strategy for ILC2s and eosinophils isolated from mouse colon. This gating strategy was used for summary data presented in f, h, i, and j and Extended Data Fig. 10c,e. Gating shown is from n = 1 SPF mouse region B. d, Representative histograms showing expression of CD28 on the surface of ILC2s gated as in c for SPF (purple) and GF (black) mice from colon region B. e, Representative histograms for the expression of surface markers ST2, IL17RB, CD25, and CD127 from left to right on ILC2s from colon region B. f, Data summarizing the median fluorescence intensity (MFI) for surface markers in e on ILC2s from regions A to D in SPF (purple) and GF (black) mice. n = 9 mice from 3 independent experiments for SPF and n = 6 mice from 2 independent experiments for GF for ST2 and IL17RB. n = 15 mice from 5 independent experiments for SPF and n = 9 mice from 3 independent experiments for GF for CD25. n = 12 mice from 4 independent experiments for SPF and n = 9 mice from 3 independent experiments for GF for CD127. Box limits are the upper and lower quartiles with the middle line representing the median; and whiskers are 1.5 times the interquartile range from the box. g, Representative flow cytometric gating for IL-5 and IL-13 on ILC2s from colon region B gated as shown in b. Box limits are the upper and lower quartiles with the middle line representing the median; and whiskers are 1.5 times the interquartile range from the box. h, Data summarizing the frequency of ILC2s from colon regions A to D expressing IL-5, IL-13, or both from left to right in SPF (purple) and GF (black) mice. n = 6 mice per group summarized from 2 independent experiments. i, Data summarizing the number of ILC2s recovered from colon regions A-D gated as in c in SPF (purple) and GF (black) mice. n = 22 mice from 7 independent experiments for SPF and n = 9 mice from 3 independent experiments for GF. j, Data summarizing MFI of CD28 on ILC2s isolated from GF colon region B following explant treatment with recombinant IL-33 and IL-25 (purple) or media control (black) for 12hrs. Data summarizing n = 6 mice from 2 independent experiments. Box limits are the upper and lower quartiles with the middle line representing the median; and whiskers are 1.5 times the interquartile range from the box. k, Data summarizing the amount of IL-5 and IL-13 detected in pg/mL in supernatants from explants of colon tissue from GF colon region B treated with IL-33 and IL-25 as in j. Data summarizing n = 6 mice from 2 independent experiments. Box limits are the upper and lower quartiles with the middle line representing the median; and whiskers are 1.5 times the interquartile range from the box. l, Data summarizing expression of selected middle colon genes by quantitative PCR from explants of GF colon region B treated with IL-4/5/13 for 6 hrs. Gene expression relative to the housekeeping gene Gapdh. n = 4 mice representative of 2 independent experiments. Box limits are the upper and lower quartiles with the middle line representing the median; and whiskers are 1.5 times the interquartile range from the box.