Multiomic sequencing characterization of ex vivo expanded human ILC2 cells. (A) Schematic of ex vivo 14 day expansion of peripheral human ILC2s from distinct donors. Cells from 2 different donors (A and B) were enriched via RosetteSep, and expanded in complete α-MEM media with 10 to 50 ng/mL IL-7, IL-2, IL-4, IL-25, and IL-33. (B) The transcriptome(s) of ILC2s generated across both sites were compared with those of ILC1s, ILC2s, and ILC precursors from the University of Lausanne in Switzerland.²⁸ Comparisons were calculated by scoring based on signatures derived from 2 previously published characterizations of ILC2s, as well as a merged score.^26,27 Gene signatures were calculated using Bioconductor’s singscore (V.1.4.0) simpleScore function. (C) Heat map depicting the expression of ILC2 lineage-defining genes between UNC-CH- and UMN-derived samples expanded after RosetteSep, and subsequent culture in α-MEM with IL-7, IL-2, IL-4, IL-25, and IL-33 for 14 days at each site. (D) Comparison of the similarity of ILC2s generated as described earlier at UNC-CH or UMN to the transcriptional landscape of human Th1, Th2, or Th17 subsets from healthy donors as presented in other PB data sets. (E-F) Representative tracks of normalized ATAC signal from samples expanded at UNC-CH on day 0 (E), or day 21 (F) at ILC1- (Tbx21 and Ifng), ILC2- (Gata3 and Il13), and ILC3-associated (Rorc and Il17a) gene loci. Tick marks indicate specific locations of differential relative chromatin accessibility (RCA) peaks. (G) Peaks detected by MACS2 and matched by HOMER to the 6 genes of interest in panels E-F (not filtered or weighted by distance to nearest transcriptional start site [TSS] or by peak score). (H) HOMER motif output for day 21 vs day 0 samples (sequence logo). Analysis was performed by finding differential expression by feature count of consensus peaks between all samples, and filtering to only include peaks that had ≥2.0 log₂ fold change in day 21 samples, then finding motifs within those peaks.