Fig 1: The heterogeneity of conventional and unconventional ILC2 subsets.a UMAP visualization of Group 2 ILC with five subpopulations. Colors indicate cell types. b Expression of the indicated genes projected to UMAP of ILC2 sub-clusters. Colors indicate the scaled gene expression level. c Violin plots show the expression of featured genes in ILC2 sub-clusters. Colors represent cell clusters. d UMAP visualization of ILC2 sub-clusters described in a. Stage (left panel) and site (right panel) information of different ILC2 sub-clusters indicated by colors. e Dot plots show the top 10 DEGs in Pre_ILC2, Kit_ILC2, CCR9_ILC2, CRTH2_ILC2, PTGS2_ILC2 clusters. Colors represent the average expression and size encodes the proportion of gene-expressing cells. f ILC-C2 cluster in human fetal thymus identified previously (GEO: GSE133341) is projected to UMAP of ILC2 sub-clusters. ILC-C2 cells are highlighted and the ILC2 cells in this study are in gray (below panel). g Representative flow cytometry results show the frequencies of CCR9_ILC2 in fetal thymus (upper) and intestine (below). CCR9_ILC2 cell is gated as Lin–CD45+CD127+CD117–CRTH2–CCR9+. h Flow cytometry results show the expression of GATA-3 and ROR?t in CD117–CRTH2–CCR9+ ILC2 (red), conventional CRTH2+ ILC2 (orange) and CD117+CRTH2– ILC3 (blue) cells.
Fig 2: Study strategy and ILC-related populations analysis overview.a Schematic overview of the strategy of this study. Cells from human fetal hematopoietic (liver), lymphoid (thymus and spleen) and non-lymphoid (intestine, skin and lung) tissues at indicated gestational stages were first labeled with cell hashing antibodies and fluorescence-labeled antibody for sorting based on the sorting strategy in e. The cells from 2–6 tissues were pooled and then loaded on a droplet-based 10× genomics scRNA-seq platform. Cells of each tissue from different samples were distinguished by cell hashing barcode. The information of individual sample was listed in Supplementary information, Fig. S1. Two replicates per developmental stage, with a total of 6 samples were used in this study. b The gating strategy to identify human mature ILCs with example from 12 PCW thymus. In the 7AAD– lineage (CD3, CD4, CD5, FceRI, CD11c, CD11b, CD14 and CD19)-negative (Lin–)CD34-CD45+ cells, there are CD56+CD127– killer ILCs and CD56–CD127+ helper ILC subsets. The helper ILCs are divided into CRTH2+ ILC2, CRTH2–CD117+ ILC3 and CRTH2–Kit– putative ILC1. c Pie charts show the proportions of three mature helper ILCs of each organ based on flow cytometry analysis. The proportions were calculated based on more than three independent experiments. d Sorting strategy of human fetal lymphoid progenitors and ILC-associated populations. In the 7-AAD–Lin–CD45+ cells, CD34+CD127+ lymphoid progenitors, CD34+CD127– HSPCs, CD34–CD161+CD127+/– ILCs and CD34–CD161– cells were sorted from each tissue, and mixed at the ratios indicated in Supplementary information, Fig. S1 for scRNA-seq. e The flow cytometry results show that the percentages of the cell populations identified in d in each tissue change from 8 to 12 PCW. The results are representative of 2–3 independent experiments in each gestational week.
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