Innate lymphoid cells (ILCs) are increasingly appreciated as important participants in homeostasis and inflammation. Substantial plasticity and heterogeneity among ILC populations have been reported. Here we have delineated the heterogeneity of human ILCs through single-cell RNA sequencing of several hundreds of individual tonsil CD127+ ILCs and natural killer (NK) cells. Unbiased transcriptional clustering revealed four distinct populations, corresponding to ILC1 cells, ILC2 cells, ILC3 cells and NK cells, with their respective transcriptomes recapitulating known as well as unknown transcriptional profiles. The single-cell resolution additionally divulged three transcriptionally and functionally diverse subpopulations of ILC3 cells. Our systematic comparison of single-cell transcriptional variation within and between ILC populations provides new insight into ILC biology during homeostasis, with additional implications for dysregulation of the immune system.
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Integrated supplementary information
Histograms showing number of reads (a), percent uniquely mapping reads (b), fraction mismatches (c), fraction exon mapping reads (d), fraction of reads mapping to a region at the 10% most 3prime end of each transcript (e) and number of mRNA reads (f). Blue bars represents empty well controls and red bars represent wells containing one cell. Black bold lines indicates filtering cutoffs where cells above/below the black line have been removed. Data were generated in three independent experiments with one tonsil donor each (n=798).
a) Detection of biologically variable transcripts over technical noise with ERCC spike-in RNAs highlighted in black, human transcripts in blue (variable) or red (non-variable). Violin plots showing distributions of: b) Forward scattering (FSC), c) ratio of cell RNA to ERCC spike-in RNA (ERCC-ratio) and d) number of detected transcripts. e) PCA based on 847 variable transcripts, before (upper panel) and after (lower panel) batch normalization, cells colored either by surface phenotype (left panel) or donor (left panel) origin. Data were generated in three independent experiments with one tonsil donor each (total number of cells per cell population: ILC1, n=112; ILC2, n=143; ILC3, n=320; NK cells, n=73).
a) Pairwise comparison of the mean expression of the top 50 differentially expressed transcripts for the two cell populations in question. Cells are colored according to cluster definition described in main Fig. 2. Cells where cluster definition and surface phenotype were in agreement are shown with a star (*). Cells that deviated in the clustering are either highlighted as cross (x) if they were defined as the other phenotype in the comparison, and as a circle (o) if the cell had another phenotype than the 2 cell populations in question. b) ERCC-ratio plots for each plot shown in a demonstrating the ratio of cellular RNA to ERCC spike-in RNA for each cell. Data were generated in three independent experiments with one tonsil donor each. Total number of cells per cell population: ILC1, n=112; ILC2, n=143; ILC3, n=320; NK cells, n=73.
Each plot shows the surface protein expression intensity (y-axis) vs. log2(RPKM) RNA expression (x-axis) as measured by flow cytometry (indexed flow cytometric sorting data collection) and scRNA-seq, respectively. Both quantities were normalized on a scale from 0-1. Correlation values, both Spearman and Pearson are shown in the titles. Data were generated in three independent experiments with one tonsil donor each (n=648).
Violin plots with expression distribution in each cell population on log2(rpkm) scale for ILC and NK specific transcripts. Coloring according to mean expression. a) Other transcripts commonly expressed by ILCs (according to SCDE; multiple-testing corrected p-value < 0.001). b) Transcription factors commonly expressed by ILCs (according to SCDE; multiple-testing corrected p-value < 0.05). c) Transcripts known to be expressed by NK cells d) Other transcripts expressed by NK cells (according to SCDE; multipletesting corrected p-value < 0.001 for both c and d). Data were generated in three independent experiments with one tonsil donor each (total number of cells per cell population: ILC1, n=112; ILC2, n=143; ILC3, n=320; NK cells, n=73).
a) Heatmap of T-cell related transcripts, expression in ILC1s (blue), ILC2s (cyan), NK cells (green) and ILC3s (red). Each vertical line in the heatmap represents the expression intensity in an individual cell. Color intensities according to log2(rpkm) values. b) Number of significantly differentially expressed genes (p-value < 0.001) in ILC1s (blue), ILC2s (cyan) and ILC3s (red) with random subsampling of 25,50,75 or 100 cells from each population. Error bars represents standard deviation from 10 iterations. Data were generated in three independent experiments with one tonsil donor each (total number of cells per cell population: ILC1, n=112; ILC2, n=143; ILC3, n=320; NK cells, n=73).
a) Top 20 transcript loadings for principal components 1 (PC1) and 2 (PC2) in PCA with ILC3s (Fig. 8a). b) t-SNE plots with the ILC3s colored according to expression intensity of some selected transcripts. Data were generated in three independent experiments with one tonsil donor each (n=320).
a) t-SNE plots based on surface marker intensities for 5 adult donors and 7 pediatric donors (n=20543) with intensities of the 4 markers used for t-SNE (NKp44, HLA-DR, CD62L and CD45RA) followed by donor distribution (red to yellow shades for pediatric, blue to green shades for adults). b) Bar charts show percentage of IL-2+, IL-22+, IL-17F+ and TNF+ cells from the indicated ILC3 subpopulations after IL-23 plus IL-1β (50 ng/ml each, for 12+6 hours) and/or PMA plus ionomycin (20 ng/ml plus 0.5 μM, for the last 6 hours). Bars show mean and SEM from 4-6 donors. **** p<0.0001, *** p<0.005, ** p<0.01 and * p<0.05 as calculated using oneway ANOVA and Tukey’s multi-comparisons test. c) Representative dot plots show intracellular IL-2 and IL-22 production by the different ILC3 subpopulations after the indicated stimulations.
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Björklund, Å., Forkel, M., Picelli, S. et al. The heterogeneity of human CD127+ innate lymphoid cells revealed by single-cell RNA sequencing. Nat Immunol 17, 451–460 (2016). https://doi.org/10.1038/ni.3368
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