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. 2018 Nov 20;49(5):971-986.e5.
doi: 10.1016/j.immuni.2018.09.009. Epub 2018 Nov 6.

High-Dimensional Single-Cell Analysis Identifies Organ-Specific Signatures and Conserved NK Cell Subsets in Humans and Mice

Adeline Crinier  1 , Pierre Milpied  1 , Bertrand Escalière  1 , Christelle Piperoglou  2 , Justine Galluso  1 , Anaïs Balsamo  1 , Lionel Spinelli  1 , Inaki Cervera-Marzal  1 , Mikaël Ebbo  3 , Mathilde Girard-Madoux  1 , Sébastien Jaeger  1 , Emilie Bollon  4 , Sami Hamed  4 , Jean Hardwigsen  4 , Sophie Ugolini  1 , Frédéric Vély  5 , Emilie Narni-Mancinelli  1 , Eric Vivier  6
Affiliations

High-Dimensional Single-Cell Analysis Identifies Organ-Specific Signatures and Conserved NK Cell Subsets in Humans and Mice

Adeline Crinier et al. Immunity. .

Abstract

Natural killer (NK) cells are innate lymphoid cells (ILCs) involved in antimicrobial and antitumoral responses. Several NK cell subsets have been reported in humans and mice, but their heterogeneity across organs and species remains poorly characterized. We assessed the diversity of human and mouse NK cells by single-cell RNA sequencing on thousands of individual cells isolated from spleen and blood. Unbiased transcriptional clustering revealed two distinct signatures differentiating between splenic and blood NK cells. This analysis at single-cell resolution identified three subpopulations in mouse spleen and four in human spleen, and two subsets each in mouse and human blood. A comparison of transcriptomic profiles within and between species highlighted the similarity of the two major subsets, NK1 and NK2, across organs and species. This unbiased approach provides insight into the biology of NK cells and establishes a rationale for the translation of mouse studies to human physiology and disease.

Keywords: ILC; NK cells; innate immunity; scRNA-seq.

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Figures

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Graphical abstract
Figure 1
Figure 1
Mouse NK Cells Have an Organ-Specific Transcriptomic Profile (A) t-SNE plot of 8,118 NK cells from mouse blood (3,936) and spleen (4,182). (B) Heatmap of the 164 total genes distinguishing between mouse blood (63) from splenic (101) NK cells tested with a Wilcoxon rank-sum test. Cells are plotted in columns, by organ source, and genes are shown in rows, ranked by adjusted p value < 0.05. Gene expression is color coded with a scale based on z-score distribution, from −2 (purple) to 2 (yellow). Squares identify the mouse organ-specific NK cell transcriptomic signature. (C) Top ten expressed total genes and top ten expressed genes encoding secreted proteins, cell membrane markers, and transcription factors differentiating significantly between spleen and blood NK cells. Genes are ranked by p value. (D) Selected Gene Ontology terms. Benjamini and Hochberg-corrected −log10 p values from hypergeometric tests. The black dotted line represents the significance threshold set at −log10(0.05).
Figure 2
Figure 2
High-Throughput scRNA-Seq Identifies Three Mouse Splenic NK Subsets (A) t-SNE plot of 1,439 mouse splenic NK cells representative of one sample. (B) Heatmap of the 159 genes tested with a Wilcoxon rank sum test separating the 4,182 splenic NK cells into subsets. White lines separate the samples. Squares identify specific transcriptomic signatures of mouse splenic NK cell subsets. (C) Left: PCA for the three mouse splenic NK cell subsets from each sample based on the mean expression of the genes with variable expression. Right panel: driving genes for each cell subset accounting for 40% of total information in each PC. (D) Top ten genes significantly distinguishing between the three splenic mouse NK cells in a representative sample. (E) Selected Gene Ontology terms. (F) Left: Module scores of CD27−CD11b+ and CD27+CD11b− gene expression programs defined by Chiossone et al. (2009) for each spleen NK cell at the single-cell level. Middle and right: Violin plots represent the distribution of module score for CD27−CD11b+ (middle) and CD27+CD11b− (right) cells for each spleen NK cell, grouped by subsets. Kruskal-Wallis with Dunn’s multiple-comparison tests and Benjamini-Hochberg adjusted p values. Error bars indicated the mean (±SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Figure 3
Figure 3
High-Throughput scRNA-Seq Identifies Two Splenic-like NK Cell Subsets in Mouse Blood (A) t-SNE plot of 1,223 mouse blood NK cells representative of one sample. (B) Heatmap of the 87 genes tested with a Wilcoxon rank sum test separating the 3,936 mouse blood-specific NK cells into subsets. Squares identify the specific transcriptomic signatures of mouse blood NK cell subsets. (C) Left: PCA for the two mouse blood NK cell subsets. Right: driving genes. (D) Top ten genes significantly differentiating between the two blood mouse NK cell subsets in a representative sample. (E) Selected Gene Ontology terms. (F) Left: Module scores for CD27−CD11b+ and CD27+CD11b− gene expression programs. Middle and right: Violin plot showing the distribution of module scores for CD27−CD11b+ (middle) and CD27+CD11b− (right) cells, for each blood NK cell, grouped by subset. Wilcoxon rank sum test with continuity correction. Error bars indicated the mean (±SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (G) Left: PCA on the two blood and three splenic mouse NK cell subsets. Right: driving genes. (H) Heatmap of the 428 genes tested with a Wilcoxon rank sum test separating the 8,118 mouse blood and splenic NK cells into subsets. Squares identify mouse-specific transcriptomic signatures of NK cell subsets.
Figure 4
Figure 4
Human NK Cells Have an Organ-Specific Transcriptomic Profile (A) t-SNE plot of 7,403 human NK cells from blood (3,200) and spleen (4,203). (B) Heatmap of the 294 total genes distinguishing human blood (106) from splenic (188) NK cells tested with a Wilcoxon rank-sum test. (C) Top ten genes significantly differentiating spleen from blood NK cells. (D) Selected Gene Ontology terms.
Figure 5
Figure 5
High-Throughput scRNA-Seq Identifies Four Human Splenic NK Subsets (A) t-SNE plot of 1,321 human splenic NK cells representative of one sample. (B) Heatmap of the 270 genes from one representative individual tested with a Wilcoxon rank sum test discriminating the human splenic NK cells into subsets. Squares identify specific transcriptomic signatures of the human splenic NK cell subsets. (C) Left: PCA on the four human splenic NK cell subsets. Right: driving genes. (D) Top ten genes significantly differentiating between the four spleen NK cell subsets in one representative sample. (E) Selected Gene Ontology terms. hNK_Sp1 and hNK_Sp4 gene signatures are grouped. (F) Left: Module scores of CD56bright and CD56dim gene expression programs defined by Hanna et al. (2004) for each of the blood NK cells at the single-cell level. Middle and right: Violin plots representing the distribution of module score for CD56dim (middle) CD56bright (right) for each blood NK cell, grouped by subset. Kruskal-Wallis with Dunn’s multiple-comparison test and Benjamini-Hochberg adjusted p values. Error bars indicated the mean (±SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Figure 6
Figure 6
High-Throughput scRNA-Seq Identifies Two Splenic-like NK Cell Subsets in Human Blood (A) t-SNE plot of 1,166 human blood NK cells representative of one sample. (B) Heatmap of the 55 genes from one representative individual tested with a Wilcoxon rank-sum test discriminating the total 3,200 human blood NK cells into subsets. Squares identify specific transcriptomic signatures of human blood NK cell subsets. (C) Left: PCA of the two human blood NK cell subsets. Right: driving genes. (D) Top ten genes significantly differentiating between the two blood NK cell subsets in one representative sample. (E) Selected Gene Ontology terms. (F) Left: Module scores of CD56bright and CD56dim gene expression programs. Middle and right: Violin plots representing the distribution of module scores for CD56dim (middle) and CD56bright (right) cells for each blood NK cell, grouped by subset. Wilcoxon rank-sum test with continuity correction. Error bars indicated the mean (±SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (G) Left: PCA on the two blood and three splenic NK cell subsets. Right: driver genes. (H) Heatmap of the 290 genes from one representative individual tested with a Wilcoxon rank-sum test separating the total 7,403 human blood and splenic NK cells into subsets. Squares identify specific transcriptomic signatures of the human NK cell subsets.
Figure 7
Figure 7
High-Throughput scRNA-Seq Reveals Transcriptomic Signatures Common to Organs and Species (A) Spleen-specific (left) and blood-specific (right) transcriptomic signature common to human and mouse NK cells. (B) Subset-specific transcriptomic signature common to human and mouse splenic NK cells. (C) Subset-specific transcriptomic signature common to human and mouse blood NK cells. (B and C) Underlined genes are common to the blood and spleen subsets. (D) Schematic representation of mouse and human spleen and blood NK cells subsets based on scRNA-seq analysis.
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