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Innate-like functions of natural killer T cell subsets result from highly divergent gene programs

Nature Immunology volume 17, pages 728–739 (2016)Cite this article

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A Publisher Correction to this article was published on 17 October 2019

This article has been updated

Abstract

Natural killer T cells (NKT cells) have stimulatory or inhibitory effects on the immune response that can be attributed in part to the existence of functional subsets of NKT cells. These subsets have been characterized only on the basis of the differential expression of a few transcription factors and cell-surface molecules. Here we have analyzed purified populations of thymic NKT cell subsets at both the transcriptomic level and epigenomic level and by single-cell RNA sequencing. Our data indicated that despite their similar antigen specificity, the functional NKT cell subsets were highly divergent populations with many gene-expression and epigenetic differences. Therefore, the thymus 'imprints' distinct gene programs on subsets of innate-like NKT cells that probably impart differences in proliferative capacity, homing, and effector functions.

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Figure 1: Transcriptional and enhancer profiles of iNKT cell subsets.
Figure 2: Bulk and single-cell RNA-Seq profiling of iNKT cell subsets.
Figure 3: NKT2-enriched transcripts.
Figure 4: NKT17-enriched transcripts.
Figure 5: NKT1-enriched transcripts.
Figure 6: Gene expression in NKT0 cells.
Figure 7: TF-binding motifs in enhancers linked to iNKT cell subset differentiation.

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Change history

  • 26 April 2016

    In the version of this supplementary file originally posted online, Supplementary Figures 7, 9, and 21 were rotated and cropped incorrectly. These errors have been corrected in this file as of 26 April 2016.

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Acknowledgements

We thank C. Kim, L. Nosworthy and K. Van Gundt for assisting with single cell sorting; J. Day for assistance with next generation sequencing; Z. Fu, A.M. Gholami and J. Greenbaum for help with processing and analysis of sequencing data; and K.M. Ansel (University of California, San Francisco) and A. Rao (La Jolla Institute for Allergy & Immunology ) for HS V–deficient mice. Supported by the Alexander von Humboldt Foundation (L.C.), The William K. Bowes Jr Foundation (P.V.) and the US National Institutes of Health (R01 HL114093 to P.V.; U19 AI100275 to P.V.; and R37 AI71922 and R01 AI105215 to M.K.).

Author information

Author notes

  1. Lukas Chavez

    Present address: Present address: Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Heidelberg, Germany.,

  2. Isaac Engel, Grégory Seumois and Lukas Chavez: These authors contributed equally to this work.

  3. Pandurangan Vijayanand and Mitchell Kronenberg: These authors jointly directed this work.

Authors and Affiliations

  1. La Jolla Institute for Allergy & Immunology, La Jolla, California, USA

    Isaac Engel, Grégory Seumois, Lukas Chavez, Daniela Samaniego-Castruita, Brandie White, Ashu Chawla, Pandurangan Vijayanand & Mitchell Kronenberg

  2. David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Dennis Mock

  3. Clinical and Experimental Sciences, Southampton NIHR Respiratory Biomedical Research Unit, Faculty of Medicine, University of Southampton, Southampton, UK

    Pandurangan Vijayanand

  4. Division of Biological Sciences, University of California San Diego, La Jolla, California, USA

    Mitchell Kronenberg

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Contributions

I.E., G.S., P.V. and M.K. conceived of the work, designed, performed and analyzed experiments, and wrote the paper; I.E. performed all cell isolation, iNKT subset phenotyping and functional experiments with wild-type and HS V–deficient mice; G.S. performed 'micro-scaled' RNA-Seq, ChIP-Seq and single-cell RNA-Seq experiments; D.S.-C. and A.C. assisted in the analysis of bulk and single-cell RNA-Seq data under the supervision of L.C.; B.W. assisted in single-cell sequencing experiments under the supervision of G.S. and P.V.; and D.M. provided statistical input and support and direction for the single-cell RNA-Seq analysis.

Corresponding authors

Correspondence to Pandurangan Vijayanand or Mitchell Kronenberg.

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Integrated supplementary information

Supplementary Figure 1 Cell-sorting strategy and scope of study.

Left: sorting strategy for isolating NKT0, NKT1, NKT2 and NKT17 subsets from the thymus. Data are representative of three individual experiments using a pool of thymocytes prepared from five five-week old C57BL/6J females. Cell suspensions were enriched for iNKT cells as described in the Online Methods section. Right: the number of samples processed that passed quality control checks for the bulk RNA-Seq, single-cell RNA-Seq and H3K27ac ChIP-Seq assays is shown.

Supplementary Figure 2 Gene expression and H3K27ac patterns for selected iNKT cell subset–specific genes.

Bulk RNA-Seq and H3K27ac enrichment tracks for sorted NKT1, NKT2 and NKT17 thymocyte subsets (data merged from 3-4 biological replicates) are shown for the indicated gene loci along with UCSC gene tracks (top row).

Supplementary Figure 3 Divergent gene expression program in NKT cell subsets.

(a) Top: unsupervised hierarchical clustering of 203 single-cell transcriptomes based on the expression of genes with the highest variance (n=1500, which accounts for 34% of the total variance); each blue, yellow, green and magenta bar represents a single NKT2, NKT0, NKT1 or NKT17 cell, respectively. Bottom: heatmap below shows row-wise Z-scores of the library size normalized read counts from 203 single-cell RNA-Seq assays for the expression of 59 cell cycle genes obtained from Reactome Pathway Database (rows). Samples (single cells) and genes were ordered as above; color key represents Z-score values of gene expression. (b) Principal component analysis of bulk RNA-Seq data of NKT subsets shows highly divergent gene expression programs.

Supplementary Figure 4 Differential gene-expression analysis of bulk RNA-Seq data from iNKT cell subsets.

‘Minus-average’ (MA) plots illustrate differentially expressed genes for pairwise comparisons of bulk RNA-Seq data from NKT1, NKT2 and NKT17 cells (Benjamini-Hochberg adjusted P < 0.05 (5% FDR) and > 2 fold change in expression, calculated using Bioconductor package DESeq. For details see Online Methods and Supplementary Table 2.

Supplementary Figure 5 Changes in enhancer strength in comparisons of iNKT cell subsets.

Row-wise Z-scores of library size normalized read counts for all differentially enriched cis-regulatory regions (DERs, rows) obtained from any of the three pairwise comparisons of NKT1, NKT2 and NKT17 subsets (raw P < 0.05 and false discovery rate (FDR) < 0.1, calculated by Benjamini-Hochberg method, using edge R integrated in Bioconductor package MEDIPS); data from each independent H3K27ac ChIP-Seq assay (n = 11) is shown in the columns.

Supplementary Figure 6 Correspondence of bulk and single-cell RNA-Seq analysis.

(a) Heatmap in the left and middle panel show row-wise Z-scores of mean RPKM values from bulk and single-cell RNA-Seq assays for the genes identified as iNKT cell subset enriched by bulk RNA-Seq analysis. The percentage of iNKT cell subset enriched genes that show concordant patterns (greater than 2-fold increase compared to other subsets) in single-cell RNA-Seq assay is shown. Individual single-cell heatmap for the same genes is shown in the right panel; Columns indicate row-wise Z-scores of RPKM values from 203 single-cell RNA-Seq assays; green, blue and magenta bars at top represent cells from the NKT1, NKT2 or NKT17 cell subsets, respectively. (b) Correlation of the expression values between bulk and single-cell RNA-Seq assays for iNKT cell subset enriched genes is shown for the corresponding iNKT cell subset. Dots are colored according to a heatmap indicating the fraction of single cells expressing each gene. r value indicates the Spearman correlation coefficient. P values were calculated using “asymptotic t approximation”.

Supplementary Figure 7 Single-cell RNA-Seq analysis of NKT2-enriched genes.

(a) PCA plots, with central plot showing the four iNKT cell subsets as in Figure 1d with NKT2 cells colored in blue. Shown surrounding the center plot are the same PCA plots with individual cells colored according to their relative expression of the indicated subset-specific transcripts mapped back to the subset PCA plot. Color key represents normalized gene expression with mean 0 and variance 1. (b) Scatter plot of single-cell RNA-Seq data showing expression values (log2 normalized counts) for the indicated genes. Each dot shown in yellow, green, blue and magenta color is a single NKT0, NKT1, NKT2 and NKT17 cell, respectively. (c) Histogram plots depict staining for ICOS and PD-1 (Pdcd1) in electronically gated iNKT thymocyte subsets from C57BL/6J mice. Data are representative of at least four independent experiments, each of which using thymocytes prepared from at least two age-matched female mice examined either as a pool or separately analyzed in parallel.

Supplementary Figure 8 Single-cell RNA-Seq analysis of NKT17-enriched genes.

(a) PCA plots, with central plot showing the four iNKT cell subsets as in Figure 1d with NKT17 cells colored in magenta. Shown surrounding the center plot are the same PCA plots with individual cells colored according to their relative expression of the indicated subset-specific transcripts mapped back to the subset PCA plot. Color key represents normalized gene expression with mean 0 and variance 1. (b) Scatter plot of single-cell RNA-Seq data showing expression values (log2 normalized counts) for the indicated genes. Each dot shown in yellow, green, blue and magenta color is a single NKT0, NKT1, NKT2 and NKT17 cell, respectively. (c) Histogram plot depicts staining for IL-12RB1 in electronically gated iNKT thymocyte subsets from C57BL/6J mice. Data are representative of two independent experiments, each of which using a pool of thymocytes prepared from three age-matched female mice.

Supplementary Figure 9 Single-cell RNA-Seq analysis of NKT1-enriched genes.

(a) PCA plots, with central plot showing the four iNKT cell subsets as in Figure 1d with NKT1 cells colored in green. Shown surrounding the center plot are the same PCA plots with individual cells colored according to their relative expression of the indicated subset-specific transcripts mapped back to the subset PCA plot. Color key represents normalized gene expression with mean 0 and variance 1. (b) Scatter plot of single-cell RNA-Seq data showing expression values (log2 normalized counts) for the indicated genes. Each dot shown in yellow, green, blue and magenta color is a single NKT0, NKT1, NKT2 and NKT17 cell, respectively. (c) Histogram plots depict staining for Bcl2 and Runx3 in electronically gated iNKT thymocyte subsets from C57BL/6J mice. Data are representative of at least two independent experiments, each of which using thymocytes prepared from at least two age-matched female mice examined either as a pool or separately analyzed in parallel.

Supplementary Figure 10 Surface expression of CCR4, CXCR3 and CD49A by NKT subsets.

Histogram plots depict staining for CCR4, CXCR3, and CD49A in electronically gated iNKT thymocyte subsets from C57BL/6J mice. Data for CCR4 and CXCR3 are representative of at least three independent experiments utilizing thymocytes pooled from 3-6 mice. Data for CD49A are representative of one experiment that used thymocytes pooled from three females, and two additional experiments, each of which separately analyzed thymocytes from two females.

Supplementary Figure 11 Gene-expression and H3K27ac patterns for differentially expressed transcripts encoding integrin subunits.

Bulk RNA-Seq and H3K27ac enrichment tracks for NKT1, NKT2 and NKT17 thymocyte subsets (data merged from 3-4 biological replicates) are shown for integrin receptor gene loci showing differential expression comparing subsets, along with UCSC gene tracks.

Supplementary Figure 12 Gene-expression and H3K27ac pattern for NKT2-enriched genes.

Bulk RNA-Seq and H3K27ac enrichment tracks for NKT1, NKT2 and NKT17 thymocyte subsets (data merged from 3-4 biological replicates) are shown for the indicated NKT2-enriched gene loci with UCSC gene tracks (top row).

Supplementary Figure 13 Expression of CXCR3, IL17RB and ICOS by T-bet+PLZFhiiNKT thymocytes.

Top plot shows the expression of T-bet and PLZF in electronically gated C57BL/6 RORγT- iNKT thymocytes. Rectangles indicate the position of electronic gates used to define the populations depicted in the lower plots, the red rectangle identifies those cells expressing relatively high levels of PLZF and T-bet. The three plots below show histogram overlays that depict the expression of CXCR3, IL17RB and ICOS within the iNKT populations defined in the top plot. Data are representative of two independent experiments examining a total of four age-matched females.

Supplementary Figure 14 Single-cell RNA-Seq analysis of NKT2-enriched genes.

PCA plots, with central plot showing the four iNKT cell subsets as in Figure 1d with NKT2 cells colored in blue. Shown surrounding the center plot are the same PCA plots with individual cells colored according to their relative expression of the indicated subset-specific transcripts mapped back to the subset PCA plot. Color key represents normalized gene expression with mean 0 and variance 1.

Supplementary Figure 15 Surface expression of IL6Rα on NKT2 thymocytes.

Histogram overlay depicting the surface expression of IL-6Rα (IL6RA) within electronically defined NKT subsets from C57BL/6J thymi. Data are representative of two independent experiments in which a pool of thymocytes from three age-matched females were analyzed.

Supplementary Figure 16 Gene-expression and H3K27ac patterns for NKT17-enriched genes.

Bulk RNA-Seq and H3K27ac enrichment tracks for NKT1, NKT2 and NKT17 subsets (data merged from 3-4 biological replicates) are shown for the indicated NKT17-enriched gene loci, along with UCSC gene tracks (top row).

Supplementary Figure 17 Single-cell RNA-Seq analysis of NKT17-enriched genes.

Scatter plot of single-cell RNA-Seq data showing expression values (log2 normalized counts) for the indicated genes. Each dot shown in yellow, green, blue and magenta color is a single NKT0, NKT1, NKT2 and NKT17 cell, respectively. Right: Box and whisker plots with the median and interquartile range of expression levels in single-cells from each iNKT cell subset.

Supplementary Figure 18 Single-cell RNA-Seq analysis of NKT1-enriched genes.

Scatter plot of single-cell RNA-Seq data showing expression values (log2 normalized counts) for the indicated genes. Each dot shown in yellow, green, blue and magenta color is a single NKT0, NKT1, NKT2 and NKT17 cell, respectively. Right: box and whisker plots with the median and interquartile range of expression levels in single-cells from each iNKT cell subset.

Supplementary Figure 19 Gene-expression and H3K27ac patterns for NKT1-enriched genes.

Bulk RNA-Seq and H3K27ac enrichment tracks for NKT1, NKT2 and NKT17 subsets (data merged from 3-4 biological replicates) are shown for the indicated NKT1-enriched gene loci, along with UCSC gene tracks (top row).

Supplementary Figure 20 NKT1 cell are more resistant to apoptosis.

Histogram plots showing expression of Annexin V in iNKT thymocyte subsets from C57BL/6J mice following four hours of culture at 37 °C. Data are representative of two independent experiments using a total of six age-matched females.

Supplementary Figure 21 Single-cell RNA-Seq analysis of NKT0-enriched genes.

Right: PCA plots, with central plot showing the four iNKT cell subsets as in Figure 1d with NKT0 cells colored in yellow. Shown surrounding the center plot are the same PCA plots with individual cells colored according to their relative expression of the indicated subset-specific transcripts mapped back to the subset PCA plot. Color key represents normalized gene expression with mean 0 and variance 1. Left: scatter plot of single-cell RNA-Seq data showing expression values (log2 normalized counts) for the indicated genes. Each dot shown in yellow is a single NKT0 cell. The data indicate that many of the sorted cells express the iNKT cell TCR and there are few if any immature DP thymocyte contaminants.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–21 (PDF 6758 kb)

Detailed description of 11 bulk RNA-Seq, 12 H3K27ac ChIP-Seq and 203 single-cell RNA-Seq assays that passed quality control checks.

For each assay, the table lists sample IDs, cell type of the sample, and experimental set. The last column contains total number of mapped reads per each assay, excluding mitochondrial reads. (XLSX 21 kb)

List of differentially enriched genes (DEGs) in iNKT cell subsets based on bulk RNA-Seq.

For each DEG the table lists the iNKT cell enriched subset, adjusted P values together with fold change for each pairwise comparison, and cell type and average RPKM value (Online Methods and Supplementary Table 3). (XLSX 792 kb)

Classification of the differentially expressed genes (DEGs) into categories.

Each DEG from the Supplementary Table 2 was assigned to one of eighteen groups based on the combined results in the three iNKT cell subset comparisons. DEGs with biologically similar patterns of outcomes were grouped into three categories: ‘NKT1-, NKT2- and NKT17-enriched’ genes (Online Methods). (XLSX 49 kb)

Biological process-enrichment analysis of NKT1-, NKT2- and NKT17- enriched genes.

The enrichment analysis was performed using software from the DAVID bioinformatic database (Online Methods). (XLSX 99 kb)

List of differentially enriched enhancer regions (DERs) obtained from any of the three pairwise comparisons of NKT1, NKT2 and NKT17 subsets.

List of differentially enriched enhancer regions (DERs) obtained from any of the three pairwise comparisons of NKT1, NKT2 and NKT17 subsets. For each DER the table lists the chromosomal location, raw counts, normalized counts (RPKM), raw P values, adjusted P value and fold change for each cell-type pairwise comparison (Online Methods). (XLSX 4583 kb)

List of the top 520 most differentially expressed genes (DEGs) from single- cell RNA-Seq assays comparing the four iNKT subsets (Online Methods).

For each DEG the table lists the Trimmed Mean of M-values (TMM) normalized log2 read counts for each of the 203 single cells. (XLSX 1059 kb)

List of the 50 most differentially expressed genes (DEGs) from single-cell RNA-Seq assays in NKT2 cells compared to other iNKT cell subsets.

For each DEG the table lists the chromosomal location, Gene ID, P values together with fold change for each cell-type pairwise comparison, Q value (false discovery rate), TMM normalized log2 read counts for each of the 203 single cells (Online Methods). (XLSX 148 kb)

List of the 50 most differentially expressed genes (DEGs) from single-cell RNA-Seq assays in NKT17 cells compared to other iNKT subsets.

For each DEG the table lists the chromosomal location, Gene ID, P values together with fold change for each cell-type pairwise comparison, Q value (false discovery rate), TMM normalized log2 read counts for each of the 203 single cells (Online Methods). (XLSX 149 kb)

Overlap of genes upregulated in analogous ILC and iNKT subsets.

A recently published study1 compared ILC1, ILC2, and ILC3 or LTi-like ILC (ILC3) subsets to identify ILC subset-specific transcripts. The list of ILC subset enriched transcripts were compared to the lists of genes with increased expression in the NKT1, NKT2, or NKT17 subsets, as compared to both of the other iNKT subsets. Table 12 indicates the numbers of genes found to be upregulated in both an ILC and an iNKT subset, with all possible ILC and iNKT cell subset comparisons listed. Numbers in parentheses indicate the total number of genes uniquely upregulated in each ILC and iNKT cell subset. Numbers in bold indicate significantly increased numbers of genes in comparison to the mean chance expectations, as determined by a hypergeometric probability test (P value for the ILC1 and NKT1 group: 8.0 * 10−40, ILC2 and NKT2: 1.9 * 10−4, ILC3 and NKT17: 3.1 * 10−28. P values for all of the other subset comparisons were ⩾.05). The genes in common between ILC1 and NKT1, ILC2 and NKT2, and ILC3 and NKT17 are shown to the right of the table. (XLSX 44 kb)

List of the 50 most differentially expressed genes (DEGs) from single-cell RNA-Seq assays in NKT1 cells compared to other iNKT subsets.

For each DEG the table lists the chromosomal location, Gene ID, P values together with fold change for each cell-type pairwise comparison, Q value (false discovery rate), TMM normalized log2 read counts for each of the 203 single cells (Online Methods). (XLSX 143 kb)

List of the 50 most differentially expressed genes (DEGs) from single-cell RNA-Seq assays in NKT0 cells compared to other iNKT subsets.

For each DEG the table lists the chromosomal location, Gene ID, P values together with fold change for each cell-type pairwise comparison, Q value (false discovery rate), TMM normalized log2 read counts for each of the 203 single cells (Online Methods). (XLSX 149 kb)

Transcription factor motif enrichment analysis. The enrichment analysis was performed using the findMotifsGenome.pl function of the HOMER software package (Online Methods).

Table shows known motifs that are significantly enriched in at least one out of six groups of differentially enriched enhancer regions (DERs) identified by pairwise comparison of the three iNKT cell subsets. (XLSX 15 kb)

Single-cell RNA-Seq analysis

Principal component analysis of 203 single-cell transcriptomes distinguished iNKT cell subsets. Data generated from two experimental replicates performed on single cells isolated from thymuses pooled from three mice in each experiment. Each dot shown in yellow (NKT0), green (NKT1), blue (NKT2) and magenta color (NKT17) is from a single cell. Percentage of variance in each PC, principal component is shown in parenthesis next to the PC. (WMV 38495 kb)

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Engel, I., Seumois, G., Chavez, L. et al. Innate-like functions of natural killer T cell subsets result from highly divergent gene programs. Nat Immunol 17, 728–739 (2016). https://doi.org/10.1038/ni.3437

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