Research ArticleT CELL DIFFERENTIATION

Helios enhances the preferential differentiation of human fetal CD4+ naïve T cells into regulatory T cells

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Science Immunology  22 Nov 2019:
Vol. 4, Issue 41, eaav5947
DOI: 10.1126/sciimmunol.aav5947
  • Fig. 1 Fetal naïve T cells have expression of a partial Treg transcriptome.

    (A) PCA plot of RNA-seq data comparing adult naïve (AN), fetal naïve (FN), adult Treg (AT), and fetal Treg (FT) cells. Boxplots show scores for PC1 (bottom) and PC2 (right). (B) Heatmap shows relative expression levels of Treg-specific differentially expressed genes in adult naïve, fetal naïve, adult Treg, and fetal Treg cells. Clusters are labeled and defined by k-means clustering. Genes associated with Treg and naïve T cell function are labeled. Boxplots (left) show the averaged trimmed mean of M values (TMM) normalized reads across all up-regulated (top; clusters 1 and 2) and down-regulated (bottom; clusters 3 and 4) genes. Kruskal-Wallis test and Dunn’s multiple comparison test with Bonferroni correction for multiple testing, ***P < 0.001; n.s., P > 0.05 (n = 4 biological replicates per group). (C) Volcano plot of differentially expressed (log2FC, >1.5; Padj < 0.05) genes in fetal and adult naïve T cells, all differentially expressed genes in light gray. Dashed lines (gray) denote FC cutoffs. Eighty-eight and 42 Treg-specific genes are up-regulated (orange) and down-regulated (blue) in fetal naïve T cells, respectively. Among Treg–up-regulated genes, genes previously associated with Treg function are labeled. Among Treg–down-regulated genes, the top five genes with the lowest Padj values are labeled. All boxplots in this figure show median (center line), interquartile range (box), and 10th and 90th percentiles (whiskers).

  • Fig. 2 Fetal iTreg cells retain expression of the partial Treg-specific transcriptome detected in fetal naïve T cells in steady state.

    (A) PCA of RNA-seq data comparing fetal and adult iTreg cells generated in IL-2 alone (IL-2–iTreg) or with added exogenous TGF-β (TGF-β–iTreg). Boxplots show scores for PC1 (bottom) and PC2 (right). (B) Heatmap shows relative expression levels of differentially expressed genes (log2FC > 1.5; FDR < 0.05) in adult and fetal IL-2–iTreg and TGF-β–iTreg. Clusters are labeled and defined by k-means clustering. Genes associated with Treg or proinflammatory/effector T cell function are labeled. (C) Preranked GSEA was used to assess overrepresentation of predefined activated Treg-associated gene sets (see fig. S3B for details of all gene sets) in fetal (orange) or adult (blue) IL-2–iTreg cells (left) or TGF-β–iTreg cells (right). n = 4 for all conditions. Barplot shows normalized enrichment scores (ES) for gene sets with FDR < 0.05, and arrows denote direction of enrichment in adult or fetal iTreg cells. (D) Preranked GSEA was used to assess overrepresentation of each of the gene clusters identified in Fig. 1B. Barplot shows normalized enrichment scores for all clusters with FDR < 0.05 for fetal (orange) or adult (blue) IL-2–iTreg cells (left) or TGF-β–iTreg cells (right).

  • Fig. 3 Fetal naïve T cells have increased ATAC and H3K27ac enrichment at two Treg-accessible SEs associated with Helios.

    (A and B) PCA performed on all SEs and TEs with mapped (A) ATAC or (B) H3K27ac reads. Boxplots show scores for PC1 (bottom) and PC2 (right). (C) Barplots show number of Treg-accessible/inaccessible enhancers with increased (orange) or decreased (blue) ATAC and H3K27ac signal in fetal naïve T cells relative to adult naïve T cells (Treg-accessible/inaccessible enhancers defined in Supplementary Materials and Methods). (D) Plots show cumulative H3K27ac signal at stitched enhancers within 12.5 kb against enhancer rank, and SEs were defined where the tangent of the plotted curve is 1. Dashed lines (gray) show cutoff for SEs for one representative sample each for adult Treg (n = 3). SEs defined as Treg accessible and meet FC > 1.5 and FDR < 0.05 cutoffs are colored red, and SEs associated with key Treg genes are labeled. (E) Plots as with (D) show one representative sample for fetal (left) and adult (right) naïve T cells (n = 3). Treg-accessible SEs identified in (D) were assessed for differential enrichment in fetal naïve against adult naïve T cells and vice versa. Differentially enriched Treg SEs in each sample with FC > 1.5 and FDR < 0.05 are colored red and labeled. TEs associated with key Treg genes in (D) are also labeled. (F and G) Scatterplots of normalized (F) ATAC-seq reads and (G) H3K27ac reads at all stitched enhancer regions of fetal naïve against adult naïve T cells. Differentially enriched SEs and TEs with FC > 1.5 and FDR < 0.05 are shown in light gray, with Treg-accessible SEs (orange) and Treg-inaccessible SEs (blue). Treg-accessible SEs associated with transcription factors are labeled; Helios (IKZF2) is labeled in bold.

  • Fig. 4 Helios expression is increased in fetal naïve T cells.

    (A) Tracks show H3K27ac and ATAC signals at two Treg-accessible SEs associated with the Helios (IKZF2) locus. Representative tracks of one replicate shown (H3K27ac ChIP-seq, n = 3; ATAC-seq, n = 2). (B) The top five genes contributing to PC2, which segregates cells by functional subtype (naïve versus Treg) are plotted for both directions; Helios (IKZF2) is highlighted in red. (C) Boxplot shows log2 trimmed mean of M values of normalized RNA-seq reads for Helios in adult naïve, fetal naïve, adult Treg, and fetal Treg cells (n = 4). (D) Helios staining intensity in sorted CD4+CD25CD27+CD45RA+ adult naïve (blue) and fetal naïve (green) and adult CD25hiCD127loFOXP3 (orange) and fetal Treg (brown). (E) Boxplot shows quantification of mean fluorescence intensity (MFI) of Helios for adult naïve (n = 8), fetal naïve (n = 10), adult Treg (n = 8), and fetal Treg (n = 10) cells. (F) Flow cytometry analyses of sorted populations in (D). Helios+ and Helios gates were set on the basis of negative and positive populations in adult Treg samples (bottom left). Adult naïve T cells were universally Helios. (G) Quantification of Helios+ cells among sorted populations in (F). All statistics were calculated by unpaired two-sided Mann-Whitney test. ***P < 0.001, **P < 0.01, *P < 0.05. All boxplots show median (center line), interquartile range (box), and 10th and 90th percentiles (whiskers).

  • Fig. 5 Helios+ fetal iTreg cells have increased FOXP3 expression.

    Sorted fetal and adult naïve T cells were stimulated with αCD3/αCD28/αCD2 tetramers and IL-2 in the absence or presence of TGF-β over 1, 3, or 5 days and analyzed by flow cytometry. The number of biological replicates for each time point and stimulation condition are specified in table S8. (A) Representative flow cytometry plots show Treg induction in the presence of IL-2 and TGF-β for fetal (top) and adult (bottom) naïve T cells respectively gated on live, CD4+ T cells. (B) Quantification of the percentage of Helios+FOXP3+ iTreg cells gated in (A) for adult and fetal naïve T cells stimulated in the presence or absence of TGF-β. Statistics calculated using two-way ANOVA with Tukey’s honest significant difference posttest, ***P < 0.001. Error bars denote means ± SD. (C) Representative flow cytometry plots shown for one fetal sample stimulated with IL-2 and TGF-β at day 1. (D) Quantification of the proportion of fetal Treg cells in the Helios+ or Helios population as gated in (C) over 1, 3, and 5 days in the absence (left) or presence (right) of TGF-β. (E) Quantification of FOXP3 mean fluorescence intensity for Helios+ and Helios iTreg cells as gated in (C) across all time points in the absence (left) or presence (right) of TGF-β. Statistics for (D) and (E) were calculated by two-sided Wilcoxon signed-rank test, ***P < 0.001, **P < 0.01. All boxplots show median (center line), interquartile range (box), and 10th and 90th percentiles (whiskers).

  • Fig. 6 CRISPR-Cas9–mediated knockout of Helios in fetal naïve T cells reduces their preferential differentiation into Treg cells.

    (A) Schematic showing experimental design. CRISPR-Cas9 editing at the Helios locus with two independent gRNAs (gRNA1 and gRNA2; see Supplementary Materials and Methods) was carried out in prestimulated fetal naïve T cells, with an NT gRNA as a control. Edited cells were stimulated with αCD3/αCD28/αCD2 tetramers in the absence or presence of TGF-β. Treg induction was assessed at 6 days. The numbers of biological replicates for each guide and stimulation condition are specified in table S9. (B) Flow cytometry plots showing Helios expression in fetal iTreg cells differentiated in IL-2 alone at day 6 after CRISPR-Cas9 editing with Helios gRNA1/2 or NT controls. Representative plots of one experiment are shown gated on live, CD4+ T cells. (C) Quantification of Helios expression after editing in fetal naïve T cells after Treg induction as in (B). Boxplots show paired samples for gRNA1 (left) and gRNA2 (right). (D) Flow cytometry plots showing FOXP3 and CD25 staining in fetal iTreg cells at day 6 after CRISPR-Cas9 editing with Helios gRNA1/2 or NT controls. Representative plots of one experiment are shown gated on live, CD4+ T cells. (E) Quantification of iTreg proportions in edited fetal naïve T cells as in (D). Boxplots show paired samples for gRNA1 (left) and gRNA2 (right). All statistics were calculated by two-sided Wilcoxon signed-rank test, ***P < 0.001, **P < 0.01. All boxplots show median (center line), interquartile range (box), and 10th and 90th percentiles (whiskers).

  • Fig. 7 Ablation of Helios in fetal iTreg results in down-regulation of Treg-specific genes and the concurrent up-regulation of proinflammatory genes.

    CRISPR-Cas9 editing was carried out in fetal naïve T cells (n = 6) with Helios gRNA1 (HeliosKO) or the NT control guide (HeliosWT). Edited cells stimulated with αCD3/αCD28/αCD2 tetramers in the absence or presence of TGF-β for 6 days, after which changes in their overall transcriptome were assessed by RNA-seq. (A) Schematic showing hypothesized expression levels (dashed lines) of a Treg-specific gene that is up-regulated with IL-2 in the absence or presence of TGF-β signaling, with proposed changes in transcription level given one of the three proposed scenarios of transcriptional control by Helios and TGF-β. Gene expression that is driven by Helios is shown, with the arrows denoting the corresponding decrease in gene expression occurring with Helios knockout. (B and C) Scatterplots show log2FC values comparing the absence or presence of exogenous TGF-β during the differentiation process (y axis) and HeliosKO against HeliosWT iTreg (x axis) that underwent differentiation in IL-2 alone (B) or with exogenous TGF-β added (C). Dashed lines in gray denote log2FC cutoffs. Only genes up-regulated (dark purple) or down-regulated (dark orange) in HeliosKO relative to HeliosWT iTreg cells are colored and shown (FC > 1.1; FDR < 0.05). Genes associated with Treg or proinflammatory immune functions are outlined and labeled. (D and E) Same scatterplots as in (B) and (C) for IL-2–only iTreg cells (D) and IL-2 + TGF-β–iTreg (E) now colored to only show shared genes meeting the same cutoffs that were up-regulated (light purple) or down-regulated (light orange) in HeliosKO relative to HeliosWT iTreg cells. Genes associated with Treg or proinflammatory immune functions within the shared Helios-regulated transcriptome are outlined and labeled.

  • Fig. 8 Fetal Helios knockout iTreg cells have decreased IL-10 and increased IFN-γ cytokine production.

    (A) Boxplots quantify IL-10 cytokine concentration within culture supernatants collected at days 3 and 5 of differentiation for adult (blue; n = 27) or fetal (orange; n = 58) IL-2–iTreg cells, as well as adult (light blue; n = 27) or fetal (light orange; n = 55) TGF-β–iTreg cells. (B) Boxplots quantify IFN-γ cytokine concentrations for samples as in (A). (C) Boxplots show log2(n + 1) ratio of IL-10 to IFN-γ cytokine concentration for samples as in (A). Dashed line marks the ratio at which cells produce IL-10 and IFN-γ at 1:1. All statistics for (A) to (C) were calculated by unpaired two-sided Mann-Whitney test, ***P < 0.001, **P < 0.01, *P < 0.05. (D) Boxplots quantify IL-10 cytokine concentrations at day 5 of differentiation for iTreg cells that received the NT guide (HeliosWT; red) or for Helios knockout (HeliosKO; blue) with gRNA1 (left; n = 24) and gRNA2 (right; n = 23). (E) Boxplots quantify IFN-γ cytokine concentrations for samples in (D). (F) Boxplots show log2(n + 1) ratio of IL-10 to IFN-γ cytokine concentration for samples as in (D). Dashed line marks the ratio where cells produce IL-10 and IFN-γ at 1:1. All statistics for (D) to (F) were calculated by two-sided Wilcoxon signed-rank test, ***P < 0.001, *P < 0.05; n.s., P > 0.05. All boxplots show median (center line), interquartile range (box), and 10th and 90th percentiles (whiskers).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/41/eaav5947/DC1

    Materials and Methods

    Fig. S1. Gating strategy and purity assessment for sorted naïve and Treg cells.

    Fig. S2. Definition of the Treg transcriptional signature.

    Fig. S3. Assessing the enrichment of Treg up-regulated or down-regulated genes in fetal and adult iTreg populations.

    Fig. S4. Fetal iTreg cells have increased sensitivity to TGF-β signaling.

    Fig. S5. Identification of Treg-accessible and -inaccessible enhancers.

    Fig. S6. Binding motifs for downstream effectors of Treg differentiation are enriched within shared Treg-accessible peaks in fetal naïve T cells.

    Fig. S7. The highest ranked SEs shared across all cell populations are associated with T cell development and function.

    Fig. S8. Chromatin accessibility and H3K27ac enrichment at the Helios locus in fetal naïve T cells correlate with increased RNA and protein expression.

    Fig. S9. Fetal naïve T cells do not have an increased proportion of CD31+ cells relative to adult naïve T cells.

    Fig. S10. A fraction of fetal naïve T cells are highly proliferative.

    Fig. S11. Fetal naïve T cells do not have demethylation at the FOXP3 conserved noncoding sequence 2 TSDR.

    Fig. S12. Fetal naïve T cells up-regulate Helios during Treg induction.

    Fig. S13. Validation of CRISPR-Cas9 editing at the Helios locus.

    Fig. S14. The effect of CRISPR-Cas9 knockout of Helios on protein expression of Treg functional markers is variable.

    Fig. S15. Fetal, but not adult, iTreg cells have suppressed IL-2 production after restimulation.

    Fig. S16. Helios knockout in fetal iTreg cells result in a subtle shift in the underlying transcriptome.

    Table S1. RNA-seq Treg up-regulated and down-regulated signature genes (Excel spreadsheet).

    Table S2. Genes up-regulated/down-regulated within iTreg populations with cluster assignment (Excel spreadsheet).

    Table S3. Statistics from pre-ranked GSEA for fetal versus adult iTreg populations (Excel spreadsheet).

    Table S4. Treg-accessible enhancers (Excel spreadsheet).

    Table S5. Treg-inaccessible enhancers (Excel spreadsheet).

    Table S6. De novo motif enrichment in Treg-accessible ATAC peaks shared between fetal naïve and adult Treg cells (Excel spreadsheet).

    Table S7. Top 25 ranked SEs in adult naïve, fetal naïve, and adult Treg cells (Excel spreadsheet).

    Table S8. Experimental setup for Treg induction time course carried out for adult and fetal naïve T cells.

    Table S9. Experimental setup for Helios CRISPR-Cas9–mediated editing for subsequent Treg induction carried out for fetal naïve T cells.

    Table S10. Up-regulated and down-regulated differentially expressed genes defined in Helios knockout versus Helios wild-type fetal iTreg cells (Excel spreadsheet).

    Table S11. Raw data file (Excel spreadsheet).

    References (95105)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. Gating strategy and purity assessment for sorted naïve and Treg cells.
    • Fig. S2. Definition of the Treg transcriptional signature.
    • Fig. S3. Assessing the enrichment of Treg up-regulated or down-regulated genes in fetal and adult iTreg populations.
    • Fig. S4. Fetal iTreg cells have increased sensitivity to TGF-β signaling.
    • Fig. S5. Identification of Treg-accessible and -inaccessible enhancers.
    • Fig. S6. Binding motifs for downstream effectors of Treg differentiation are enriched within shared Treg-accessible peaks in fetal naïve T cells.
    • Fig. S7. The highest ranked SEs shared across all cell populations are associated with T cell development and function.
    • Fig. S8. Chromatin accessibility and H3K27ac enrichment at the Helios locus in fetal naïve T cells correlate with increased RNA and protein expression.
    • Fig. S9. Fetal naïve T cells do not have an increased proportion of CD31+ cells relative to adult naïve T cells.
    • Fig. S10. A fraction of fetal naïve T cells are highly proliferative.
    • Fig. S11. Fetal naïve T cells do not have demethylation at the FOXP3 conserved noncoding sequence 2 TSDR.
    • Fig. S12. Fetal naïve T cells up-regulate Helios during Treg induction.
    • Fig. S13. Validation of CRISPR-Cas9 editing at the Helios locus.
    • Fig. S14. The effect of CRISPR-Cas9 knockout of Helios on protein expression of Treg functional markers is variable.
    • Fig. S15. Fetal, but not adult, iTreg cells have suppressed IL-2 production after restimulation.
    • Fig. S16. Helios knockout in fetal iTreg cells result in a subtle shift in the underlying transcriptome.
    • Table S8. Experimental setup for Treg induction time course carried out for adult and fetal naïve T cells.
    • Table S9. Experimental setup for Helios CRISPR-Cas9–mediated editing for subsequent Treg induction carried out for fetal naïve T cells.
    • References (95105)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. RNA-seq Treg up-regulated and down-regulated signature genes (Excel spreadsheet).
    • Table S2. Genes up-regulated/down-regulated within iTreg populations with cluster assignment (Excel spreadsheet).
    • Table S3. Statistics from pre-ranked GSEA for fetal versus adult iTreg populations (Excel spreadsheet).
    • Table S4. Treg-accessible enhancers (Excel spreadsheet).
    • Table S5. Treg-inaccessible enhancers (Excel spreadsheet).
    • Table S6. De novo motif enrichment in Treg-accessible ATAC peaks shared between fetal naïve and adult Treg cells (Excel spreadsheet).
    • Table S7. Top 25 ranked SEs in adult naïve, fetal naïve, and adult Treg cells (Excel spreadsheet).
    • Table S10. Up-regulated and down-regulated differentially expressed genes defined in Helios knockout versus Helios wild-type fetal iTreg cells (Excel spreadsheet).
    • Table S11. Raw data file (Excel spreadsheet).

    Files in this Data Supplement:

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