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. 2018 Jul 9;9(1):2650.
doi: 10.1038/s41467-018-05026-6.

TCR signal strength controls thymic differentiation of iNKT cell subsets (VSports最新版本)

Kathryn D Tuttle  1   2 , S Harsha Krovi  1   2 , Jingjing Zhang  1   2 , Romain Bedel  1   2   3 , Laura Harmacek  4   5 , Lisa K Peterson  5   6   7 , Leonard L Dragone  5   8 , Adam Lefferts  1   2 , Catherine Halluszczak  1   2 , Kent Riemondy  9 , Jay R Hesselberth  9   10 , Anjana Rao  11   12   13 , Brian P O'Connor  4   5 , Philippa Marrack  1   2   14 , James Scott-Browne  11   12 , Laurent Gapin  15   16
Affiliations

TCR signal strength controls thymic differentiation of iNKT cell subsets

Kathryn D Tuttle et al. Nat Commun. .

Abstract

During development in the thymus, invariant natural killer T (iNKT) cells commit to one of three major functionally different subsets, iNKT1, iNKT2, and iNKT17 VSports手机版. Here, we show that T cell antigen receptor (TCR) signal strength governs the development of iNKT cell subsets, with strong signaling promoting iNKT2 and iNKT17 development. Altering TCR diversity or signaling diminishes iNKT2 and iNKT17 cell subset development in a cell-intrinsic manner. Decreased TCR signaling affects the persistence of Egr2 expression and the upregulation of PLZF. By genome-wide comparison of chromatin accessibility, we identify a subset of iNKT2-specific regulatory elements containing NFAT and Egr binding motifs that is less accessible in iNKT2 cells that develop from reduced TCR signaling. These data suggest that variable TCR signaling modulates regulatory element activity at NFAT and Egr binding sites exerting a determinative influence on the dynamics of gene enhancer accessibility and the developmental fate of iNKT cells. .

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"VSports在线直播" Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Thymic iNKT subsets have different avidities for the PBS57–CD1d tetramer that correlate with expression of surrogate markers reflective of strength of signaling during selection. a Cells from the thymus of BALB/c IL-4 reporter KN2 mice were stained with anti-TCRβ mAbs and PBS57–CD1d tetramers. iNKT cells were then electronically placed on a grid consisting of 30 gates, allowing for the analysis of iNKT cells expressing different TCR levels and/or different binding to the PBS57–CD1d tetramer for a given level of TCR. b The proportion of iNKT1 (PLZFlo, Rorγt−, Tbet+), iNKT17 (PLZFint, Rorγt+, Tbet−), iNKT2 (PLZFhi, Rorγt–, Tbet–, or hCD2+) as well as the proportion of CD44hi cells in each gate was recorded and displayed as a heatmap. c Representative histograms for the expression of CD5, CD6, Ly6C, and Egr2 in each iNKT cell subset (as defined by the gating strategy shown on the left) is shown. Data are representative of n > 3 mice for each staining
Fig. 2
Fig. 2
Development of iNKT cell subsets in DO11.10 TCRβ transgenic mice on the C57BL/6 or C57BL/6 × BALB/c F1 background. a The thymi of 8-week-old C57BL/6, C57BL/6 DO11.10 TCRβ transgenic, BALB/c, C57BL/6 × BALB/c F1, and BALB/c × C57BL/6 DO11.10 TCRβ transgenic mice were stained with anti-CD24 mAbs, PBS57–CD1d tetramers and mAbs specific for the transcription factors PLZF, Rorγt, and Tbet. Representative flow cytometry plots for the frequency of total mature iNKT cells and iNKT subsets in each genotype is shown. b Summary of the data shown in a with three mice per group. Data are mean ± SD. Significance was assessed using multiple comparison one-way analysis of variance (ANOVA) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. For clarity purposes, the only statistics shown are between mice with or without expression of the DO11.10 TCR transgene for a given genetic background
Fig. 3
Fig. 3
Thymic iNKT subsets with different TCR levels express different proportions of Vβ usage but discrete Egr2 levels. Cells from the thymus of 8-week-old BALB/c mice were stained with anti-TCRβ mAbs, PBS57–CD1d tetramers, mAbs specific for the transcription factors PLZF, Rorγt, and Tbet and the indicated anti-Vβ mAbs. a iNKT cells were subdivided based on expression of different TCR levels. The proportion of iNKT cell subsets for each level of TCR expression (TCRlow (blue), TCRint (orange) and TCRhigh (green)) is shown. b The percentage of Vβ usage found in each iNKT subset expressing three distinct TCR levels is shown with the fluorescence minus one control (FMO). Data are representative of three independent mice analyzed. c Example of Egr2 expression in each iNKT cell subsets expressing different levels of TCR. d The gMFI of Egr2 expression was recorded from three independent staining of iNKT cell subsets expressing different TCR levels from one individual mouse as gated in a. Three independent mice were analyzed in this way with similar results
Fig. 4
Fig. 4
Cell-intrinsic deficiency in iNKT2 and iNKT17 subsets development in mice with Zap70 hypomorphic mutations. a Representative flow cytometry plots showing the frequency of total iNKT cells and iNKT cell subsets in the thymi of 8-week-old C57BL/6, C57BL/6 YYAA, BALB/c, and BALB/c SKG mice. Each genotype was analyzed independently and compared to wild-type control mice of the appropriate background. b, c Summary of the data shown in a with percentages and cell numbers of total iNKT cells and iNKT cell subsets in the various genotypes analyzed. Data are mean ± SD. Significance was assessed using the unpaired t-test. d Analysis of thymic iNKT cell subsets in competitive bone marrow chimera. Congenically marked bone marrow cells from BALB/c wild-type mice (CD45.1) were mixed in a 1:1 ratio with (CD45.2) BALB/c or SKG bone marrow cells and injected into lethally irradiated F1 (CD45.2 × CD45.1) BALB/c mice. The thymi of the chimera were analyzed for iNKT cell subset composition 8–10 weeks post reconstitution and representative flow cytometry plots are shown. After gating on total iNKT cells, the proportion of iNKT cell subsets derived from either CD45.2+ or CD45.1+ bone marrow cells is shown. e Summary of the data shown in d with ≥5 mice per group. Data are represented as mean ± SD. Significance was assessed using one-way analysis of variance (ANOVA) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant
Fig. 5
Fig. 5
Differential gene expression in iNKT cells of BALB/c and SKG mice. a Gating strategy used to identify and sort stage 0 iNKT cells in BALB/c (top) and SKG (bottom) mice. Enrichment of iNKT cells from the thymi of BALB/c or SKG mice was achieved after staining with APC-labeled PBS57–CD1d tetramer and enrichment using anti-APC magnetic beads. Stage 0 iNKT cells were defined as TCRβ+ PBS57–CD1d tetramer+ CD44− CD24+ CD4lo CD8− CD69+. As seen on the figure, these cells are also Egr2+ (red) in contrast with the CD4+CD8+ (blue) population that invariably contaminates the cell preparation. b Mean average plot of genes expressed differentially in stage 0 iNKT cells isolated from SKG mice relative to their expression in such cells from BALB/c control mice. Data are representative of one experiment with three biological replicates per genotype. 238 genes (displayed in black) were found to be differentially expressed >1.5-fold with a false discovery rate <0.1. c Analysis of transcription factor (PLZF, Rorγt, and Tbet) expression in stage 1 (CD44− CD24−) and stage 2 (CD44+) iNKT cells from the thymi of BALB/c and SKG mice. Data are representative of >5 mice analyzed for each genotype. d Analysis of Egr2 expression level in BALB/c (orange) and SKG (purple) iNKT cells at different developmental stages. Results from three mice of each genotypes analyzed independently are shown. The mean ± SD of Egr2 gMFI in each iNKT cell developmental stage in BALB/c and SKG mice is shown. Significance was assessed using the unpaired t-test. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant
Fig. 6
Fig. 6
Induction of Egr2 and PLZF in pre-selection DP thymocytes stimulated with plate-bound anti-CD3 and anti-Ly108 mAbs is affected by the SKG mutation. a Expression of Egr2 and PLZF proteins in pre-selection BALB/c or SKG DP thymocytes stimulated with media alone (no stim), plate-bound anti-CD3, anti-Ly108, or anti-CD3 + anti-Ly108 for 48 h. At the end of the stimulation, cells were stained using anti-CD4, -CD8, -Egr2, -PLZF mAbs, and a viability dye. The percentages of live cells that are Egr2+ and PLZF+ are indicated for each stimulatory condition. b Time course of CD4, CD8, Egr2, and PLZF expression in pre-selection BALB/c or SKG DP thymocytes stimulated with plate-bound anti-CD3 + anti-Ly108 mAbs. Data are shown for live cells in each conditions. c Summary of the data shown in b for each stimulatory condition. The percentage of Egr2+ cells, the gMFI of Egr2 expression, and the percentage of PLZF+ cells are shown. Each experimental condition was tested in duplicate wells and the data are representative of greater than three independent experiments with similar results
Fig. 7
Fig. 7
Genome-wide chromatin landscapes of iNKT1 and iNKT2 subsets are affected by the SKG mutation. a, c Scatterplots of mean ATAC-seq counts per peak comparing iNKT1 and iNKT2 subsets from BALB/c (a) or SKG (c) mice. b, d Boxplots of ATAC-seq counts per plots from the indicated samples (labeled at the bottom) at common or differentially accessible regions from the comparison labeled above. Box indicates interquartile range with whiskers ± 1.5 times this range and outlier points. e Scatterplots of mean ATAC-seq counts per peak comparing iNKT1 from BALB/c vs SKG mice and iNKT2 subsets from BALB/c vs SKG mice. f k-means clustered log2 fold-change from mean ATAC-seq signal for all differentially accessible regions compared to all differentially accessible regions ini NKT1 and iNKT2 cells from BALB/c and SKG mice. The enrichment of known motifs within each cluster of differentially accessible regions compared to all differentially accessible regions in iNKT1 and iNKT2 cells from BALB/c and SKG mice. All motifs with an enrichment log p-value <−35 and found in 10% or more regions in at least one cluster are shown. g Mean ATAC-seq coverage at the Pdcd1 locus with a scale 0–1000 for ATAC-Seq tracks. NFAT ChIP-seq coverage from CD8 T cells stimulated or not with PMA + ionomycin for 1 h are also shown. h Flow cytometry analysis of PD1 expression on thymic iNKT cell subsets from BALB/c (red) and SKG (blue) mice. i Mean ATAC-seq coverage at the Egr2 locus with a scale 0–1000 for ATAC-Seq tracks. j Mean ATAC-seq coverage at the Runx1 locus with a scale 0–1000 for ATAC-Seq tracks. For g, i, and j, ATAC peaks with differential signals where BALB/c > SKG mice are highlighted in blue, while peaks with signals SKG > BALB/c mice in at least one subset are in red. k ATAC-Seq of chromatin accessibility at the transcription start site of Klf2 in thymic iNKT1 and iNKT2 subsets from BALB/c and SKG mice
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