A Gain-of-Function HDAC7 Mutant, HDAC7-ΔP, Arrests Thymic iNKT Development.(A) Representative flow cytometric plots of iNKT cells and conventional αβ T-cells, identified by staining with TCRβ and CD1D tetramer, empty or loaded with αGalCer as indicated, in thymocytes from WT (top) and HDAC7-ΔP (bottom) mice. Staining after magnetic enrichment of 2 × 107 cells with loaded tetramer is shown at right. (B) Quantification of iNKT cell frequency in Thymus (left), spleen (center), and liver (right) of WT (black symbols) and HDAC7-ΔP (blue symbols) mice (C) Representative flow plots showing conventional staging of iNKT development by CD44 and NK1.1 expression (left) and CD24 expression (right) in magnetically enriched Tet+ TCRβ+ thymic iNKT cells from WT and HDAC7-ΔP mice as indicated. (D) Quantification of difference in frequency of magnetically enriched iNKT cells at the indicated stages, as defined in (C), for five littermate pairs WT and HDAC7-ΔP mice. Difference is expressed as (% of live cells / % of live cells) * 100 for HDAC7-ΔP/WT. Numbers above each column indicate P-value by 2-tailed Student’s T-test. (E) Representative flow cytometric plots and of iNKT from thymus in WT (CD45.1): HDAC7-ΔP (CD45.2) mixed bone-marrow chimeras. Data in (B) are combined from eight independent experiments involving 1–3 littermate pairs; data in (C) are representative of 5 WT: HDAC7-ΔP littermate pairs. Data in (E) are representative of 3 sets of chimeras with 3–6 mice per group. Statistical significance was determined using unpaired two-tailed t tests; ****p≤0.0001.10.7554/eLife.32109.007Figure 1—source data 1.Multi-sheet Microsoft Excel workbook containing numerical data matrices for all figure panels (on separate sheets) in which individual data points are not represented graphically.Figure 1D, Figure 1—figure supplement 1C, and Figure 1—figure supplement 2A–B.Multi-sheet Microsoft Excel workbook containing numerical data matrices for all figure panels (on separate sheets) in which individual data points are not represented graphically.Figure 1D, Figure 1—figure supplement 1C, and Figure 1—figure supplement 2A–B.Supporting Data on iNKT Phenotype of HDAC7-ΔP Transgenic Mice.(A) Representative flow scatter plots showing expression of CD44 vs. TCRβ (left), CD4 vs. CD8α (center), and CD8α vs. CD8β (right) in intra-epithelial lymphocytes isolated from small intestines of WT littermate (top) and HDAC7-ΔP mice (bottom). Data are representative of 3 independent experiments. Plots at center and right show the populations indicated by arrows in the plots to their left. (B) Representative flow scatter plots showing staining of splenocytes (left) or liver-resident lymphocytes (right) from WT littermate (top) or HDAC7-ΔP (bottom) mice with αGalCer-loaded or empty CD1D tetramers, as indicated. (C) Data from five independent littermate pair comparisons showing the percent ratio (as percent of live cells/percent of live cells) of the frequency of total tetramer-positive cells or tetramer-positive cells at the indicated stages, based on the gating shown in Figure 1C. Graph at top shows percent ratios for empty/loaded CD1D tetramer in HDAC7-ΔP mice. Graph at bottom shows percent ratios for loaded tetramer in thymocytes from HDAC7-Δp/WT littermate mice. Numbers above columns indicate P-values by 2-tailed, paired Student’s T-test. (D) Representative flow plot showing CD44 vs. NK1.1 staining of tetramer-positive thymocytes from WT littermate (top) or HDAC7-ΔP (bottom) mice with αGalCer-loaded or empty CD1D tetramers, as indicated. Numbers at upper left of each plot indicate the total number of cells falling in the iNKT gate for approximately 2 million total events.Supporting Data on the iNKT Phenotype of WT: HDAC7-ΔP Mixed Hematopoietic Chimeras.(A) Log2 fold ratio of HDAC7-ΔP-derived (CD45.2) to WT-derived (CD45.1) cells at the indicated thymocyte stages. A composite DN1-4 engraftment ratio (Lin-CD4-CD8-) was calculated per mouse to normalize the ratio at each successive stage. (B) Proportion of T-cell subsets plotted as percentage of parent from HDAC7-ΔP-derived (CD45.2) or WT-derived (CD45.1) in mixed chimeras. CD4 naïve are defined as CD44loCD4+, CD4 memory as CD44hiCD4+, CD8 naïve as CD44loCD62LhiCD8+, CD8 central memory (TCM) as CD44hiCD62LhiCD8+, and CD8 effector memory (TEM) as CD44hiCD62LloCD8+. (C) Representative flow plots showing TCRβ vs. PBS-57 tetramer staining for WT and HDAC7-ΔP-derived populations in thymus (left) or spleen (right) of WT: HDAC7-ΔP Mixed Hematopoietic Chimeras. Frequency of iNKT cells and Tconv is indicated. (D) Quantification of iNKT cell frequency in mixed WT: HDAC7-ΔP mixed bone-marrow chimeras from thymus, spleen and liver. Bars on graphs indicate mean ±SEM; symbols represent individual mice. (E, F) Representative flow cytometric plots (E) from liver and total quantification (F) from liver and spleen of type II iNKT cells (Tet- CD44hi NK1.1+ TCRβ+) in mixed bone-marrow chimeras depending on bone marrow of origin. Data in (D) are derived from 3 sets of chimeras with 3–6 mice per group. Data in (F) are derived from 2 sets of chimeras with 3 and 4 mice. Statistical significance was determined using unpaired two-tailed t tests; ***p≤0.001, ****p≤0.0001.Full Gating Strategy for Detection and Staging of iNKT Cells in HDAC7-ΔP Transgenic Mice and WT: HDAC7-ΔP Mixed Hematopoietic Chimeras.(A) Full gating for representative panels from thymus shown in Figure 1A and C. Bottom row of plots is as shown in Figure 1A and C. From left to right, top row shows gating for leukocytes by FSC area vs. SSC area, gating for singlets using chained FSC height vs. width and SSC height vs. width, then gating for live cells using eFluor 520 fixable viability dye vs. SSC. This scheme was employed for all data shown in Figures 1–4, Figure 8, and their associated supplements. (B) Full gating for analysis of liver-resident lymphocytes WT: HDAC7-ΔP Mixed Hematopoietic Chimeras. Last two panels in bottom row are as shown in Figure 1E. Top row shows gating for live single leukocytes as for (A). First panel in bottom row shows gating for WT and HDAC7-ΔP -derived cells using CD45.1 and CD45.2. HDAC7-ΔP cells are 45.2+/45.1-, WT cells 45.1+/45.2-, and host-derived cells are 45.1/45.2 double-positive. less

Supporting Data on iNKT Phenotype of Vα14/Jα18 X HDAC7-ΔP Transgenic Mice.(A, B) Restoration of iNKT cells (Tet + TCRβ+) in Hdac7-KO mice by expression of the Vα14-Jα18 TCR transgene (A, top row), as well as representative CD44/NK1.1 staging for each genotype (bottom row). Representative plots for the indicated genotypes shown in (A) with total quantification shown in (B) for liver (left), thymus (center), and spleen (right) for at least 3 pairs of littermate mice. (C) Proportion of iNKT cells expressed as percent of total TCRβ+T cells in thymus (left) and spleen (right) from WT, HDAC7-ΔP, Vα14, and Vα14 x HDAC7-ΔP mice. (D) Overlaid histograms of CD24 expression in the CD44-NK1.1- populations from thymocytes of the indicated genotypes, as shown in Figure 3B. (E) Proportion of iNKT cells expressed as percent of total TCRβ+T cells in thymus, spleen and liver from WT and Vα14 x HDAC7-ΔP mice. (F) Fold enrichment of iNKT cells in liver (% total TCRβ+) over spleen (% total TCRβ+) in WT and Vα14 x HDAC7-ΔP mice. Statistical significance was determined using unpaired two-tailed t tests; ***p≤0.001, ****p≤0.0001. less