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. 2018 Apr 17;7:e32109. doi: 10.7554/eLife.32109

Figure 2. Deletion of HDAC7 in thymocytes Reduces iNKT Numbers and Expands an Innate-Memory CD8 Population.

(A) Representative flow plots showing CD4/CD8 expression (left), loaded and empty CD1D tetramer reactivity (center), and CD44/NK1.1 expression of magnetically enriched iNKT cells (right) from thymus of WT (top) and Hdac7-KO (bottom) thymocytes. (B) Representative flow plots showing an expanded CD44hi Eomes+ innate memory population in mature CD8SP thymocytes from Hdac7-KO mice. Mature CD8 SP thymocytes are identified as TCRβ+CD8+CD4-. (C) Expression of CD44 and CD62L in CD8 T-cells from spleens of WT and Hdac7-KO littermate mice. Data are representative of 3 independent experiments with N = 2–4 mice per group. (D, E) Representative flow plots (D) and total quantification (E) of peripheral naive, central memory (TCM), and effector memory (TEM) CD8 T-cell populations from WT (CD45.1) and Hdac7-KO (CD45.2) derived bone marrow in mixed hematopoietic chimeras. (F, G, H) Representative flow plots (F) and total quantification (G, H) of IFNγ secretion in ex vivo stimulated CD8 T-cells. Splenocytes were harvested from mixed WT (CD45.1)/Hdac7-KO (CD45.2) hematopoietic chimeras, and stimulated ex vivo for 4 hr with PMA/Ionomycin. Percent of cells secreting IFNγ (G) and median fluorescence intensity (MFI) of IFNγ secretion (H) are shown. Bars on graphs indicate mean ±SEM (error bars). Data in (E) are combined from three independent experiments with at least three mice per group; data in (G, H) are combined from three independent experiments with two mice per group. Statistical significance was determined using either unpaired two-tailed T-test (E, H) or two-way ANOVA (G); ***p≤0.001, ****p≤0.0001. A Bonferroni post-test was used for pairwise comparisons in (E).

Figure 2—source data 1. Microsoft Excel workbook containing numerical data matrices for all figure panels (on separate sheets) in which individual data points are not represented graphically.
Figure 2E, Figure 2G–H, Figure 2—figure supplement 1B–C, and Figure 2—figure supplement 2A,C.
elife-32109-fig2-data1.xlsx (21.5KB, xlsx)
DOI: 10.7554/eLife.32109.011

Figure 2.

Figure 2—figure supplement 1. Supporting Data on T Cell Phenotypes of Hdac7-KO Mice.

Figure 2—figure supplement 1.

(A) Representative flow scatter plots showing analysis of WT (top) and HDAC7-ΔP TG (bottom) thymocytes for the frequency of γδ T cells (second column), and their Vδ6.3-positive (third column) and PLZF-positive (fourth column) subsets. (B) Quantification of CD44+/Eomes + cells among mature CD8SP thymocytes. Data are combined from four independent experiments with 1–2 mice per group. ****: p≤0.0001, 2-tailed Student’s T-test. (C) Quantification of total γδ T cells, as well as Vδ6.3-positive and PLZF-positive subsets, as represented in (A), based on six independent comparisons of WT and Hdac7-KO mice. Based on a 2-tailed Student’s T-test, p>0.05 for all comparisons except total γδ T cells (p=0.043). (C) Surface expression of Ly6C and CXCR3 from peripheral CD8 T-cells. Black unfilled histograms correspond to WT, blue-filled to Hdac7-KO. Plots represent three independent experiments with 2–4 mice per group.
Figure 2—figure supplement 2. Supporting Data on Memory Markers and Cytokine Production in WT: Hdac7-KO Mixed Hematopoietic Chimeras.

Figure 2—figure supplement 2.

(A) Bar chart showing log2 ratios of Hdac7-KO/WT cells present in WT: Hdac7-KO hematopoietic chimeras at the indicated thymic developmental stages. (B) Representative flow plots showing gating for analysis of memory markers and cytokine secretion in WT: Hdac7-KO mixed hematopoietic chimeras. (C) Quantification for 8 WT: Hdac7-KO mixed chimeras of total CD4/CD8 prevalence and expression of memory markers in CD4 cells (left), or for 6 chimeras of expression of IL-4 and IFNγ in CD4 cells (right). *: p=3.07×10−8, **: p=0.0083. ***: p=0.016.