Figure 2.

Loss of Ezh2 results in increased iNKT cell numbers and development into NKT2 cell. (A) iNKT cells in thymus, spleen, and peripheral lymph nodes of Ezh2^fl/fl; CD4-cre (Ezh2^−/−) and littermate controls were detected as CD1d-Tetramer⁺ and TCRβ⁺ cells. (bottom) Percentage (left) or absolute number (right) of CD1d-Tetramer⁺ and TCRβ⁺ cells in the thymus. Representative plots of >5 independent experiments with >15 mice in total are shown. Each dot represents a single mouse. (B) Flow cytometry histograms show levels of H3K27me3 in DP, CD4⁺ single positive (CD4⁺ SP), and iNKT cells (top) and Ezh2, H3K27me3, and PLZF in subpopulations of developing iNKT cell stages defined as: stage 0 (CD1d-Tetramer⁺, TCRβ⁺, and CD24⁺); stage 1 (CD1d-Tetramer⁺, TCRβ⁺, CD24⁻, CD44^lo, and NK1.1⁻); stage 2 (CD1d-Tetramer⁺, TCRβ⁺, CD24⁻, CD44⁺, and NK1.1⁻); and stage 3 (CD1d-Tetramer⁺, TCRβ⁺, CD24⁻, CD44⁺, and NK1.1⁺). Representative plots of three independent experiments with more than five mice in total are shown. Gray shaded area, WT; red line, Ezh2-deficient cells. (C) Deficiency in Ezh2 leads to altered iNKT cell development and increased number of IL-4–producing cells. The subpopulations of developing iNKT cells were defined by expression of CD44 and NK1.1 on CD1d-Tetramer⁺ TCRβ⁺ CD24⁻ cells. (middle) Absolute number of iNKT cells. (right) Absolute number of IL-4–producing iNKT cells. Representative plots of more than three independent experiments with three or more mice in total are shown. Each dot represents a single mouse. Significance for all data was determined by unpaired Student’s t test: *, P ≤ 0.05; **, P ≤ 0.01. ≤ 0.05.