Figure 4.

Accumulation of Ezh2-deficient iNKT and iNKT-like cells results in the development of bystander CD8⁺ T cells. (A) Generation of memory-like (bystander) CD8⁺ T cells was quantified by flow cytometry. Numbers indicate percentage of cells of defined surface phenotype (left). Histograms (right) show surface expression of the memory T cell markers (CD24^lo, CD44^hi, and CD122^hi) on thymic and splenic CD8⁺ T cells derived from Ezh2^fl/fl; CD4-cre (red line) and Ezh2^fl/fl (WT) littermate controls (shaded area). Data are representative of 5–7 experiments with 2–3 mice per genotype each. (B) Absolute number of CD4⁺ CD8⁺ double positive (DP), CD4⁺ CD8⁻ single positive (CD4⁺ SP), and CD4⁻ CD8⁺ single positive (CD8⁺ SP) thymocytes from Ezh2^fl/fl control (WT, black symbols) or Ezh2^fl/fl; CD4-cre (red symbols) mice are shown. Data represents three independent experiments with eight mice per genotype analyzed. Each dot represents a single mouse. (C) Expression of Eomes and T-bet in ex vivo isolated CD8⁺ splenic T cells (top) and IFN-γ production after PMA/ionomycin stimulation (middle), were measured by intracellular FACS. (bottom) Quantification of percentage of IFN-γ positive CD8⁺ splenocytes isolated from Ezh2^fl/fl control (WT, black diamonds) or Ezh2^fl/fl; CD4-cre (red circles) mice. Data represents two independent experiments with four or more mice analyzed. Each dot represents a single mouse. Significance was determined for all data by unpaired Student’s t test: **, P ≤ 0.01; ***, P ≤ 0.001.