Fig. 3. Altered iNKT cell development in PLZF deficient mice.

Thymocytes from the indicated mouse lines were stained with CD1d:α-GalCer tetramers and antibodies against CD3, CD44, CD69, CD4 and CD8. (A) PLZF deficient mice have an approximately 9-fold decrease in the number of iNKT cells. Numbers indicate the percentage of cells within the gate. The frequency of iNKT cells in 13 wild type and 14 PLZF deficient mice is shown in the scatter plot. Analysis was done by determining the percentage of tetramer positive cells among the CD3^hi thymocytes. Percentages were used rather than absolute numbers to normalize for differences in the total cellularity of the thymus due to the age or sex of the mice. P values are indicated in the figure. (B) iNKT cells from PLZF deficient mice do not upregulate CD44. Both WT and KO iNKT cells upregulate CD69. (C) BrdU incorporation into iNKT cells in the thymus 16-hours after injection into 10-13 day old mice. Both total iNKT cells and only CD44^lo iNKT cells are shown. Experiment was repeated twice. (D) iNKT cells from PLZF deficient mice are skewed towards CD4⁺. Only iNKT cells are shown in the FACS plot. Numbers indicate the percentage of cells in the region. The increase in the percentage of CD4⁺ iNKT cells and the corresponding decrease in the percentage of CD4⁻ iNKTs in the in PLZF deficient mice in 8 wild type and 9 PLZF deficient mice are shown as a scatter plots. Data set comparisons and P values are indicated in the figure. (E) PLZF deficient iNKT cells have reduced expression of many cell surface markers typical of iNKT cells such as NK1.1, DX5, NKG2D, 2B4 (CD244) and CD122. Change in expression of each marker was confirmed in at least three independent experiments. P values were derived from two-tailed, unpaired Student's t-test. Choices of statistical tests are explained in the methods section.