Fig. 5. PLZF deficient iNKTs accumulate in the lymph nodes and spleen.

Lymph node (axillary, brachial, inguinal and mesenteric) and spleen cells 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 approximately 2-fold more lymph node iNKT cells and approximately 5-fold fewer spleen iNKT cells. Numbers indicate the percentage of cells within the gate. The frequency of iNKT cells among lymph node cells from 6 wild type and 8 PLZF deficient mice and spleen cells from 8 wild type and 10 PLZF deficient mice are shown in the scatter plots. Analysis was done by determining the percentage of tetramer positive cells among the total CD3⁺ T cells. Percentages were used rather than absolute numbers to normalize for differences in the total cellularity of the tissues due to the age of the mice. Cellularity of the lymph nodes and the spleens from age matched WT and PLZF KO mice were, however, nearly identical. Data set comparisons and P values are indicated in the figure. (B) iNKT cells from the lymph nodes and spleens of PLZF deficient mice do not upregulate CD44 and tend to be CD69^lo. Data is representative of at least five independent experiments. (C) Lymph node PLZF deficient iNKT cells are skewed towards expressing CD4. The increase in the percentage of CD4⁺ iNKT cells and the corresponding decrease in the percentage of CD4⁻ iNKTs in the lymph nodes and spleens from wild type and PLZF deficient mice are shown in the scatter plots. P values are shown in the figure. (D) PLZF deficient iNKT cells have markedly reduced expression of several markers typically found on wild type iNKT cells from the spleen. P values were derived from two-tailed, unpaired Student's t-test. Choices of statistical tests are explained in the methods section.