Figure 5. Gene expression microarray analysis indicates impaired terminal maturation of Itk^−/− γδ NKT cells.

(A) Scatter plot showing expression of consolidated probe sets by CD24(HSA)^lo V6 γδ T cells in thymocytes from adult WT and Itk^−/− mice. Nomenclature is according to ImmGen (immgen.org): MatV6.WT= CD24(HSA)^lo V6 γδ T cells from adult WT thymus; TotalV6.Itk^−/−, total V6 γδ T cells from adult Itk^−/− thymus, which are 95–98% CD24^lo. Each dot represents one gene (mean of all probe sets and mean of replicates); red and blue indicate genes with expression increased or decreased, respectively, by more than twofold (P < 0.05 (Student's t-test); coefficient of variation < 0.5; mean expression value (MEV) > 120 in one subset); numbers in parentheses above plot indicates total number of these genes. The numbers within the plot indicate the number of up-regulated (red) or down-regulated (blue) genes in the comparison. Data are from three independent experiments with 4–30 mice each.

(B) The 124 genes increased in MatV6.WT versus TotalV6.Itk^−/− cells were classified into functional pathways using the KEGG analysis program (DAVID). The top five pathways enriched in the dataset are shown along with the number of genes, the gene names, the P-value, and the Benjamini P-value for each pathway.

(C) Venn diagram comparing the list of genes that were increased or decreased in iNKT (NKT.44⁺NK1.1⁺) cells versus MatCD4 (CD24^lo) and MatCD8 (CD24^lo) single positive thymocytes (identified previously in (21)) with the list of 124 genes down-regulated in TotalV6.Itk^−/− versus MatV6.WT cells. Roughly 40% of the genes decreased in TotalV6.Itk^−/− versus MatV6.WT cells are normally increased in NKT.44⁺NK1.1⁺ cells versus mature single-positives.

(D) Heat map showing relative expression of the 51 genes identified in Figure 5C that were decreased in TotalV6.Itk^−/− versus MatV6.WT cells and increased in NKT.44⁺NK1.1⁺ cells versus MatCD4 and MatCD8 single positive thymoctyes in populations of NKT and MatCD4 cells from WT mice and V6 cells from WT or Itk^−/− mice. Data were log transformed, centered by gene row and hierarchically clustered by gene and subset. The clustering dendrogram for samples is shown. Color coding reflects relative expression levels of a given gene in each subset, and does not provide information about absolute expression levels of each gene.