Gene expression analysis of Tec family kinases in B- and T-lymphocytes

The microarray technology is a powerful tool used in many different research areas. The technique has been around for more than a decade and has revolutionized the molecular biology field. In this thesis the Affymetrix GeneChip® arrays were used to study the transcriptomes of Tec family kinase mutant mouse models. Bruton s tyrosine kinase (Btk), IL-2 inducible T-cell kinase (Itk) and Tyrosine kinase expressed in hepatocellular carcinoma (Tec) are protein tyrosine kinases belonging to this Tec kinase family. Mutations in Btk cause a primary immunodeficiency disease called Xlinked agammaglobulinemia (XLA) in humans and X-linked immunodeficiencydisease (Xid) in mice. Gene expression profiling was performed on whole splenic Bcells as well as Transitional 1 (T1) B-cells from Xid, Btk knockout (Btk KO) and control mice. This was done in order to study differences and similarities between Btkdefective mice (Xid and Btk KO together) compared to control. Small differences were distinguished between the Btk-defective mouse strains in the whole splenic B-cell population; only seven genes differed (>2-fold) between the two. A number of potentially interesting genes were found to be differentially expressed in the Btkdefective groups compared to the controls. We also show that the Btk defect is already manifested at the T1 B-cell stage. Itk is important for the T-cell development where it has a role in regulating the conventional versus innate T-cells. Itk-deficient T-cells are of an innate, memory-like type. Tec is poorly expressed in resting T-cells, but is expressed 2-3 days after stimulation. Tec-deficient mice show no phenotypic alterations and the mice develop normally. In Papers III and IV we looked at the transcriptomes of Itk-, Tec- and Itk/Tec-deficient mice by studying unstimulated as well as stimulated CD3+ T-cells. The Itk-deficiency was also studied in CD4+ and CD8+ T-cell subpopulations. The gene expression patterns of Tec-deficient mice compared to control mice showed small differences, further supporting earlier findings. More differences were seen in Itkdeficient as well as Itk/Tec-deficient mice compared to controls. We also investigated if the Itk-deficiency could mimic calcineurin inhibition by treating Wt T-cells with cyclosporin A (CsA). CsA was shown to have a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Greater differences were