Gene expression analysis of Tec family kinases in Band 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 immunodeficiency disease (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-, Tecand 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 seen in CD4 + and CD8 + T-cells in comparison to total CD3 + T-cells. In Paper IV we found Zbtb26, an interesting transcription factor, being up-regulated in Itk-deficient T-cells, while normalized in Itk/Tec-deficient cells compared to Wt. By the use of a combination of the microarray technology and quantitative RT-PCR analysis, we have evaluated the transcriptomes of Btk-, Itk-, Tecand Itk/Tec-deficient mice. LIST OF PUBLICATIONS This thesis is based on the following articles and manuscript, which will be referred to in the text by their Roman numerals. I. Lindvall JM, Blomberg KEM, Berglöf A, Yang Q, Smith CIE and Islam TC. Gene expression profile of B cells from Xid mice and Btk knockout mice. Eur J Immunol. 2004 Jul;34(7):1981-91. II. Lindvall JM, Blomberg KEM, Berglöf A and Smith CIE. Distinct gene expression signature in Btk-defective T1 B-cells. Biochem Biophys Res Commun 2006 Jul 28;346(2):461-9. III. Blomberg KEM, Boucheron N, Lindvall JM, Yu L, Raberger J, Berglöf A, Ellmeier W and Smith CIE. Transcriptional signatures of Itk-deficient CD3 + , CD4 + and CD8 + Tcells. BMC Genomics in press. IV. Blomberg KEM, Boucheron N, Lindvall JM, Vargas L, Raberger J, Berglöf A, Ellmeier W and Smith CIE. Tec and Itk regulate overlapping signaling pathways in T lymphocytes. Manuscript Paper I: Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission. Publications by the author which are not included in the thesis: Raberger J, Schebesta A, Sakaguchi S, Boucheron N, Blomberg KEM, Berglöf A, Kolbe T, Smith CIE, Rülicke T, Ellmeier W. The transcriptional regulator PLZF induces the development of CD44 high memory phenotype T cells. Proc Natl Acad Sci U S A. 2008 Nov 12. Nawaz HM*, Blomberg KEM*, Lindvall JM, Kurosaki T, Smith CIE. Expression profiling of chicken DT40 lymphoma cells indicates clonal selection of knockout and gene reconstituted cells. Biochem Biophys Res Commun. 2008 Dec 12;377(2):584-8. Epub 2008 Oct 16. Yu L, Mohamed AJ, Simonson OE, Vargas L, Blomberg KEM, Bjorkstrand B, Arteaga HJ, Nore BF, Smith CIE. Proteasome dependent auto-regulation of Bruton's tyrosine kinase (Btk) promoter via NF-kappaB. Blood. 2008 Feb 21. Hasan M, Lopez-Herrera G, Blomberg KEM, Lindvall JM, Berglöf A, Smith CIE, and Vargas L. Defective TLR9 mediated cytokine production in B cells from Btk deficient mice. Immunology. 2008 Feb;123(2):239-49. Epub 2007 Aug 28. Blomberg KEM, Smith CIE and Lindvall JM. Gene expression signatures in primary immunodeficiencies; the experience from human disease and mouse models. Curr Mol Med. 2007 Sep;7(6):555-66. Review. Lindvall JM, Blomberg KEM, Christensson B, Sander B, Smith CIE. Gene expression in lymphomas and immunodeficiencies provides new insights into B-lymphocyte development. In: New Messenger RNA Research Communications (ed. Kwang LB), Nova Science Publishers, 2007. Book chapter. Lindvall JM*, Blomberg KEM*, Wennborg A, Smith CIE. Differential expression and molecular characterisation of Lmo7, Myo1e, Sash1, and Mcoln2 genes in Btk-defective B-cell. Cell Immunol. 2005 May; 235(1): 46-55. Lindvall JM, Blomberg KEM, Väliaho J, Vargas L, Heinonen JE, Berglöf A, Mohamed AJ, Nore BF, Vihinen M, Smith CIE. Bruton tyrosine kinasecell biology, sequence conservation, mutation spectrum, siRNA modifications and expression profiling. Immunological Reviews. 2005 Feb; 203:200-15. Lindvall JM, Blomberg KEM, Smith CIE. In silico tools for signal transduction research. Briefings in Bioinformatics. 2003 Dec; 4(4): 315-24. * These authors contributed equally to this paper