Genetic approaches to dissect the function of the slam- and sap- family members

Resumen

The nine SLAM-family genes, SLAMF1-9, a subfamily of the immunoglobulin superfamily, encode differentially expressed cell surface receptors of hematopoietic cells. Engagement with their ligands, which are predominantly homotypic, leads to distinct signal transduction events, for instance those thar occur in the T or NK cell immune synapse. Upon phosphorylation of one or more copies of a unique tyrosine-based signalling motif in their cytoplasmic tails, six of the SLAM receptors recruit the highly specific single SH2-domain adapters SLAM-associated protein (SAP) and EAT-2A. These adapters in turn bind to the tyrosine kinase Fyn and/or other protein tyrosine kinases connectin the receptors to signal transduction networks. Individuals deficient in the SAP gene, SH2D1A, develop an immunodeficiency syndrome: X-linked Lympho Proliferative (XLP) disease. The SLAM-family of cell surface receptors is quickly emerging as an integral component of both innate and adaptive immunity. Althought first recognized as co-receptors on lymphocytes and NK cells, the SLAM-family is clearly involved in development and function of a multitude of hematopoietic cells. These include early stages of hematopiesis, development of NKT, T-CD4+and innate-like CD8+ T cells and macrophage and platelet functions. Furthermore, extensive polymorphisms and co-expression of isoforms of the SLAM-family receptors in epistatic pathogenic networks leadas to systemic lupus erithematosus. The main goal of this thesis was to unravel the functional redundancies and the specific ontributions of each of the SLAM-family members as well as the SAP-related adaptors in immune functions, by the disruption of individual genes through gene targeting. We have generated the targeted deletion in ES cells of a 400-kb region on mouse chromosome 1, which codes for seven SLAM family genes. Althought ew have been unable to generate mice harbouring the mutation, we have presented alternative approaches, that together with our preliminary data, expertise and current materials will ultimately lead to the generation of competent ES cells that will give rise to mice. We have also generated CD244-deficient mice in a C57BL/6 background. Although the murine CD244 was originally described as an activating receptor on NK and T cells, later studies showed that CD244 also has an inhibitory function in the IEL compartment; a highly specialized group of effector memory T cells that provides the most effective immune protection and preserves the integrity and vital functions of the intestinal mucosal epithelium. In this study we also report that, unlike in humans, the mouse and rat Eat-2 genes are duplicated with an identical genomic organization. The coding regions of the mouse Eat2a and Eat2b genes share 91% identity at the nucleotide level and 84% at the protein level; similary, segments of introns are highly conserved. Whereas expression of mouse Eat2a mRNA was detected in multiple tissues, Eat2b was only detectable in mouse natural killer cellsa, CD8+T cells, and ovaries, suggesting a very restricted tissue expression of the latter. Both the EAT-2A and EAT-28 coimmunoprecipitated with mouse SLAM and CD244 in transfeccted cells and augmented tyrosine phosphorylation of the cytoplasmic tail of the receptors. Botch EAT-2A and EAT-2B bind to the Src-like kinases Fyn, Hck, Lyn, and Fgr, as determined by a yeast two-hybrid assay. However, unlike SAP, the EAT-2 proteins bind to their kinase domains and not to the SH3 domain of these kinases. Finally, we created mice lacking EAT-2A, EAT-2B and both. Study of the phenotype of these mice will provide important clues to the adaptor function of these molecules in regulating downstream signalling of SLAM-family receptors and/or other cell surface receptors.