Defects of Thymic Output in Wiskott-Aldrich Syndrome

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DESCRIPTION (provided by applicant): The Wiskott-Aldrich syndrome (WAS) is a rare inherited platelet/ immunodeficiency disease characterized by thrombocytopenia, eczema, infections and frequency of autoimmune disorders and malignancies. The defective gene encodes WASP, a blood cell protein that coordinates cytoskeletal changes in endocytotic and proliferative responses of various blood cells. In a recent cross-sectional study of blood specimens, we found that WAS patients have decreased numbers of T and B lymphocytes. The deficit of cells was most pronounced for infant patients and for T cells primarily reflected a decrease of naive cells. Preliminary findings showed normal survival properties for naive T cells, suggesting that the cell deficit is due to deficient output. In the currently accepted paradigm for WAS, disease pathology is due to defective functions of peripheral blood cells. In the proposed R21 project, we will establish "proof of concept" for the novel hypothesis that defective thymocyte development and deficient output contribute significantly to the pathology of severe WAS. We will test for deficient thymic output by quantifying T cell receptor excision circles (TRECs) by real time PCR in CD4 and CDS cells of infant patients compared to normal infants. TREC values will be compared for WASP+ patients and WASP- patients, for whom frequency of autoimmune disease and malignancies is substantially higher. Measurement of the marker CD31 on CD4 naive cells (CD4+CD45RA+) will provide independent quantization of recent thymic emigrant cells. To eliminate other mechanisms as significant contributors to the cell deficit, patient naive CD4 and CD8 cells will be compared to normal cells for survival in vitro, levels of survival factors Bcl-2, Bcl-xL and IL7 receptor, and altered proliferation by analyzing nuclear antigen Ki-67 and ex vivo BrdU incorporation. Finally, we will determine whether naive cells of WAS patients have restricted/skewed antigen receptor repertoire. TCR repertoire diversity of sorted naive CD4 cells will be examined by CDR3 length analysis (spectratyping). Each T cell receptor (TCR) Vbeta fragment will be amplified with one of the 24 Vbeta-specific primers and a fluorescently labeled Cbeta. The results of the proposed 2 year proof-of-concept pilot project combined with our existing findings on mouse thymocytes would form the basis of a coordinated RO1 application combining study of patient T cells and WASP-null mouse models to delineate the role of WASP in thymocyte maturation.