GENETIC ANALYSIS OF THYMOCYTE DEVELOPMENT

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Research is directed at investigating the cellular and genetic events that control normal T cell development. Transgenic and gene-targeting approaches are used to analyze the function of known genes and various molecular techniques (e.g.,RT-PCR, gene cloning) are being employed to identify novel genes that participate in thymocyte development. Current studies are focused in three major areas: (I) Role of T cell antigen receptor (TCR) signal transduction in thymocyte maturation. In mature T cells, the TCR transduces signals important for T cell activation and cell mediated immunity and in immature T cells TCR signals are required for thymocyte development and for thymic (positive and negative) selection. The TCR is composed of multiple potential signal transducing subunits (the CD3 chains and one or more members of the zeta-family of proteins; zeta, eta and Fcgamma) that couple the TCR to intracellular signal transduction pathways via conserved functional sequences (Immunoreceptor Tyrosine based Activation Motifs; ITAMs). To determine if the TCR signal transducing subunits perform distinct or analogous functions in development we have: a) examined their role in T cell ontogeny by generating zeta/eta/Fcgamma deficient mice and CD3-gammadeltaepsilon deficient mice by gene targeting, b) genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient forms of the individual zeta-family or CD3 proteins, and c) examined the function of the 3 zeta-chain ITAMs in thymocyte development and selection by transgene reconstitution of zeta-deficient mice. These studies reveal that expression of zeta-family and CD3 chains is required for normal T cell development but that zeta chain signals are not specifically required. Thus the CD3 subunit ITAMs can transduce all of the signals necessary for thymocyte maturation. However, a direct relationship was observed between the total number of TCR-(zeta) ITAMs and the efficiency of thymocyte selection demonstrating that the multiple ITAMs within the TCR function to amplify the signaling response. These results demonstrate a previously unrealized role for multiple TCR ITAMs in thymocyte selection and identify a function for signal amplification in selection of the T cell repertoire. (II) The role of other signal transducing proteins in T cell development is being examined by the generation of transgenic/knockout mice. These include CD5, a surface receptor distinct from the TCR that also contains an ITAM-like sequence but acts as a negative regulator of TCR signaling. (III) Novel genes that have potential functions in thymocyte development or T cell activation are being identified. A new lymphoid-specific kinase, Txk, and phosphatase PTPK1 have been cloned and biochemical and transgenic approaches are being employed to analyze the function of these proteins. A similar approach is being employed to identify members of the homeobox gene family that are expressed during T cell ontogeny.