In order to understand the development and functioning of the thymus, both in terms of T cell differentiation and stromal cell environmental support, we have undertaken a molecular approach to identify genes that are uniquely expressed in this organ. We have created an anchored RT-PCR based cDNA library from 14-day fetal thymuses after culturing them in deoxyguanosine and treating them with an anti-CD45 antibody to deplete the cell population of hematopoietic cells. The cDNA library was subtracted with poly A+ RNA prepared first from a fibroblast cell line and then from whole spleen. Three prime sequencing of 250 random cDNAs revealed 137 which were not in the databases of known sequences. The novel cDNAs were then screened by Northern blotting for expression in various tissues and in a set of SV40 transformed thymic epithelial cell lines. Four genes were selected for further analysis because they were limited in their expression to the thymus or to one of the stromal cell lines. All four were completely sequenced by obtaining full length cDNA clones from a SCID thymus library. During the past year the lab has made progress in understanding the structure and function of three of these genes. 1. The lC12 gene, encoding a 12 transmembrane spanning co- transporter glycoprotein called TSCOT, was transfected into rat basophilic leukemia cells and used to immunize a rabbit. The antiserum obtained partially inhibited T cell development in fetal thymic organ cultures by depleting double negative thymocytes at the CD44- CD25+ stage. This result suggests that TSCOT function may be required for early thymocyte development. 2. We completed the sequence of the 1C10 cDNA . The putative protein has weak homology to transcription factors containing a POU domain and it possesses a nuclear localization signal. Antibodies directed against a predicted c-terminal peptide immunoprecipitated a long (36kD) and a short (32kD) form of the protein from thymocytes. These corresponded to two forms of the mRNA detected in the cDNA library and on Northern blots. Preliminary data from genomic clones suggest that these two forms of mRNA arise from alternative splicing. Interestingly, during embryonic development, the short form of the mRNA is expressed earlier (day 10) than the long form (day 14). 3. One cDNA we cloned from our library was subsequently described in the literature as the gamma 2 chain of lamin 5. Knowing that it is expressed in the thymus, we obtained monoclonal antibodies against lamin 5 and tested their effect on thymocyte development in fetal thymus organ cultures. A blocking antibody, but not a nonblocking antibody, directed against the a chain of lamin 5 inhibited T cell development at the double negative stage, with some depletion of the earliest CD44+ CD25- thymocytes and a large effect on the CD44- CD25+thymocytes. The observations suggest that interaction with lamin 5 may be critical for thymocyte development and/or survival.
