ITM2A is induced during thymocyte selection and T cell activation and causes downregulation of CD8 when overexpressed in CD4(+)CD8(+) double positivethymocytes

To identify novel genes that are involved in positive selection of thymocyt es, we performed polymerase chain reaction (PCR)-based subtractive hybridiz ation between selecting and nonselecting thymi. OT-1 T cell receptor (TCR) transgenic thymocytes on a recombination activating gene (RAG) null backgro und are efficiently selected into the CD8 lineage in H-2(b) mice (RAG-2(-/- )OT-1, selecting thymi), but are not selected on a transporter associated w ith antigen processing (TAP) null background (RAG-2(-/-)TAP-1(-/-)OT-1, non selecting thymi). We report here our studies of one gene, ITM2A, whose expr ession is dramatically higher in T cells in the selecting thymus. The expre ssion pattern of ITM2A in thymocyte subsets correlates with upregulation du ring positive selection. In addition, ITM2A expression is higher in the thy mus than in either the spleen or lymph nodes, but can be upregulated in per ipheral T cells upon activation. ITM2A expression was also induced in RAG-2 (-/-) thymocytes in vivo upon CD3 cross-linking. We demonstrate that ITM2A is a type II membrane glycoprotein that exists as two species with apparent M-r of 45 and 43 kD and appears to localize primarily to large cytoplasmic vesicles and the Golgi apparatus, but is also expressed on the cell surfac e. Expression on the surface of EL4 cells increases with activation by phor bol myristate acetate (PMA) and ionomycin. Finally, overexpression of ITM2A under control of the lck proximal promoter in mice results in partial down regulation of CD8 in CD4(+) CD8(+) double positive (DP) thymocytes, and a c orresponding increase in the number of CD4(+)CD8(lo) thymocytes. Possible r oles for this novel activation marker in thymocyte development are discusse d.