MOLECULAR-CLONING OF A NOVEL T-CELL-DIRECTED CC CHEMOKINE EXPRESSED IN THYMUS BY SIGNAL SEQUENCE TRAP USING EPSTEIN-BARR-VIRUS VECTOR

Precursors of most secreted and cell surface molecules carry signal se quences at their amino termini. Here we describe an efficient signal s equence trap method and isolation of a novel CC chemokine. An expressi on library was constructed by inserting 5' portion-enriched cDNAs from phytohemagglutinin-stimulated peripheral blood mononuclear cells into upstream of signal sequence deleted CD4 cDNA in an Epstein-Barr virus shuttle vector. After electroporation into Raji cells, CD4 antigen-po sitive cells were enriched by repeated cell sorting and plasmids were recovered in Escherichia coli. Out of 100 plasmid clones examined, 42 clones directed expression of CD4 antigen on the cell surface. Among t hem were signal sequences of CD6, beta(2)-microglobulin, MGC-24, and T cell receptor epsilon-chain, and at least four novel potential signal sequences. A cDNA clone encoding a novel CC chemokine was isolated by using one of the trapped fragments. The gene designated as TARC from Thymus and Activation-Regulated Chemokine was expressed transiently in phytohemagglutinin-stimulated peripheral blood mononuclear cells and constitutively in thymus. Radiolabeled recombinant TARC specifically b ound to T cell lines and peripheral T cells but not to monocytes or gr anulocytes. The binding of radiolabeled TARC to the high-affinity rece ptor (K-d, 2.1 nM) on Jurkat was displaced by TARC but not by interleu kin-8, MIP-1 alpha, RANTES, or MCP-1. TARC also bound to the promiscuo us chemokine receptor on erythrocytes (K-d, 17 nM). TARC induced chemo taxis in T cell lines Hut78 and Hut102. Pretreatment of Hut78 with per tussis toxin abolished the TARC-induced cell migration. Collectively, T cells express a highly selective receptor for TARC that is coupled t o pertussis toxin-sensitive G-protein. TARC may a factor playing impor tant roles in T cell development in thymus as well as in trafficking a nd activation of mature T cells.