Molecular Cloning of a Novel T Cell-directed CC Chemokine Expressed in Thymus by Signal Sequence Trap Using Epstein-Barr Virus Vector

Abstract

Precursors of most secreted and cell surface molecules carry signal sequences at their amino termini. Here we describe an efficient signal sequence trap method and isolation of a novel CC chemokine. An expression 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-positive 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 them were signal sequences of CD6, beta2-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 bound to T cell lines and peripheral T cells but not to monocytes or granulocytes. The binding of radiolabeled TARC to the high-affinity receptor (Kd, 2.1 nM) on Jurkat was displaced by TARC but not by interleukin-8, MIP-1alpha, RANTES, or MCP-1. TARC also bound to the promiscuous chemokine receptor on erythrocytes (Kd, 17 nM). TARC induced chemotaxis in T cell lines Hut78 and Hut102. Pretreatment of Hut78 with pertussis toxin abolished the TARC-induced cell migration. Collectively, T cells express a highly selective receptor for TARC that is coupled to pertussis toxin-sensitive G-protein. TARC may a factor playing important roles in T cell development in thymus as well as in trafficking and activation of mature T cells.

C-C Chemokine Receptor 4 Expression Defines a Major Subset of Circulating Nonintestinal Memory T Cells of Both Th1 and Th2 Potential

“…The restricted expression of CCR4 on SP CD4 ϩ thymocytes correlates with the chemotaxis of this subset of thymocytes to MDC and further demonstrates that expression of CCR4 and chemotaxis to MDC correlate (44). TARC and MDC are also reported to be expressed in the thymus (45,46) and by APCs (24,47). It is possible that TARC and MDC are expressed on macrophages, dendritic cells, and epithelial cells in the thymus and attract mature CCR4 ϩ CD4 SP thymocytes to modulate positive and negative selection during T cell development.…”

IL‐4 induces expression of TARC/CCL17 via two STAT6 binding sites

“…It encodes a highly basic 94 amino acid residues long preprotein with a cleavage site between Ala23 and Ala24. The mature TARC/CCL17 protein is 71 amino acid residues in length and was shown to be constitutively expressed in thymus [2] and, upon activation with different inducers, in several cell types including PBMC [5], monocytes [6], dendritic cells [7], endothelial cells, and bronchial epithelial cells [8].…”

Chemokines: signal lamps for trafficking of T and B cells for development and effector function

“…Chemokines are good candidates to regulate the ordered localization and migration of T cell progenitors. Thymus expresses a number of chemokines including MDC/STCP-1 [24,25], TECK [26], TARC [27], SDF-1 [28], LARC/MIP-3␣/Exodus [29][30][31], murine thymusderived chemotactic agent 4 (TCA4) [32], and CK␤-11/MIP-3␤/ ELC [31,33] at high levels ( Table 3). TECK is expressed by thymic dendritic cells but not by bone marrow dendritic cells [26].…”