T-cell Growth Factor: Complete Nucleotide Sequence and Organization of the Gene in Normal and Malignant Cells

Using a cloned cDNA copy of T-cell growth factor (TCGF) mRNA from the Jurkat leukemic T-cell line, we have isolated three overlapping TCGF genomic clones from a human DNA library. The entire TCGF gene is contained within two adjacent EcoRI fragments spanning about 8 kilobases. The complete nucleic acid sequence was determined. The gene is divided into four exons. The 5\u27 untranslated region and the first 49 amino acids of the protein, 20 of which constitute a signal polypeptide and are not present in the secreted protein, are encoded by the first exon. Exons 2 and 3, separated from each other by a long intervening sequence, contain coding information for the next 20 and 48 amino acids, respectively. The remaining 36 amino acids and the 3\u27 untranslated region are contained in the fourth exon. A promoter sequence T-A-T-A-A-A is present 77 base pairs (bp) upstream from the translation initiation site, and a CAT homology region occurs 104 bp upstream from the initiation site. A putative site for initiation of mRNA transcription was identified 53 bp 5\u27 of the translation initiation codon. The organization of the gene was shown by Southern blot analysis to be identical in normal peripheral blood lymphocytes and in a variety of malignant lymphoid cell types. Restriction analysis of these cellular DNAs produced results exactly as predicted by the map for the cloned genomic TCGF, indicating that there is only a single copy of the human TCGF gene

Lipoteichoic Acid Inhibits Interleukin-2 (IL-2) Function by Direct Binding to IL-2

Lipoteichoic acid (LTA) is associated with the cell envelope of most gram-positive bacteria. Although previously thought to act mainly as a virulence factor by virtue of its adhesive nature, evidence is now provided that LTA can also suppress the function of interleukin-2 (IL-2), an autocrine growth factor for T cells. LTA from four separate bacterial strains lowered the levels of detectable IL-2 during a peripheral blood mononuclear cell response to the antigen tetanus toxoid (TT). T-cell proliferation in response to TT was similarly inhibited by LTA. In contrast, levels of detectable gamma interferon increased. In addition, LTA inhibited IL-2 detection by enzyme-linked immunosorbent assay (ELISA) and blocked the proliferative response of an IL-2-dependent T-cell line to soluble IL-2. Further studies using ELISA demonstrated that LTA blocks IL-2 detection and function by binding directly to IL-2. Flow cytometric analysis revealed that IL-2 binding to T cells is inhibited in the presence of purified LTA but not LTA plus anti-LTA monoclonal antibody. In summary, these studies demonstrate a novel effect of LTA on the immune response through direct binding to IL-2 and inhibition of IL-2 function. Importantly, gram-positive organisms from which LTA is obtained not only play an important role in the pathology of diseases such as bacterial endocarditis, septic shock, acute respiratory distress syndrome, and multiple organ failure but also comprise a significant portion of commensal populations within the human host. Inhibition of IL-2 function by LTA may represent yet another mechanism by which gram-positive bacteria dampen the host immune response and facilitate survival. Thus, LTA provides a potential target for therapeutic intervention when gram-positive organisms are involved

Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A

Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response. In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response. The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases. Binding of the drug inhibits isomerase activity, but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase, and not from inhibition of isomerase activity. A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-kappa B. Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT. FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit