T-cell activation and induction of interleukin-2 (IL-2) expression in human T lymphocytes require both interaction of foreign antigen with the T-cell antigen receptor and protein kinase C (PKC) stimulation. Agents such as phorbol 12-myristate 13-acetate (PMA) that stimulate PKC augment the effects of antigen but are not sufficient for activation. By analysis of deletion mutants, we identified three DNA sequences extending from -73 to -89, -217 to -255, and -263 to -279, designated IL-2 sites A, D, and E, respectively, that are required for maximal induction of IL-2 expression. One of these regions, site E, interacted with a protein (NF-IL-2E) present only in the nuclei of cells which have been stimulated. The other two sequences interacted with a protein (NF-IL-2A) that is constitutively expressed in T cells. When multiple tandem copies of either the E site or the A site were placed upstream of the gamma-fibrinogen promoter, they activated expression via this promoter in response to signals initiated at the antigen receptor but not following PMA stimulation. For this reason, we denoted them antigen receptor response elements. The uncoupling of antigen receptor and PKC requirements in these studies indicates that these signal pathways are, at least in part, distinct and integrated at the level of the gene.Exposure of T lymphocytes to foreign antigen in the proper histocompatibility context induces a complex series of events that lead to cellular division and differentiation.
Using a transient transfection assay, we have defined the sequences required for the activation of the IL-2 gene in response to signals from the T cell antigen receptor. To do so we have transfected the human T cell line Jurkat with hybrid DNA constructs in which fragments from the IL-2 gene are linked to an indicator gene. The indicator gene product, as well as IL-2 production from the endogenous IL-2 gene were assayed after activation of the transfected Jurkat cells by various stimuli. We have demonstrated that a 275 bp fragment stretching from 52 to 326 bp upstream of the IL-2 gene transcription initiation site is required for expression of the linked indicator gene. This IL-2 gene fragment has several of the characteristics of a transcriptional enhancer element, in that it functions in both orientations and will enhance the expression from the promoter of an unrelated gene. Such enhancement occurred only after activation of Jurkat cells through the T cell antigen receptor. More specifically, this 275 bp fragment activated the expression of a linked gene after binding of a monoclonal antibody to the Jurkat T cell antigen receptor in the presence of PMA. In addition, calcium ionophore, which circumvents antigen receptor binding in T cell activation, induced the expression of the linked gene through this 275 bp sequence, in the presence of PMA. Finally, in a mutant Jurkat cell line lacking T3/antigen receptor complexes at the cell surface, no expression due to the IL-2 5' flanking region was seen after exposure to antibody to the T cell antigen receptor plus PMA or to PHA plus PMA. In contrast, calcium ionophore plus PMA did induce the expression of a linked gene through the IL-2 5' flanking region in the mutant Jurkat cell line. The responsiveness of the transfected hybrid genes containing the IL-2 regulatory region paralleled the expression of the endogenous IL-2 gene, as determined by IL-2 bioassays. We conclude that the 275 bp IL-2 sequence (-326 to -52 bp) is a target for the signal pathway originating at the T cell antigen receptor. Definition of this 275 bp target sequence should now permit the isolation of the molecules that bind to and activate the IL-2 gene.
The family of an obstetrical patient with an unusually weak Du phenotype has been studied. Strong positive reactions in the Du test were observed with the cells of four siblings who are of the DucE phenotype whereas very weak reactions were obtained with the proposita and one sib who are DuCe. The father is Rh negative thus proving the mother to be homozygous Du (DuCe/DucE). The two Du phenotypes and the homozygous Du mother were compared by titration scores and by the uptake of labeled anti‐D. The correlation was good between the titration score and the number of D antigen sites. However, red blood cells with normal D antigen content and one of the two weakly reactive DuCe/dce cells had titration scores that were too low in comparison to the number of D sites.
In a study of anti-N-like antibodies, we tested sera from 93 hemodialysis patients for hemagglutination reactions with untreated and formaldehyde-treated reagent red blood cells. Six of 22 sera from patients who had been dialyzed with formaldehyde-sterilized membranes had anti-N-like activity and 20 (91%) specifically agglutinated formaldehyde-treated red blood cells. Sera from 71 patients dialyzed with disposable membranes neither had anti-N-like activity nor agglutinated formaldehyde-treated red blood cells. The agglutination of formaldehyde-treated red blood cells by sera from hemodialysis patients was unrelated to MNU phenotypes and, therefore, identified a second serologic specificity, provisionally termed "anti-formaldehyde." "Anti-formaldehyde" was absorbed by and eluted from NN red blood cells as well as from formaldehyde-treated red blood cells regardless of MNU phenotype. All eluates and sera containing anti-N-like activity also agglutinated formaldehyde-treated red blood cells, typically after the addition of anti-human serum. These findings are consistent with the hypothesis that anti-N-like reactions of hemodialysis patients' sera represent cross reactions of formaldehyde related antibodies with N antigens of normal red blood cells.
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