Growth of human T lymphocytes: an analysis of interleukin 2 and its cellular receptor.
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Animals↗
Biomedical subjects
W C Greene
Publications and source records attributed to W C Greene.
Selective killing of human T-lymphotropic virus-I infected leukemic T-cells by monoclonal anti-interleukin 2 receptor antibody-ricin A chain conjugates: potentiation by ammonium chloride and monensin.
Adult T-cell leukemia is a progressive disease produced by infection of mature T-cells with the human T-lymphotropic virus-I (HTLV-I). These retrovirus infected T-cells express large numbers of receptors for interleukin 2 (or T-cell growth factor). Due to the presence of these receptors, these leukemic T-cells can be selectively killed in vitro by monoclonal anti-interleukin 2 receptor antibody covalently linked to the A chain of the plant toxin, ricin (anti-TAC-A), suggesting that such immunotoxins may be useful in the therapy of this disease. In this report we demonstrate that the lysosomotropic agent ammonium chloride and the carboxylic ionophore monensin substantially potentiate the cytotoxicity of anti-TAC-A on HUT 102 cells, a long-term cultured HTLV-I infected T-cell line. Anti-TAC-A alone produces half-maximal inhibition of protein synthesis in HUT 102 cells at a concentration of 2.2 X 10(-10) M (the 50% inhibitor, concentration). Addition of ammonium chloride or monensin augments the potency of anti-TAC-A killing 100-fold (50% inhibitory concentration = 2.5 X 10(-12) M) and 400-fold (50% inhibitory concentration = 8 X 10(-13) M), respectively; furthermore, these agents accelerate rate of anti-TAC-A intoxication and increase the specific killing of interleukin 2 receptor-bearing leukemic cells. At concentrations which cause only minor harm to colony forming hematopoietic progenitor cells (granulocyte-erythroid, monocytic, megakaryocytic colony forming unit, granulocyte-macrophage colony forming unit, macrophage colony forming unit, and granulocyte colony forming unit), anti-TAC-A alone is able to eliminate greater than 99.99% of an HTLV-I infected T-cell population. In the presence of ammonium chloride or monensin, respectively, a 3.5- and 20-fold greater cytoreduction of HTLV-I infected T-cells is achieved. Combined treatment with anti-TAC-A and monensin may offer an efficient and highly selective means of purging bone marrow of patients with adult T-cell leukemia, which may then be used for autologous marrow transplantation.
Ammonium Chloride↗
The arrangement of immunoglobulin, T cell antigen receptor, and interleukin 2 receptor genes in human lymphoid neoplasms.
Ig and T cell antigen receptor genes in their germ line form are separated DNA segments that are joined by recombinations during lymphocyte development. The analysis of Ig and T cell receptor gene arrangements has been of value in the study of lymphoid neoplasms. The identification of T cell receptor gene rearrangements taken in conjunction with studies of Ig gene rearrangements aids in the elucidation of the lineage (T cell or B cell) and the clonality of lymphoid populations of all series. The application of this molecular genetic approach has great potential for complementing conventional marker analysis, cytogenetics, and histopathology, thus broadening the scientific basis for the classification, diagnosis, and monitoring of the therapy of lymphoid neoplasia. IL 2 is a lymphokine synthesized by some T cells following activation. Resting T cells do not express IL 2 receptors, but receptors are rapidly expressed on T cells following the interaction of antigens, mitogens, or monoclonal antibodies with the antigen-specific T cell receptor complex. Normal resting T cells and most leukemic T cell populations do not express IL 2 receptors; however, the leukemic cells of all patients with HTLV-I-associated adult T cell leukemia examined expressed the Tac antigen. The constant display of large numbers of IL 2 receptors that may be aberrant may play a role in the uncontrolled growth of these leukemic T cells. Patients with the Tac antigen positive adult T cell leukemia are being treated with the anti-Tac monoclonal antibody directed toward this growth factor receptor.
Adult↗
Structure of the human interleukin-2 receptor gene.
The gene encoding the human interleukin-2 (IL-2) receptor consists of 8 exons spanning more than 25 kilobases on chromosome 10. Exons 2 and 4 were derived from a gene duplication event and unexpectedly also are homologous to the recognition domain of human complement factor B. Alternative messenger RNA (mRNA) splicing may delete exon 4 sequences, resulting in a mRNA that does not encode a functional IL-2 receptor. Leukemic T cells infected with HTLV-I and normal activated T cells express IL-2 receptors with identical deduced protein sequences. Receptor gene transcription is initiated at two principal sites in normal activated T cells. Adult T cell leukemia cells infected with HTLV-I show activity at both of these sites, but also at a third transcription initiation site.
Amino Acid Sequence↗
Qualitative analysis of immune function in patients with the acquired immunodeficiency syndrome. Evidence for a selective defect in soluble antigen recognition.
We studied purified subpopulations of lymphocytes from patients with the acquired immunodeficiency syndrome (AIDS) in order to determine whether intrinsic defects in lymphocyte function, aside from those due to alterations in lymphocyte numbers, were present. Mitogen-stimulated DNA synthesis, production of gamma interferon, production of interleukin-2, and expression of interleukin-2 receptors, although variably decreased in unseparated cell populations, were normal in populations of purified T-cell subsets. In contrast, DNA synthesis in response to the soluble protein antigen tetanus toxoid was decreased in both unseparated and purified T-cell subpopulations. Cell-mixing experiments demonstrated that the hyporesponsiveness of the unfractionated lymphocytes from patients with AIDS was not due to active suppression. We conclude that the lymphocytes of patients with AIDS, although capable of undergoing a normal degree of blast transformation and lymphokine production after mitogenic stimulation, have an intrinsic defect in their ability to recognize and respond to soluble antigen.
Acquired Immunodeficiency Syndrome↗
Stable expression of cDNA encoding the human interleukin 2 receptor in eukaryotic cells.
Human interleukin 2 (IL-2) receptor cDNA derived from HUT 102B2 cells was stably expressed in murine L cells. These L cell transfectants (a) displayed surface receptors of the aberrant size of the IL-2 receptors on HUT 102B2 cells, (b) did not respond to exogenous IL-2 with augmented proliferation, and (c) expressed low affinity but not high affinity receptors for IL-2.
Animals↗
Localization of the gene encoding the human interleukin-2 receptor on chromosome 10.
The human interleukin-2 receptor is an inducible growth factor receptor present on the surface of activated T lymphocytes. The receptor is required for a normal T-cell immune response. High-resolution fluorescence-activated chromosome sorting and DNA spot-blot analysis with complementary DNA's for the interleukin-2 receptor indicated that the receptor gene was located on chromosome 9, 10, 11, or 12. In situ hybridization studies showed that the interleukin-2 receptor gene is on the short arm of chromosome 10, p14----15.
Cell Line↗
Deregulation of interleukin-2 receptor gene expression in HTLV-I-induced adult T-cell leukemia.
Infection of human T cells by human T-lymphotropic virus, type I (HTLV-I), a retrovirus, is uniformly associated with the constitutive expression of large numbers of cellular receptors for interleukin-2 (IL-2). Comparison with normal T cells shows that neither IL-2 receptor gene organization nor IL-2 receptor messenger RNA processing are altered in the leukemic cells. However, mitogenic stimuli activate IL-2 receptor gene expression in normal T cells, whereas these stimuli paradoxically inhibit IL-2 receptor gene transcription in HTLV-I-infected leukemic T cells.
Cell Nucleus↗
Sequential expression of genes involved in human T lymphocyte growth and differentiation.
Nuclear transcription assays were performed with isolated nuclei from human peripheral blood T lymphocytes stimulated with phytohemagglutinin and phorbol myristate acetate to determine the kinetics of transcriptional activity of various genes occurring in T cell activation. Although silent in resting T cells, the genes encoding c-myc and the interleukin 2 (IL-2) receptor were induced early, preceding gamma interferon (IFN-gamma), IL-2, and transferrin receptor gene transcription. Transcriptional activity of these genes fell after their respective peaks, indicating that the expression of these genes is a transient event during T cell activation. With the exception of the transferrin receptor gene, the kinetics of induction of these genes were not altered by concentrations of cycloheximide that inhibited protein synthesis. These data indicate that the induction of genes encoding c-myc, IL-2, IL-2 receptor, and IFN-gamma occur independently of the sequential production of the proteins they encode.
Cell Differentiation↗
The human receptor for T-cell growth factor. Evidence for variable post-translational processing, phosphorylation, sulfation, and the ability of precursor forms of the receptor to bind T-cell growth factor.
The T-cell growth factor (TCGF) receptor on phytohemagglutinin-activated normal peripheral blood T-cells is characterized as a glycoprotein with an apparent Mr = 55,000 that contains N-linked and O-linked carbohydrate with only approximately 33,000 daltons of peptide structure (p33) as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There are two N-linked glycosylated intermediate precursor forms (apparent Mr = 35,000 (p35) and 37,000 (p37]. This receptor differs from the TCGF receptor on HUT-102B2 cells (apparent Mr = 50,000) because of differences in post-translational processing. Experiments with the carboxylic ionophore monensin demonstrate blockade of the transition of the p35 and p37 receptor precursor forms to the mature receptor, presumably secondary to inhibition of Golgi-associated receptor processing. We identify the primary translation product of TCGF receptor mRNA as intermediate in size between the p33 and the p35/p37 forms. We further demonstrate that the p33, p35, and p37 precursor forms, but not the primary translation product, are all capable of binding TCGF. Thus, the removal of the signal peptide and/or conformational changes of the primary translation product are necessary for ligand binding; however, the extensive post-translational modifications are not. Lastly, we demonstrate that at least some TCGF receptors are phosphorylated and sulfated, and that TCGF receptors on phytohemagglutinin-activated normal T-cells are more heavily sulfated than those on HUT-102B2 cells.
Carbohydrate Conformation↗
Activators of protein kinase C and 5-azacytidine induce IL-2 receptor expression on human T lymphocytes.
Resting human T lymphocytes do not express receptors for interleukin-2, but expression is rapidly induced by exposure to PHA. After maximal expression 2-3 days after stimulation, a progressive decline in receptor number is observed. Receptor expression can be augmented by reexposure to PHA. In this study we show that activators of protein kinase C including phorbol diester, phospholipase C, and the diacylglycerol congener diC8 also increase IL-2 receptor expression. Moreover, 5-azacytidine, which inhibits cytosine methyltransferase, and hydroxyurea, which inhibits ribonucleotide reductase, also increased receptor number. These studies demonstrate that IL-2 receptor expression can be altered in vitro, and that IL-2 receptor number, in combination with IL-2 secretion, may contribute to the regulation of IL-2-dependent immune responses.
Azacitidine↗
Receptors for T-cell growth factor: structure, function and expression on normal and neoplastic cells.
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Amino Acid Sequence↗
Molecular cloning of cDNA for the human interleukin-2 receptor.
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Amino Acid Sequence↗
Interleukin 2 (IL-2) augments transcription of the IL-2 receptor gene.
We demonstrate that purified interleukin 2 (IL-2) can directly upregulate IL-2 receptor expression on phytohemagglutinin-activated T lymphocytes maintained in culture until IL-2 receptor expression had markedly declined. The IL-2-induced increase in IL-2 receptor number is maximal within 12 hr, requires new RNA and protein synthesis, and is mediated by an interaction of ligand with the high-affinity receptors for IL-2. IL-2 stimulation results in increased accumulation of IL-2 receptor mRNA within 4 hr, while an increase in IL-2 receptor gene transcription is detected within 30 min in isolated nuclei. In addition, IL-2 incubation results in increased amounts of c-myc and transferrin receptor mRNA, but it does not augment levels of mRNA encoding the beta chain of the T-cell receptor for antigen. These results demonstrate that IL-2 can directly upregulate transcription and expression of its own receptor and, therefore, indicate that IL-2 may regulate IL-2-dependent immune responses, in part, by influencing the expression of IL-2 receptors.
Cells, Cultured↗
Interleukin 2 receptor gene expression in normal human T lymphocytes.
We have used cDNAs for the human interleukin 2 (IL-2) receptor to study IL-2 receptor gene expression in normal activated T cells. Resting T cells do not contain detectable IL-2 receptor mRNA. Within 1 hr after stimulation with phytohemagglutinin (PHA), a large, presumably nuclear precursor RNA species is seen, which then gradually disappears. Mature IL-2 receptor mRNA forms appear within 8 hr after stimulation, reach peak levels between 8 and 24 hr, and then decline. Thus, in PHA-activated lymphocytes the rise and fall in IL-2 receptor mRNA levels precede by more than 24 hr the peak and decline of IL-2 receptor protein expression occurring at the cell surface. 4 beta-Phorbol 12-myristate 13-acetate (PMA) also stimulates IL-2 receptor mRNA and protein expression by T cells. Combinations of optimal concentrations of PHA and PMA produce an additive effect on IL-2 receptor mRNA levels, suggesting that PHA and PMA may induce IL-2 receptor gene expression through different, complementary mechanisms. Nuclease S1-protection assays indicate that IL-2 receptor mRNAs may differ in length due to the use of three different polyadenylylation signals. Further, these assays demonstrate the presence of transcripts that lack a 216-base segment within the protein-coding region and thus do not encode a functional IL-2 receptor. Nuclear transcription assays indicate that the increase in IL-2 receptor mRNA is reflected at the level of transcription. Thus, IL-2 receptor gene regulation controls IL-2 receptor expression at the cell surface and is intimately linked to the control of T-cell proliferation.
Cell Division↗
The human interleukin-2 receptor.
Complementary DNAs corresponding to the human receptor for interleukin-2 (IL-2) have been molecularly cloned, sequenced, and expressed in both COS-1 and L cells. The human genome appears to contain a single structural gene for this receptor located on the short arm of chromosome 10 (band 14-15). However, when transcribed, at least two families of mRNAs are produced, which vary in length due to the use of at least three different polyadenylation signals. Sequence analysis of the cloned cDNAs and S1 nuclease protection assays indicate an alternative pathway of mRNA processing for this receptor whereby a 216 base-pair segment contained within the protein coding region is spliced, resulting in an mRNA unable to encode a functional IL-2 receptor. In contrast, cDNAs corresponding to mRNA retaining this 216 base-pair region code membrane receptors that bind both IL-2 and anti-Tac (monoclonal anti-IL-2 receptor antibody). Analysis of the deduced amino acid sequence reveals that the receptor is composed of 272 amino acids including a signal peptide 21 amino acids in length. Hydrophobicity analysis suggests a single, 19 amino acid transmembrane domain. A short intracytoplasmic domain composed of 13 amino acids is present and contains two potential phosphate acceptor sites (serine and threonine but not tyrosine) as well as positively charged residues presumably involved in cytoplasmic anchoring. Two sites for N-linked glycosylation sites and numerous extracytoplasmic O-linked glycosylation sites are present.
Amino Acid Sequence↗
Adult T cell leukemia: a potential target for ricin A chain immunotoxins.
Adult T cell leukemia (ATL) is an almost uniformly fatal malignancy of mature T cells associated with human T cell leukemia/lymphoma virus type 1 (HTLV-1) infection. Cells from this leukemia are characterized by the expression of large numbers of receptors for interleukin 2 (IL-2). In an attempt to prepare an immunotoxin with selective cytotoxicity for ATL cells, we conjugated anti-Tac, a monoclonal anti-IL-2 receptor antibody, to purified ricin A chains. Although unmodified anti-Tac had no effect on the protein synthesis of these cells, anti-Tac-ricin A chain conjugates produced half-maximal inhibition of protein synthesis in HTLV-1-infected leukemic T cell lines at concentrations of 2 to 6 X 10(-10) mol/L (ID50). An essentially identical ID50 was obtained with leukemic peripheral blood T lymphocytes isolated from two patients with ATL. In contrast, half-maximal inhibition of protein synthesis in HTLV-uninfected, IL-2 receptor-negative T and B cell lines required 200- to 1,000-fold higher concentrations of anti-Tac-ricin A chain conjugates. Both unconjugated anti-Tac and immunoaffinity-purified IL-2 completely inhibited the toxic effects of anti-Tac-ricin A, confirming the specificity of the conjugate-IL-2 receptor interaction. Clonogenic assays demonstrated that anti-Tac-ricin A chain was able to eliminate greater than 99.9% of an HTLV-1-infected T cell population at concentrations only marginally affecting IL-2 receptor-negative cells. The data presented demonstrate that anti-Tac-ricin A is selectively cytotoxic for HTLV-1-infected leukemic T cells in vitro and raises the future possibility of specific therapeutic intervention with immunotoxins in this disease.
Adult↗
Interleukin 2 receptor (Tac antigen) expression in HTLV-I-associated adult T-cell leukemia.
Interleukin-2 (IL-2) is a lymphokine synthesized by some T-cells following activation. Resting T-cells do not express IL-2 receptors, but receptors are rapidly expressed on T-cells following interaction of antigens, mitogens, or monoclonal antibodies with the antigen-specific T-cell receptor complex. Using anti-Tac, a monoclonal antibody that recognizes the IL-2 receptor, the receptor has been purified and shown to be a Mr 33,000 peptide that is posttranslationally glycosylated to a Mr 55,000 mature form. Normal resting T-cells and most leukemic T-cell populations do not express IL-2 receptors; however, the leukemic cells of the 11 patients examined who had human T-cell lymphotropic virus-associated adult T-cell leukemia expressed the Tac antigen. In human T-cell lymphotropic virus-I infected cells, the Mr 42,000 long open reading frame protein encoded in part by the pX region of this virus may act as a transacting transcriptional activator that induces IL-2 receptor gene transcription, thus providing an explanation for the constant association of IL-2 receptor expression with adult T-cell lymphotropic virus-I infection of lymphoid cells. The constant expression of large numbers of IL-2 receptors which may be aberrant may play a role in the uncontrolled growth of adult T-cell leukemia cells. Two patients with Tac-positive adult T-cell leukemia have been treated with the anti-Tac. One of the patients had 6- and 3-mo remissions of his leukemia following two courses of therapy with this monoclonal antibody directed toward this growth factor receptor.
Adult↗