An exon 5-deleted mRNA encodes a functional interleukin 2 receptor alpha-subunit.

Two polypeptides are involved in interleukin 2 binding: a low-affinity receptor of 55 kD (IL2-R alpha) and an intermediate affinity component of 75 kD (IL2-R beta). We describe the cloning by the Polymerase Chain Reaction of the coding region of IL2-R alpha from a human T-cell lymphoma cell line. One clone presented a 72-bp deletion that precisely corresponds to exon 5. The deleted form and the normal IL2-R alpha cDNA were expressed CHO cells. Stable transfected cellular clones were compared for their immunoreactivity to monoclonal antibodies directed against IL2-R alpha and for their ability to bind radiolabeled IL2. The presence or absence of the protein region encoded by exon 5 did not modify the IL2-binding capacity of the receptor.     

Reconstitution of a functional IL-2 receptor by the beta-chain cDNA. A newly acquired receptor transduces negative signal

The high-affinity IL-2R results from the noncovalent association between at least two subunits; alpha (p55) and beta (p70), both of which are capable of binding IL-2 with a low and intermediate affinity, respectively. Although the alpha-chain itself has been shown to be nonfunctional, suggestions have been made that the beta-chain mediates an IL-2 signal. To directly study the role of the beta-chain in the signal transduction, we transfected with the cDNA encoding the IL-2R beta-chain a human T lymphotropic virus-I-transformed T cell line, MT-1 originally expressing low-affinity alpha-chain alone, and established a stable transformant (designated MT-beta 7) which expressed both alpha- and beta-chains simultaneously. We showed 1) MT-beta 7 manifested the high-affinity IL-2 binding, which was completely disrupted by the anti-beta chain mAb (Mik-beta 1), 2) the 125I-IL-2 crosslinking patterns of MT-beta 7 were indistinguishable from those of cells expressing the native high-affinity IL-2R, 3) MT-beta 7, but not parental MT-1, internalized the bound IL-2 and responded to IL-2 with a negative signal, i.e., inhibition of the de novo DNA synthesis. These results clearly demonstrate that the beta-chain not only participates in forming the high-affinity IL-2R with the alpha-chain but also is directly involved in the IL-2 signal transduction.     

The released interleukin 2 receptor binds interleukin 2 efficiently

The released interleukin 2 receptor (IL 2R) molecule was characterized in order to clarify its biochemical structure and to determine its functional capacity. Enzymatic digestions demonstrated that the released IL 2R, like the cell surface IL 2R, is a complex glycoprotein, modified by the addition of both N- and O-linked carbohydrates and sialic acid residues. It has a peptide backbone that is approximately 10 Kd smaller than that of its membrane-associated counterpart. Affinity chromatography demonstrated that released IL 2R from either an HTLV-I-positive T cell line (HUT-102) or PHA-activated normal peripheral lymphocytes binds efficiently to purified recombinant IL 2. Furthermore, the interaction between the growth factor and the released receptor does not appear to require any accessory molecules. These observations suggest a potentially significant role for the released IL 2R in the regulation of IL 2-dependent lymphocyte functions.     

A soluble interleukin 2 receptor produced by a normal alloreactive human T cell clone binds interleukin 2 with low affinity

Several alloreactive human T cell clones derived from a rejected kidney graft were found to produce in their culture supernatants soluble interleukin 2 receptors (IL-2R) upon specific antigenic challenge (irradiated B cell line from the graft's donor). Among them, the 2B11, a high producer clone, was used to purify a soluble IL-2R preparation which was analyzed, in comparison with the high and low affinity cell-surface IL-2R expressed by 2B11 cells, for its parameters of interaction with a set of anti-IL-2R monoclonal antibodies (mAb) and IL-2. This soluble receptor purified by affinity chromatography (anti-IL-2R mAb column) and sodium dodecyl sulfate gel electrophoresis is composed of a single chain of 35,000 to 45,000 Da. Immunoradiometric assays (IRMA) at equilibrium were set up, using pairs of mAb directed against two separate epitopes on the Tac antigen of the human IL-2R, to measure the respective dissociation constant of these mAb for the soluble IL-2R. They were found to be identical to those found on the cell-surface IL-2R. A 1:1 stoichiometry between the two epitopes were found both on the membrane and soluble species. Competition experiments between membrane and soluble IL-2R for binding the mAb allowed the quantitative analysis of the concentration of soluble IL-2R without the need of amino acid analysis on purified material and set up a quantitative IRMA for the human soluble IL-2R (detection limit 5 pM). The affinity of the soluble IL-2R for IL-2 was determined by various techniques including an IRMA using an anti-IL-2R mAb and radiolabeled IL-2. The results obtained led us to conclude that the soluble IL-2R binds IL-2 with a dissociation constant (KD = 30 nM) identical to that found for the binding of IL-2 to low affinity cell-surface IL-2R (Tac antigen). Whereas 2.5% of cell-surface IL-2R expressed 2 days after antigenic stimulation of 2B11 cells were of high affinity for IL-2 (KD = 25 pM), no (less than 0.07%) high affinity binding sites could be detected on the purified soluble IL-2R. This soluble IL-2R therefore likely corresponds to a truncated, extracellular part of the membrane Tac antigen. The amounts of soluble Tac antigen produced by the 2B11 alloreactive human T cell clone did not exceed 1 nM and, as expected from the binding studies, did not affect IL-2-induced T cell proliferation. The physiologic and pathologic implications of our results are discussed.     

Expression of interleukin 2 and the interleukin 2 receptor in aging rats

Lymphocytes of aged animals exhibit a marked decrease in proliferative capacity in response to mitogen stimulation when compared to those of younger animals. In humans and mice the decreased proliferation is due at least in part (i) to the inability of lymphocytes to synthesize sufficient interleukin 2 (IL-2) and (ii) to decreased expression of IL-2 receptors (IL-2R) on the surface of aged lymphocytes. We compared proliferative abilities, IL-2 production, and IL-2R expression in splenocyte cultures of 4- to 5- and 22- to 24-month-old Fischer 344 rats stimulated with either concanavalin A (Con A) or A23187 and phorbol myristate acetate (PMA). Proliferation was significantly decreased in aged lymphocytes (30-50%) with both treatment protocols. However, unlike mice and humans we observed no difference in IL-2 activity, IL-2 mRNA levels, or IL-2R cell surface expression of lymphocytes from young and aged rats stimulated with either Con A or A23187 and PMA. These results indicate that factors other than decreased expression of IL-2 and IL-2R are responsible for the diminished proliferative capacity of aged rat lymphocytes following mitogen stimulation.     

Reconstitution of in vivo cell-mediated lympholysis responses in aged mice with interleukin 2

Advancing age is accompanied by declining immune potential. Both humoral and cellular immune responses are diminished in aged humans and experimental animals. A major lesion preventing effective T cell-mediated responses is the lack of interleukin 2 (IL 2) synthesis in aged mice. Addition of IL 2 can effectively reconstitute in vitro T cell-dependent humoral and cell-mediated immunity. These results have been extended, demonstrating that IL 2-containing lymphokine preparations when administered with antigen can restore the ability of aged mice to generate cytotoxic T lymphocytes in vivo. Furthermore, IL 2 has been identified as the active component of these lymphokine preparations through the use of purified human IL 2 prepared by recombinant DNA and gene cloning technology. The cloned IL 2 is highly effective in enhancing the immune responses of aged animals both in vitro and in vivo.     

Reconstitution of a purified acetylcholine receptor

Dialysis of the purified acetylcholine receptor from Torpedo californica electroplax with lipids from the same organ results in a vesicular membrane system in which the receptor is embedded in the bilayer and oriented so that most of the neurotoxin-binding sites appear to be on the outer surface. The constituted vesicles are chemically excitable by acetylcholine and carbamylcholine, as measured by ²²Na⁺ efflux. The excitability is specifically blocked by the antagonist α-bungarotoxin. These results demonstrate that the purified reconstituted receptor system not only can specifically bind neurotransmitter but also can trigger ion translocation. It therefore has the properties necessary to effect postsynaptic depolarization in vivo.     

In vitro induction and expression of interleukin 2 receptor in a clonal T helper cell differentiation model

Induction and expression of interleukin 2 (IL 2) receptor have been studied using a poly( Glu60 Ala30 Tyr10 ) (GAT)-specific T cell clone of mouse origin. This clone (52-3) has been characterized and it exhibits functional properties of T helper (TH) cells: it leads to a specific anti-DNP response in the presence of DNP-GAT and DNP-primed B cells and it secretes biological activities which can induce polyclonal B cell proliferation and IgM secretion. In vitro this clone mimics the activation stages of normal T lymphocytes and can be obtained under two states of differentiation. depending on the antigen-specific signal provided by antigen-presenting cells (APC). The expression of IL 2 receptor by this clone has been studied by (i) its response to IL 2, (ii) its capacity to absorb IL 2 bioactivity, and (iii) its reactivity with monoclonal antibody 7D4 specific for mouse IL 2 receptor. All the results indicate that the unstimulated state does not express the IL 2 receptor while the activated state does. Clone 52-3 has been compared with clone 14-1.6 that derives from a TH cell line and expresses the IL 2 receptor constitutively. 52-3 offers a good experimental model for studying in vitro, in a clonal TH cell population, the detailed mechanism of IL 2 receptor induction.     

Inhibitory effects of anti-CD2 monoclonal antibodies on interleukin 2 production and interleukin 2 receptor expression in anti-CD3-induced T cell activation

The effects of anti-CD2 monoclonal antibodies (mAb) on anti-CD3-driven interleukin 2 (IL2) production and IL2 receptor (IL2R) expression were investigated. Two anti-CD2 mAb, which had previously been shown to inhibit in vitro anti-CD3-induced T cell proliferation, also inhibited anti-CD3-induced IL2 production. However, it seemed unlikely that this was the crucial mechanism in the inhibition of anti-CD3-driven proliferation, since anti-CD2 mAb also partially inhibited T cell proliferation induced by the anti-CD3 mAb 446 which does not induce detectable IL2 levels. Anti-CD2 mAb also inhibited anti-CD3-induced surface IL2R expression as measured by immunofluorescence staining with an anti-IL2R mAb against the p55 chain. Inhibition of IL2R expression paralleled inhibition of proliferation. This anti-CD2-mediated inhibition involved a block in the generation of normal numbers of IL2R+ cells rather than a direct inhibitory effect on the IL2R+ cells themselves, since IL2R+ cells isolated from anti-CD2-containing cultures responded normally to IL2. Exogenous IL2 and IL4, singly or in combination, could reverse neither the anti-CD2-mediated inhibition of anti-CD3-induced proliferation nor the anti-CD2-mediated inhibition of anti-CD3-induced IL2R expression. Taken together, these observations suggest that anti-CD2 mAb inhibit anti-CD3-driven proliferation by inhibiting the generation of IL2R+ cells at a maturational stage proximal to their expression of surface IL2R. This inhibition cannot be overcome by exogenous IL2 or IL4, suggesting that the underlying biochemical mechanism involves an IL2- and IL4-independent pathway.     

T cell receptor triggering induces responsiveness to interleukin 1 and interleukin 2 but does not lead to T cell proliferation

The antigen-like activity of monoclonal antibodies directed at the T3-Ti antigen receptor complex of human T lymphocytes was employed to study activation requirements of resting T cells. Efficient antigen recognition (signal 1) by T lymphocytes requires multimeric antigen receptor triggering because under appropriate experimental conditions soluble ligands do not produce this initial signal for T cell activation. The latter leads to receptiveness for both interleukin 1 (IL 1) and interleukin 2 (IL 2). Importantly, induction of proliferation requires an additional signal (signal 2), namely IL 1, which appears to be required to enable optimal secretion of IL 2. In contrast, presensitized T lymphocytes do not require IL 1 for IL 2 production. In this case, antigen receptor oligomerization is in itself sufficient to induce IL 2 receptor expression, and IL 2 secretion as well.     

The murine interleukin 2 receptor. Irreversible cross-linking of radiolabeled interleukin 2 to high affinity interleukin 2 receptors reveals a noncovalently associated subunit

The murine interleukin 2 (IL-2) receptor is a 55- to 60-kDa glycoprotein (p58) that binds IL-2 at a high and low affinity. In this investigation, we have identified sublines of EL4 that vary in their capacity to express high affinity IL-2 receptors after transfection of the IL-2 receptor cDNA. These and other cell populations were used to determine whether unique membrane molecules were specifically associated with the high affinity IL-2 receptor. Irreversible chemical cross-linking of [125I]IL-2 to only high affinity IL-2 receptors resulted in detection of IL-2 cross-linked to p58 as a 70- to 75-kDa band and other complexes of 90 to 95 kDa, 115 kDa, 150 kDa, 170 to 190 kDa, and 245 kDa. Antibodies specific for p58 resulted in precipitation of each of these complexes. However, disruption of noncovalent interactions prior to immunoprecipitation resulted in an inability to detect the material at 90 to 95 kDa. Therefore, we conclude that this complex most likely represented IL-2 cross-linked to a 75- to 80-kDa subunit that was noncovalently associated with p58. The other complexes greater than 150 kDa may represent these subunits cross-linked to each other. The detection of all the cross-linked complexes larger than 75 kDa appeared to be directly related to formation of high affinity IL-2 receptors because IL-2 was cross-linked only to p58 for three cell lines that exclusively expressed low affinity IL-2 receptors. Thus, high affinity murine IL-2 receptors are comprised of at least one alpha (p58)- and beta (p75)-subunit. Our data also raise the possibility of a more complex subunit structure.     

Monocyte interleukin 2 receptor gene expression and interleukin 2 augmentation of microbicidal activity

Activation of human peripheral blood monocytes results in the expression of interleukin 2 (IL 2) receptors, which are absent on resting monocytes. In a population of purified monocytes, the appearance of IL 2 receptors occurs on the majority of cells in association with increased levels of HLA-DR. Lipopolysaccharide (LPS) induces maximum numbers of IL 2 receptors within 12 hr, whereas IFN-gamma requires 48 hr. We used cDNA encoding for the human IL 2 receptor to evaluate IL 2 receptor gene expression in resting and activated monocytes. Within 4 hr after LPS stimulation, IL 2 receptor mRNA species of 3500 and 1500 bases appear, reaching peak levels between 8 and 12 hr and declining thereafter. The LPS-activated monocyte IL 2 receptor protein is expressed on the cell surface within a few hours after the detection of IL 2 receptor mRNA. The addition of IL 2 to IL 2 receptor-positive monocytes augments their generation of reactive oxygen intermediates and their cytotoxic activity. Thus monocytes when activated undergo a series of morphologic, phenotypic, and functional changes, including the expression of IL 2 receptors, which may provide an important immunoregulatory pathway.     

Construction of rat-mouse T cell hybridomas that express regulatable rat interleukin 2 receptor

Spleen cells obtained from Lewis rats were cultured with 4 micrograms/ml Con A for 96 hr, and activated cells were fused with BW5147 mouse T lymphoma cells. Seven clones obtained by fusion expressed rat IL 2R. The expression of rat IL 2R on those hybrid cells could be up-regulated by IL 2 itself, ATL-derived factor, and TPA and CA++ ionophore. Those IL 2R could be down-regulated by murine MoAb ART-18 that detects rat IL 2R. All hybrid clones produced IL 2 constitutively. IL 2 produced by hybrid cells bound to its receptor and promoted the proliferation of hybrid cells in some clones. Incubation of cells with exogenous IL 2 resulted in the proliferation of hybrid cells, whereas the proliferation of some clones was inhibited by exogenous IL 2, indicating that IL 2 had bifunctional properties on cell growth. Rat IL 2R from 6H2-F9 hybrid cells was studied by both one- and two-dimensional SDS-PAGE with ART-18. The IL 2R derived from 6H2-F9 cells had 72,000 to 77,000 and 40,000 to 48,000 m.w. major components under nonreducing conditions, and had 50,000 to 56,000 m.w. major and 35,000 to 38,000 m.w. minor components under reducing conditions. The 110,000 m.w. component, the third component of IL 2R, was constantly observed in 6H2-F9 hybrid cells.     

Reconstitution of a functional core polycomb repressive complex

The opposing actions of polycomb (PcG) and trithorax group (trxG) gene products maintain essential gene expression patterns during Drosophila development. PcG proteins are thought to establish repressive chromatin structures, but the mechanisms by which this occurs are not known. Polycomb repressive complex 1 (PRC1) contains several PcG proteins and inhibits chromatin remodeling by trxG-related SWI/SNF complexes. We have defined a functional core of PRC1 by reconstituting a stable complex using four recombinant PcG proteins. One subunit, PSC, can also inhibit chromatin remodeling on its own. These PcG proteins create a chromatin structure that has normal nucleosome organization and is accessible to nucleases but excludes hSWI/SNF.     

Signaling domains of the interleukin 2 receptor

Interleukin (IL-)2 and its receptor (IL-2R) constitute one of the most extensively studied cytokine receptor systems. IL-2 is produced primarily by activated T cells and is involved in early T cell activation as well as in maintaining homeostatic immune responses that prevent autoimmunity. This review focuses on molecular signaling pathways triggered by the IL-2/IL-2R complex, with an emphasis on how the IL-2R physically translates its interaction with IL-2 into a coherent biological outcome. The IL-2R is composed of three subunits, IL-2Ralpha, IL-2Rbeta and gammac. Although IL-2Ralpha is an important affinity modulator that is essential for proper responses in vivo, it does not contribute to signaling due a short cytoplasmic tail. In contrast, IL-2Rbeta and gammac together are necessary and sufficient for effective signal transduction, and they serve physically to connect the receptor complex to cytoplasmic signaling intermediates. Despite an absolute requirement for gammac in signaling, the majority of known pathways physically link to the receptor via IL-2Rbeta, generally through phosphorylated cytoplasmic tyrosine residues. This review highlights work performed both in cultured cells and in vivo that defines the functional contributions of specific receptor subdomains-and, by inference, the specific signaling pathways that they activate-to IL-2-dependent biological activities.     

Expression and the functional role of a p70/75 interleukin 2-binding molecule in human B cell

Expression of p70/75 IL-2-binding molecules and their functional roles in induction of Ig secretion by IL-2 were examined in human B cells. IL-2, at high concentrations induced higher levels of Ig secretion in Staphylococcus aureus strain Cowan I (SAC)-activated B cells than at low concentrations. About 50% of SAC-activated B cells, lacking Tac antigen, were also responsive to Ig secretion by IL-2, although the required dose of IL-2 was higher than that for Tac-positive B cells. H-31 antibody which recognizes Tac antigen did not inhibit the induction of Ig secretion by high concentrations of IL-2 in both Tac-negative and Tac-positive B cells, suggesting that IL-2 might induce Ig secretion through a receptor distinct from Tac antigens. In contrast, IL-2 was ineffective in the absence of SAC stimulation even at high concentrations. Upon analysis by SDS-PAGE, p70/75 IL-2-binding molecules were detected on Tac-negative SAC-activated B cells. Similar IL-2-binding molecules distinct from Tac antigen (p55) were detected in both Tac-positive B and T cells. However, neither p55 nor p70/75 IL-2-binding molecules could be detected in the absence of SAC stimulation. These observations suggest that p70/75 IL-2 binding molecules are induced in human B-cells in the presence or absence of Tac antigen by SAC stimulation and these determinants play an important function in the transduction of IL-2 associated signal for B cell differentiation.     

Soluble interleukin 2 receptor in atopic eczema.

OBJECTIVE--To determine whether serum soluble interleukin 2 receptor concentrations are related to disease activity in atopic eczema. DESIGN--Single cohort longitudinal study with controls. SETTING--Outpatient and general medicine departments in secondary referral centre. PATIENTS--Of 15 patients aged 17-57 with severe atopic eczema, all with acute exacerbations of disease, 13 were admitted to hospital and two treated as outpatients until the skin lesions had resolved or greatly improved. Nineteen controls gave single blood samples. INTERVENTIONS--Daily skin dressing with betamethasone valerate (0.025%) and ichthammol paste and tubular dressings. END POINT--Resolution of or considerable improvement in skin lesions. MEASUREMENTS AND MAIN RESULTS--Enzyme linked immunosorbent assays (ELISA) were used to measure serum soluble interleukin 2 receptor concentrations in blood samples taken on admission, at intervals subsequently, and on discharge. Clinical scores of disease activity were also made. Median concentrations on admission were significantly higher (770 U/ml) in the patients than the controls (300 U/ml). Concentrations fell significantly during treatment. In 25 assessments made at different times in 13 patients serum soluble interleukin 2 receptor concentration correlated significantly (R = 0.73) with clinical disease activity. CONCLUSIONS--Cellular immunopathogenic mechanisms contribute to atopic eczema. Immune activation can be measured in atopic eczema by measurements of soluble interleukin 2 receptor, and this should facilitate assessment of response to treatment.     

Cytochalasin B enhances T cell mitogenesis by promoting expression of an interleukin 2 receptor

Low concentrations of cytochalasin B enhanced the T cell mitogenesis induced by concanavalin A (Con A) and interleukin 2 (IL-2). Mitogenesis was augmented by cytochalasin B given in the Con A-dependent early phase, or through T cell mitogenesis. Cytochalasin B did not enhance T cell mitogenesis when given only in the IL-2-dependent late phase. Use of the monoclonal antibody that directs the IL-2 receptor showed that cytochalasin B increased the expression of the IL-2 receptor induced by Con A. We concluded that cytochalasin b acts on an early phase of T cell mitogenesis and augments the expression of IL-2 receptor which enables certain nonresponsive T cells to respond to IL-2.     

Reconstitution of p53 expression in a nonproducer Ab-MuLV-transformed cell line by transfection of a functional p53 gene

L12 are Ab-MuLV-transformed cells that express the abl p120 oncogene product but lack the cellularly encoded p53. The functional p53 gene in these cells has been inactivated by the insertion of Moloney virus-like sequences into the first p53 intron. Transfection of L12 cells with a functional p53 gene, contained in a 16 kb Eco RI genomic cloned fragment gave rise to L12-derived cell lines with novel p53 sequences of various sizes and copy number. A high percentage of L12-derived clones efficiently transcribed p53 mRNA and synthesized the p53 protein. Whereas injection of L12 parental cells into syngeneic mice caused the development of local tumors that later regressed, L12-derived clones that expressed p53 caused lethal tumors in syngeneic mice, thus behaving similarly to other Ab-MuLV-transformed p53-producer cell lines. These results suggest that the expression of p53 is essential for tumor cells to exhibit a fully transformed phenotype, manifested in lethal tumors in syngeneic mice.