Intermediate-affinity Interleukin-2 Receptor Research Articles

Differential inhibition of lymphokine-activated killing, proliferation, and cytokine secretion by humanized antibodies against the low- and intermediate-affinity interleukin-2 receptors: A novel model for activation of human peripheral blood mononuclear cells by interleukin 2

Human PBMCs generate LAK, proliferate, and secrete secondary cytokines in response to IL-2. We assessed the roles of the various receptor complexes in these responses to IL-2 using antibodies that block the interaction of IL-2 with the intermediate-affinity IL-2R (humanized Mikβ1) and high- and low-affinity interactions (HTac). The HTac antibody produced dramatic reductions in proliferation (27%) and in the secretion of cytokines (TNF)-α and -β and IFN-γ inhibited 68%, 72%, and 80%, respectively) with no decrease in LAK. HMik inhibited LAK activation by 65% and proliferation by 60% but had little effect on cytokine secretion. The combination of the two antibodies completely inhibited all proliferative, cytolytic, and secretory activity. These data suggest a novel model for the activation of human PBMCs by IL-2. Whereas induction of LAK activity is mediated through the intermediate-affinity IL-2R and cytokine secretion is mediated through the high-affinity complex, interaction between IL-2 and both of these receptor complexes is required for the induction and maintenance of proliferative activities in human PBMCs.

Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor.

Interleukin 15 (IL-15) is a novel cytokine that has recently been cloned and expressed. Whereas it has no sequence homology with IL-2, IL-15 interacts with components of the IL-2 receptor (IL-2R). In the present study we performed a functional analysis of recombinant IL-15 on phenotypically and functionally distinct populations of highly purified human natural killer (NK) cells. The CD56bright subset of human NK cells constitutively expresses the high affinity IL-2R and exhibits a brisk proliferative response after the binding of picomolar amounts of IL-2. Using a proliferation assay, IL-15 demonstrated a very steep dose-response curve that was distinct from the dose-response curve for IL-2. The proliferative effects of IL-15 could be abrogated by anti-IL-2R beta (p75), but not by anti-IL-2R alpha (p55). The proliferative effects of IL-2 on CD56bright NK cells could be inhibited by both antibodies. CD56dim NK cells express the intermediate affinity IL-2R in the absence of the high affinity IL-2R. Activation of CD56dim NK cells by IL-15 was similar to that of IL-2 as measured by enhanced NK cytotoxic activity, antibody-dependent cellular cytotoxicity, and NK cell production of interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-15-enhanced NK cytotoxic activity could be completely blocked by anti-IL-2R beta monoclonal antibody. The binding of radiolabeled IL-2 and IL-15 to CD56dim NK cells was inhibited in the presence of anti-IL-2R beta. Scatchard analysis of radiolabeled IL-15 and IL-2 binding to NK-enriched human lymphocytes revealed the presence of high and intermediate affinity receptors for both ligands. IL-15 is a ligand that activates human NK cells through components of the IL-2R in a pattern that is similar but not identical to that of IL-2. Unlike IL-2, IL-15 is produced by activated monocytes/macrophages. The discovery of IL-15 may increase our understanding of how monocytes/macrophages participate in the regulation of NK cell function.

Mutagenic analysis of a receptor contact site on interleukin-2: preparation of an IL-2 analog with increased potency.

Interleukin-2 (IL-2) is a 133 amino acid alpha-helical protein secreted by activated T-cells. Combinatorial cassette mutagenesis was used to investigate the functional role of a continuous five amino acid region of IL-2 suspected to interact with the intermediate-affinity IL-2 receptor. A limited random library of IL-2 mutants was constructed in which residues 17-21 (Leu-Leu-Leu-Asp-Leu) were simultaneously mutated. The proteins were produced in an Escherichia coli expression system and screened in a biological assay for their ability to mediate the proliferation of a murine IL-2-dependent cell line. From the over 2600 clones examined, only 42 exhibited significant activity, confirming the functional importance of this region. Selected clones were purified and further characterized by biological and receptor binding assays. Viewed in the context of the recently revised 2.5-A crystal structure for IL-2, these results suggest the following conclusions: both Asp20 and Leu21, as shown by their sensitivity to mutation, are the functionally more important residues in this region, but for different reasons. Asp20 is solvent-accessible and likely plays a direct receptor contact role as previous studies have indicated. Leu21, in contrast, is completely buried in the hydrophobic core of the protein. Substitutions at this position, even a conservative Leu-->Val substitution, were found to perturb the precise hydrophobic packing arrangements that are critical for activity, resulting in a significant loss of function. In addition, one of the analogs identified in the screen was found to be 2-3 times more potent than the wild-type protein.

Induction of lymphokine-activated killing with reduced secretion of interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma by interleukin-2 analogs.

Interleukin-2 (IL-2)-based immunotherapy has been shown to effect clinical responses in 15-35% of patients with metastatic renal cell carcinoma or melanoma. Despite its clinical efficacy, many clinicians refrain from using IL-2 because of the associated toxicity. This toxicity is believed to be mediated by such secondary cytokines as IL-1, tumor necrosis factor (TNF), and interferon (IFN)-gamma, which are produced by the patient's IL-2-stimulated peripheral blood mononuclear cells (PBMCs). Human PBMCs were stimulated with 1 nM wild-type recombinant IL-2 (rIL-2) or IL-2 analogs (R38A or F42K) that preferentially bind to the intermediate affinity IL-2 receptor (IL-2R). PBMCs were activated for lymphokine-activated killer (LAK) activity in 4-h 51Cr-release assays, using Daudi target cells. Cytokine content in the culture supernatants was determined by enzyme-linked immunosorbent assay. Both R38A and F42K were capable of generating substantial LAK activity. Maximal specific lysis was 54% for PBMCs activated by R38A and 52% for F42K-stimulated cells, in contrast to 64% for rIL-2. In addition, analog-stimulated PBMCs secreted 59% of the IL-1 beta, 25% of the TNF-alpha, and only 8% of the IFN-gamma produced in response to rIL-2 (all p < 0.01 compared with rIL-2-stimulated secretion; one-way ANOVA). IL-2 analogs that preferentially bind the intermediate-affinity IL-2R retain the capacity to induce substantial LAK activity despite a greatly reduced secondary cytokine production. Therefore, such IL-2 analogs may provide an effective, yet less toxic means of cancer immunotherapy.

Immunological characterization of human decidual mononuclear cells: natural killer activity, response to interleukin-2 and distribution of interleukin-2 receptor subunits.

We investigated the immunological character of mononuclear cells obtained from the decidua in the first trimester of normal pregnancy. These cells showed in vitro cytotoxicity against NK cell targets, although with lower activity than that of the peripheral blood mononuclear cells. Response to IL-2 was dose-dependent. Since decidual CD56-positive cells express two IL-2 receptor subunits, p55 and p75, it is concluded that these cells have high-affinity receptors to IL-2. Peripheral blood CD56-positive cells, which express p75 alone, might have intermediate-affinity IL-2 receptors. These results indicate that the decidual mononuclear cells have a function resembling that of the peripheral blood NK cells in vitro, moreover, that even low levels of IL-2 can affect the character of the decidual mononuclear cells through the high-affinity IL-2 receptor. It is considered that the NK activity of decidual mononuclear cells is suppressed in conditions of low IL-2 levels to permit the maintenance of pregnancy, but can be rapidly elicited by intrauterine infections or abortion, both of which elicit the secretion of IL-2.

Interleukin-2 receptor gamma chain: a functional component of the interleukin-4 receptor.

The interleukin-2 (IL-2) receptor gamma chain (IL-2R gamma) is an essential component of high- and intermediate-affinity IL-2 receptors. IL-2R gamma was demonstrated to be a component of the IL-4 receptor on the basis of chemical cross-linking data, the ability of IL-2R gamma to augment IL-4 binding affinity, and the requirement for IL-2R gamma in IL-4-mediated phosphorylation of insulin receptor substrate-1. The observation that IL-2R gamma is a functional component of the IL-4 receptor, together with the finding that IL-2R gamma associates with the IL-7 receptor, begins to elucidate why deficiency of this common gamma chain (gamma c) has a profound effect on lymphoid function and development, as seen in X-linked severe combined immunodeficiency.

Interleukin-2 Receptor γ Chain: a Functional Component of the Interleukin-7 Receptor

The interleukin-2 receptor gamma chain (IL-2R gamma) is a necessary component of functional IL-2 receptors. IL-2R gamma mutations result in X-linked severe combined immunodeficiency (XSCID) in humans, a disease characterized by the presence of few or no T cells. In contrast, SCID patients with IL-2 deficiency and IL-2-deficient mice have normal numbers of T cells, suggesting that IL-2R gamma is part of more than one cytokine receptor. By using chemical cross-linking, IL-2R gamma was shown to be physically associated with the IL-7 receptor. The presence of IL-2R gamma augmented both IL-7 binding affinity and the efficiency of internalization of IL-7. These findings may help explain the defects of XSCID. Given its role in more than one cytokine receptor system, the common gamma chain (gamma c) is proposed as the designation for IL-2R gamma.

Interleukin 2 activates extracellular signal-regulated protein kinase 2.

Interleukin 2 (IL-2) stimulated activation of the 42-kD extracellular signal-regulated kinase 2 (Erk2) in murine IL-3-dependent cells, expressing either high or intermediate affinity IL-2 receptors. Activation was both rapid, occurring within 5 min of IL-2 addition, and prolonged, remaining elevated for 30 min. Activation of Erk2 appeared to be necessary for IL-2 stimulation of proliferation, as deletion of a region of the cytoplasmic domain of the IL-2 receptor beta chain, essential for IL-2 stimulation of proliferation, abolished Erk2 activation by IL-2. Furthermore, cells that had been deprived of cytokine for 24 h were then refractory to IL-2 stimulation of both Erk2 activity and proliferation. However, elevation of Erk2 activity was not sufficient to stimulate proliferation, as protein kinase C activation stimulated Erk2 activity but not DNA synthesis. Also, cells exposed to IL-2 in the presence of rapamycin showed full Erk2 activation but not DNA synthesis. These data suggest that IL-2 must stimulate both Erk2 activity and a further pathway(s) to trigger cell proliferation.

Characterization of the Human Interleukin-2 Receptor β-Chain Gene Promoter: Regulation of Promoter Activity by ets Gene Products

The interleukin-2 receptor (IL-2R) beta chain (IL-2R beta) is an essential signaling component of high- and intermediate-affinity IL-2Rs. Our laboratory previously reported that a DNA fragment containing 857 bp of 5'-flanking sequence of the human IL-2R beta gene exhibited promoter activity. We have now further characterized the promoter and delineated cis-acting regulatory regions. The region downstream of -363 is critical for basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and contains at least three enhancer-like regions. Among them, the -56 to -34 enhancer was the most potent and had high-level activity in two T-cell lines but not in nonlymphoid HeLaS3 and MG63 cells. This enhancer contains a GGAA Ets binding site which bound two Ets family proteins, Ets-1 and GA-binding protein in vitro. Mutation of the Ets motif strongly diminished both promoter and enhancer activities. We conclude that this Ets binding site plays a key role in regulating basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and may also contribute to tissue-specific expression of the IL-2R beta gene.

Characterization of the human interleukin-2 receptor beta-chain gene promoter: regulation of promoter activity by ets gene products.

The interleukin-2 receptor (IL-2R) beta chain (IL-2R beta) is an essential signaling component of high- and intermediate-affinity IL-2Rs. Our laboratory previously reported that a DNA fragment containing 857 bp of 5'-flanking sequence of the human IL-2R beta gene exhibited promoter activity. We have now further characterized the promoter and delineated cis-acting regulatory regions. The region downstream of -363 is critical for basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and contains at least three enhancer-like regions. Among them, the -56 to -34 enhancer was the most potent and had high-level activity in two T-cell lines but not in nonlymphoid HeLaS3 and MG63 cells. This enhancer contains a GGAA Ets binding site which bound two Ets family proteins, Ets-1 and GA-binding protein in vitro. Mutation of the Ets motif strongly diminished both promoter and enhancer activities. We conclude that this Ets binding site plays a key role in regulating basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and may also contribute to tissue-specific expression of the IL-2R beta gene.

Kinetic study of interleukin-2 binding on the reconstituted interleukin-2 receptor complexes including the human gamma chain.

To gain an insight into roles of the interleukin-2 (IL-2) receptor gamma chain in IL-2 binding mechanisms, we examined association and dissociation rate constants for IL-2 binding with a series of fibroblastoid L929 cell lines expressing IL-2 receptor complexes reconstituted by transfection with the alpha, beta and gamma genes. The association rate constant (k) with the alpha beta gamma complex on L cells was fourfold larger than that with the alpha beta complex on L cells, and the dissociation rate constant (k') was one fifth of that with the alpha beta complex. These results indicate that the gamma chain is involved in both mechanisms by which IL-2 associates with and dissociates from receptors, resulting in the generation of the high-affinity IL-2 receptor along with the alpha and beta chains. During the course of this study, we found that the IL-2 dissociation from alpha beta gamma complex on lymphoid cells was a lot slower than that from the alpha beta gamma complex on fibroblast cells. A similar difference was observed with the beta gamma complex. These observations may indicate functional and constitutional differences of the high- and intermediate-affinity IL-2 receptor complexes between lymphoid and fibroblastoid cells.

Characterization of cDNAs encoding the murine interleukin 2 receptor (IL-2R) gamma chain: chromosomal mapping and tissue specificity of IL-2R gamma chain expression.

The IL-2R gamma chain (IL-2R gamma) is an essential component of high- and intermediate-affinity IL-2Rs, playing critical roles for ligand binding and internalization. Recently, our laboratory has demonstrated that IL-2R gamma mutation results in X chromosome-linked severe combined immunodeficiency in humans, suggesting that IL-2R gamma plays a vital role in thymic maturation of human T cells. We now report the isolation and characterization of cDNAs encoding murine IL-2R gamma. The open reading frame encodes 369 aa, identical in length to that encoded by the human IL-2R gamma cDNA. Murine IL-2R gamma and human IL-2R gamma have 69% and 70% identity at the nucleotide and amino acid levels, respectively. As expected, the murine IL-2R gamma retains the WSXWS motif and four cysteine residues characteristic of cytokine receptor superfamily members. IL-2R gamma mRNA distribution shows significant tissue specificity, with particularly high-level expression in spleen and thymus, and higher expression in single-positive (CD4+8- or CD4-8+)-enriched thymocytes than in double-negative (CD4-8-) thymocytes. Finally, we have localized the murine IL-2R gamma gene, Il2rg, to the X chromosome between Rsvp and Plp and demonstrated that a defect in IL-2R gamma is not responsible for the X chromosome-linked xid mutation, which maps to this same region. The cloning of the murine IL-2R gamma cDNA will facilitate the investigation of the role of this protein in lymphocyte function and thymic development.

Expression of a functional c-kit receptor on a subset of natural killer cells.

Natural killer (NK) cells are large granular lymphocytes thought to be important in the host's early immune response to viral infection and malignant transformation. NK cells proliferate and display enhanced cytotoxic activity in response to the T cell growth factor, interleukin 2 (IL-2). Stem cell factor or steel factor (SF) is the ligand for the c-kit receptor, and when combined with other hematopoietic growth factors, SF synergistically promotes the proliferation and differentiation of bone marrow stem cells. In the present study we show the c-kit receptor to be uniquely expressed on a subset of resting human NK cells (CD56bright) which constitutively expresses both the high affinity IL-2 receptor (IL-2R) and the intermediate affinity IL-2R. Other lymphocyte populations, including CD56dim NK cells, did not appear to express the c-kit receptor. Within the CD56bright NK cell subset, SF alone had no obvious effect on proliferation or cytotoxic activity. SF was shown to significantly augment the proliferative effect of IL-2, and caused a marked shift in the dose-response curve at IL-2 concentrations that selectively saturate the high affinity IL-2R. The potentiating effect of SF on NK cell proliferation was dependent on IL-2 binding to the high affinity IL-2R, and was blocked by a monoclonal antibody directed against the c-kit receptor. SF did not enhance proliferation at higher IL-2 concentrations that saturate the intermediate affinity IL-2R, nor did SF enhance IL-2-induced cytotoxic activity. Together, these data indicate that SF and IL-2 act synergistically to directly augment the proliferative capacity of a unique human NK cell subset constitutively expressing the high affinity IL-2R and the c-kit receptor. The implications of these findings on NK cell development and the host's early immune response to pathogen invasion are discussed.

Characterization of the human interleukin-2 receptor gamma chain gene

The interleukin-2 (IL-2) receptor gamma chain is an essential component of high and intermediate affinity IL-2 receptors (IL-2Rs), playing critical roles for ligand binding and internalization. We report here the isolation and characterization of the genomic locus for human IL-2R gamma, which, like IL-2R beta, is a member of the cytokine receptor superfamily. The IL-2R gamma gene is composed of eight exons and seven introns and spans approximately 4.2 kilobases. Analogous to the IL-2R beta gene, the two pairs of conserved cysteines typical of cytokine receptor superfamily proteins are located in adjacent exons, and the conserved WSXWS motif is located in the exon preceding the one that encodes the transmembrane domain and a small part of the cytoplasmic domain. In each gene, the remainder of the cytoplasmic domain is encoded by the final two exons. Southern blot analysis suggests that IL-2R gamma is encoded by a single copy gene. Cross-hybridizing sequences were detected in DNA derived from a number of other mammalian species but not from yeast. Primer extension analysis and ribonuclease protection assays revealed that there are three principal transcription initiation sites located 32-38 nucleotides 5' to the translation initiation AUG codon. These sites are upstream of the 5' end of the published IL-2R gamma cDNA sequence. The region 5' to the transcription initiation sites exhibited promoter activity when cloned upstream of the luciferase reporter gene. With this study, the organization of the genes encoding all three chains (alpha, beta, and gamma) of the IL-2 receptor has been determined and promoters for each identified.

Interleukin-2 receptor γ chain mutation results in X-linked severe combined immunodeficiency in humans

The interieukin-2 (IL-2) receptor γ chain (IL-2Rγ) is a component of high and intermediate affinity IL-2 receptors that is required to achieve full ligand binding affinity and internalization. We have localized the IL-2Rγ gene to human chromosome Xq13. Genetic linkage analysis indicates that the IL-2Rγ gene and the locus for X-linked severe combined immunodeficiency (XSCID) appear to be at the same position. Moreover, we demonstrate that each of three unrelated patients with XSCID has a different mutation in his IL-2Rγ gene resulting in a different premature stop codon and predicted C-terminal truncation. These data establish that XSCID is associated with mutations of the IL-2Rγ gene product. Since XSCID is characterized by absent or markedly reduced numbers of T cells, our findings imply that IL-2Rγ plays a vital role in thymic maturation of T cells. These results also have important implications for prenatal and postnatal diagnosis, carrier female detection, and gene therapy for XSCID.

Delta-9-Tetrahydrocannabiol (THC) decreases the number of high and intermediate affinity IL-2 receptors of the IL-2 dependent cell line NKB61A2

Treatment of the cloned NK-cell line (NKB61A2) with the major psychoactive marijuana component, delta-9-tetrahdyrocannabinol (THC), for 24 h suppressed IL-2-induced proliferation of these cells in the cytokine concentration range of 0.25 – 10 pM suggesting that the drug inhibits the functional activity of the high affinity IL-2R. The proliferation inhibitory effect of THC was accompanied by a decrease in the number of high and intermediate affinity IL-2 binding sites as measured by equilibrium binding studies. However, the expression of Tac protein on the surface of these cells was increased as determined by flow cytometry analysis. THC was also shown to decrease proliferation and the number of IL-2 binding sites of cells previously pulsed with IL-2 and then treated with the drug in the absence of IL-2. These results suggest that THC inhibits IL-2-induced proliferation by modulating the expression of high affinity IL-2 receptors (α/β) required for cell activation and also suppresses the ongoing process of functional receptor expression and clonal expansion of cells previously activated by IL-2. Because the number of intermediate binding sites is decreased following drug treatment along with an increase in the expression of Tac protein (α chain), the lowering of high affinity sites possibly results from a drug-induced depression of β chain expression.

Characterization of Lymphokine-Activated Killing by Human Peripheral Blood Mononuclear Cells Stimulated with Interleukin 2 (IL-2) Analogs Specific for the Intermediate Affinity IL-2 Receptor

Interleukin 2-stimulated human peripheral blood mononuclear cells (PBMC) generate lymphokine-activated killing (LAK). Using the IL-2 analogs R38A and F42K, which interact primarily with the β and γ subunits of the IL-2 receptor, we assessed the roles of IL-2Rβγ and the high-affinity IL-2 receptor complex in LAK activation. Although the kinetics of LAK activation were identical, lytic activity was approximately 30% lower and proliferation was up to 55% lower in those PBMC stimulated by R38A or F42K than in those exposed to wild-type IL-2. The percentage of cells expressing cell-surface markers such as CD3, CD4, CD8, and CD16 was not significantly different after treatment with wild-type IL-2, R38A, or F42K; however, the proportion of cells expressing IL-2Rα increased dramatically in response to stimulation by F42K (30%) compared to stimulation by either rIL-2 or R38A (15%). In addition, by Day 7 the concentration of soluble IL-2Rα in analog-stimulated LAK culture supernatants was 50-75% less than that from wild-type IL-2-cultured cells. These findings suggest that interaction of IL-2 with IL-2Rβγ alone is sufficient for both proliferation and the generation of LAK, and that stimulation with subunit-specific IL-2 analogs results in differential regulation of the IL-2Rα on human LAK cells.

In vitro and in vivo suppression of interleukin-2-activated killer cell activity by chimeric proteins between interleukin-2 and Pseudomonas exotoxin

The biological effects of IL-2 are mediated through high (complex of α and β chain) or intermediate (β chain) affinity IL-2 receptors. Previously, chimeric proteins composed of IL-2 and Pseudomonas exotoxin (IL-2-PE) were shown to be specifically cytotoxic to cells bearing IL-2 receptors. It has also been shown that IL-2-PE chimeric proteins can abrogate T cell-mediated immune response in vitro. In the current study, we have investigated the effects of IL-2-PE on LAK activity both in vivo and in vitro. We administered either IL-2-PE40 (comprised of IL-2 and 40-kDa portion of PE) or IL-2-PE66 (comprised of IL-2 and 66-kDa molecule of PE) to normal C57BL/6 mice for 3 or 8 days and LAK activity was assessed in various organs of mice. We found that IL-2-PE40 generated LAK activity in various compartments of mice and the level of activity was slightly lower than that observed with an equivalent amount of recombinant (r) IL-2 alone. However, IL-2-PE66 failed to generate LAK activity which would have been induced due to an equivalent concentration of rIL-2. IL-2-PE66 also did not induce LAK activity from the splenocytes during in vitro culture while IL-2-PE40 generated good LAK activity. An equivalent amount of IL-2 also generated potent LAK activity. The suppression of LAK activity by IL-2-PE66 was also evident in cells preactivated with IL-2: however, this inhibition was partial. The suppressive activity of IL-2-PE66 was shown to be mediated through IL-2 receptor interactions as excess amounts of rIL-2 were able to abrogate its effect. Both IL-2 toxins were equivalently cytotoxic to IL-2 receptor-bearing HUT 102 cells and both were able to compete from high and intermediate affinity IL-2 receptors. Taken together, our data indicate that IL-2-PE66 is highly cytotoxic to LAK cells while IL-2-PE40 is less cytotoxic. Thus, data from our study and from other published reports indicate that IL-2-PE66 is a more potent immunosuppressive agent than IL-2-PE40.

Characterization of the interleukin 2 receptors (IL-2R) expressed on human natural killer cells activated in vivo by IL-2: association of the p64 IL-2R gamma chain with the IL-2R beta chain in functional intermediate-affinity IL-2R.

Interleukin 2 (IL-2) receptors expressed on the surface of activated T cells and natural killer (NK) cells exhibit a variety of affinity states depending on their subunit composition. Low-affinity binding is associated with a 55-kDa alpha chain, intermediate-affinity binding with a 70-75-kD beta chain, and high-affinity binding with a bimolecular complex of the alpha and beta subunits. In a previous study of the IL-2 receptors expressed on NK cells obtained from cancer patients after in vivo IL-2 therapy, we documented a discrepancy between the level of beta chain and the level of intermediate-affinity IL-2 binding sites expressed on the cell surface. Based on this result, we postulated that formation of intermediate-affinity receptor sites required a component in addition to the beta chain, and that this component was present at limiting levels in the patient NK cells. In the present study we have examined the structure of the intermediate-affinity receptor complex using monoclonal antibodies that recognize the beta chain, but that do not interfere with its ability to bind IL-2. Evidence is presented establishing the physical association of a novel protein of 64 kD with the beta chain in intermediate-affinity IL-2 binding sites. This molecule, termed IL-2R gamma chain, coprecipitated with beta chains prepared from cells that had been incubated with IL-2, but was undetectable in immunoprecipitates prepared in the absence of IL-2. Examination of gamma chain expression in post-IL-2 therapy NK cells, where only low levels of intermediate-affinity IL-2 binding were detectable, revealed that the gamma chain was associated with, on average, only 10-12% of the beta chains expressed on such cells. This contrasted with approximately equal levels of beta and gamma chain expression on YT cells, a cell line that has both high levels of cell surface beta chain expression and high levels of IL-2 binding. Thus, the ratio of gamma chain to beta chain present in the immunoprecipitates roughly correlated with the proportion of beta chain involved in intermediate-affinity receptor sites. This result suggests that the 64-kD gamma chain is the component responsible for regulating the affinity of IL-2 association with the beta subunit. By further defining the structural components necessary for IL-2 receptor formation, these studies provide additional insight into mechanisms whereby lymphocytes might regulate their responsiveness to IL-2.

An associated molecule, p64, with IL-2 receptor beta chain. Its possible involvement in the formation of the functional intermediate-affinity IL-2 receptor complex.

We identified previously a membrane molecule, p64, which co-precipitates with the IL-2R beta-chain in human T cells. We have now investigated the biologic significance of p64 in the formation of the functional IL-2R complex with cell lines transfected with cDNA of IL-2R alpha- and/or beta-chains. Two functional parameters associated with IL-2R, IL-2 binding ability and association of p64 with the beta-chain, were examined. Two subclones, MOLT beta-11 and MOLT beta-12, of an IL-2R beta cDNA-transfected MOLT4 clone expressed similar numbers of IL-2R beta molecules on cell surfaces and bound to IL-2 with intermediate affinity. However, the numbers of IL-2 binding sites were significantly lower than those of IL-2R beta molecules and considerably different between the two subclones. The amount of p64 co-precipitated with IL-2R beta was proportional to numbers of the IL-2 binding sites in the two subclones. In addition, neither p64 co-precipitation nor IL-2 binding was detected in HeLa and COS7 cells transfected with IL-2R beta, and no p64 precipitation was seen even in those transfectants with both IL-2R alpha and beta cDNAs, which bind to IL-2 with high affinity but are not able to transduce intracellular signals. These results suggest the possibility that p64 associates with IL-2R beta and has an important role in formation of the functional IL-2R complex.