IL-2R Subunits Research Articles

Biosensor analysis of the interleukin-2 receptor complex.

Surface plasmon resonance (SPR) biosensor technology has been a significant addition to the evolution and refinement of methods to study macromolecular interactions. Prior to the advent of SPR, we employed a variety of biochemical and biological techniques to study the interleukin-2/interleukin-2 receptor system (IL-2/IL-2R). By combining site-directed mutagenesis, equilibrium and kinetic radioligand binding, and competitive biological assays, we and others had begun to understand many aspects of the structure-activity relationships of the IL-2/IL-2R system. Due to the complexity of the IL-2R, cell-based assays proved limited in their ability to provide quantitative information on the binding characteristics of subclasses of the IL-2 receptor. SPR technology promised to be a new and powerful approach to the quantitative analysis of complex receptor systems. To demonstrate the feasibility of this technology, we employed Biacore analysis to investigate the ligand binding characteristics of novel, pre-assembled, IL-2R coiled-coil complexes. The results of these studies, although limited by instrumentation and data analysis, clearly established the utility of this method. Subsequently, by incorporating advancements in both of these areas, we have been able to carry out detailed kinetic analyses of the binding properties of individual IL-2R subunits as well as heteromeric complexes on the surface of a biosensor. Therefore, SPR biosensor analysis combined with other established analytical methods has proven to be a powerful tool for the analysis of complex hematopoietic receptor systems. Published in 1999 by John Wiley & Sons, Ltd.

Biosensor analysis of the interleukin‐2 receptor complex

Surface plasmon resonance (SPR) biosensor technology has been a significant addition to the evolution and refinement of methods to study macromolecular interactions. Prior to the advent of SPR, we employed a variety of biochemical and biological techniques to study the interleukin-2/interleukin-2 receptor system (IL-2/IL-2R). By combining site-directed mutagenesis, equilibrium and kinetic radioligand binding, and competitive biological assays, we and others had begun to understand many aspects of the structure–activity relationships of the IL-2/IL-2R system. Due to the complexity of the IL-2R, cell-based assays proved limited in their ability to provide quantitative information on the binding characteristics of subclasses of the IL-2 receptor. SPR technology promised to be a new and powerful approach to the quantitative analysis of complex receptor systems. To demonstrate the feasibility of this technology, we employed Biacore® analysis to investigate the ligand binding characteristics of novel, pre-assembled, IL-2R coiled-coil complexes. The results of these studies, although limited by instrumentation and data analysis, clearly established the utility of this method. Subsequently, by incorporating advancements in both of these areas, we have been able to carry out detailed kinetic analyses of the binding properties of individual IL-2R subunits as well as heteromeric complexes on the surface of a biosensor. Therefore, SPR biosensor analysis combined with other established analytical methods has proven to be a powerful tool for the analysis of complex hematopoietic receptor systems. Published in 1999 by John Wiley & Sons, Ltd.

Signal for IL-2 internalization located in the endocellular domain of IL-2R γ subunit only

Interleukin-2(IL-2) is an important immunoregulatory factor. After the interaction between IL-2 and its receptor, intracellular signal molecules were activated and pro-oncogenes, e.g.c-Myc, c-Fos, c-Jun andBcl-2 were induced, then cells stimulated with IL-2 would proliferate or differentiate. During these processes, IL-2 were internalized. The internalization of IL-2 mediated by IL-2R decreases the number of IL-2R on cell membranes, and makes extracellular IL-2 enter cells and degrade. It will down-regulate IL-2 biological activities. In order to identify the IL-2R subunit responsible for IL-2 internalization, chimeric IL-2Rs which consisted of different endocellular domains and extracellular domains from various IL-2R subunits were constructed. After they were transfected into L929 cell line, the IL-2 internalization was detected. Results showed that intracellular domains from IL-2R a and β subunlts could not mediate IL-2 internalization alone; the signal for IL-2 internalization was located only in the endocellular domain of IL-2R γ subunit. Furthermore, IL-2 internalization extent would be affected by affinity between IL-2 and IL-2R.

Role of IL-15, IL-2, and their receptors in the development of T cell alveolitis in pulmonary sarcoidosis.

Recent data suggest that the newly discovered cytokine IL-15 cooperates with IL-2 in driving T cell-mediated immune responses. The aim of this study was to determine the role of IL-15 in the regulatory networks leading to the development of T cell alveolitis in the lung of patients with sarcoidosis. We demonstrated that alveolar macrophages (AMs) isolated from the bronchoalveolar lavage of patients with active sarcoidosis expressed IL-15 mRNA and membrane and cytoplasmic IL-15, while AMs from healthy subjects and patients with inactive sarcoidosis did not. Pulmonary CD4+ T cells from sarcoid patients were equipped with the IL-2R subunits, which are able to bind IL-15, i.e., the IL-2R beta/IL-2R gamma complex, and proliferated in response to IL-15. Interestingly, the T cell proliferation elicited by IL-15 was comparable with that determined by IL-2. Following the addition of graded amounts of IL-15, IL-2-pulsed T cells showed a significant increase in their stimulation. TNF-alpha up-regulated the IL-15-mediated proliferative response of bronchoalveolar lavage T lymphocytes. Following the block of the IL-2R beta- and gamma-chains with specific mAbs, the stimulatory activity of IL-15 was abolished. The evaluation of the IL-2R on sarcoid AMs demonstrated the constitutive expression of alpha- and gamma-chain mRNA and proteins. Taken together, these findings demonstrate that IL-15 triggers the growth of sarcoid T cells through the IL-2R beta/IL-2R-gamma complex and raise the possibility that AMs may deliver proliferative signals for the development of the T cell alveolitis. Modulation of IL-2R on AMs could represent a critical variable in regulating local inflammatory responses.

Substitutions at the Glu62 residue of human interleukin-2 differentially affect its binding to the alpha chain and the beta gamma complex of the interleukin-2 receptor.

Human interleukin-2 (IL-2) alpha helix B is more conserved than the whole molecule, but has been less studied than other alpha helices of IL-2. Using site-directed mutagenesis, several IL-2 mutants in this helix were obtained. We found that the IL-2 mutant containing Leu at position 62 (Leu62-IL-2) loses its ability to bind IL-2 receptor subunit alpha (IL-2R alpha), but retains binding affinity to IL-2R subunit beta gamma as well as some bioactivity; nevertheless, another substitution at the same residue, Arg62IL-2, loses its binding ability to both IL-2R alpha and IL-2R beta gamma, and can no longer stimulate IL-2-dependent cell growth, showing that Glu62 not only takes part in IL-2R alpha binding, but can also affect IL-2 binding to IL-2R beta gamma. In this regard, Glu62 may be a key site in the IL-2/IL-2R alpha interaction, and can facilitate IL-2R ternary-complex formation, leading to IL-2R alpha-mediated, IL-2-stimulated signal transduction.

Formation of high affinity IL-2 receptors is dependent on a nonligand binding region of the alpha subunit.

The IL-2R is composed of three subunits, alpha, beta, and gamma, each of which individually contributes to binding IL-2 in the high affinity IL-2R. The molecular mechanism by which the high affinity IL-2R assembles has not been established. Previous studies have shown that human IL-2R subunits form high affinity hybrid IL-2R when mixed with appropriate mouse IL-2R subunits, but the efficiency of this process is not known. In this study, we produced stable cell lines that expressed hybrid human/mouse IL-2R, comprised of heterologous alpha- and beta-chains, to determine the contribution of individual receptor subunits in stabilizing the high affinity IL-2R. Quantitative ligand binding studies and FACS analysis were performed for EL4J cells that expressed hybrid mouse/human IL-2R, and these data were compared with those of cells that expressed homologous mouse IL-2R subunits. EL4J cells that expressed human beta/mouse alpha formed high affinity IL-2R, comparable with cells that express homologous mouse alpha and beta subunits. In contrast, EL4J cells that expressed homologous mouse IL-2R contained an approximately 10-fold higher level of high affinity IL-2R than did EL4J cells that expressed heterologous human alpha/mouse beta IL-2R. This difference was not accounted for by lower levels of human alpha-chains in these cell lines or lower expression of the mouse IL-2R beta or gamma subunits. These results suggest that amino acid sequences within the alpha subunit, distinct from the IL-2 binding site, are important in the assembly and stabilization of the high affinity IL-2R.

Serotonin-stimulated aortic endothelial cells secrete a novel T lymphocyte chemotactic and growth factor.

Atherosclerotic lesions contain multiple cell types including smooth muscle cells, macrophages, and T lymphocytes. The development of an extralymphatic T lymphocyte focus of inflammation in this condition requires chemoattractant-induced cell migration and growth factor-induced cell activation. In a previous study, we described a novel 13-15-kDa T lymphocyte-specific chemotactic cytokine, endothelial cell-derived lymphocyte chemoattractant activity (ED-LCA), secreted by serotonin-stimulated bovine aortic endothelial cells that is distinct from previously identified endothelial cell-derived interleukins (IL) 1, 6, and 8. Because of the association between T lymphocyte chemotactic and growth factor activity, in the current study we investigated the effect of ED-LCA on T cell growth. We assessed its capacity to induce markers of the passage of T cells from the resting (G0) state into the G1 phase of the cell cycle, such as receptors for IL-2 (IL-2R) and transferrin (TFR) and class II major histocompatibility complex antigens (HLA-DR). Incubation of G0 freshly isolated human T lymphocytes for 48 h with chromatographically resolved, partially purified ED-LCA resulted in a threefold increase in expression of the p55 subunit of IL-2R, a threefold increase in TFR, and a twofold increase in HLA-DR. Passage into the G1 phase of the cell cycle was confirmed by cell cycle analysis employing acridine orange. Evaluation of CD4+ and CD8+ T cell subsets by double-antibody labeling demonstrated that the p55 subunit of IL-2R was induced in both T cell subsets. Although incubation of human T cells with ED-LCA alone did not induce proliferation, addition of exogenous IL-2 to T cells pulsed with ED-LCA for 24 h caused a proliferative response with a stimulation index of 3. By up-regulating functional cell surface receptors for IL-2, ED-LCA is a competence growth factor for T lymphocytes and primes them to respond to IL-2. By virtue of its effect on T cells, as a chemotactic and competence factor, this endothelial cell-derived mitoattractant could participate with other T cell growth factors like IL-2 in the recruitment and amplification of the extralymphatic T cell component of atherosclerosis.

Differential cytokine expression in maternal blood and placenta during murine gestation.

To evaluate the influence of gestation on peripheral blood T cells, we measured the mRNA production of several cytokines (IL-2, IL-4, IL-10, IFN-gamma) and the p55 subunit of IL-2R at different time points in the blood of pregnant mice and in the placenta. This was made possible by the use of a new PCR technique that is precise and quantitative. Our results show that pregnancy induces profound changes in the expression of these genes in peripheral blood cells. During the first week of gestation, there is an increase in the levels of all the cytokines, followed by a state of immunodepression characterized by levels of cytokines below normal (nonpregnant mice). In the placenta low levels of IL-2 and IL-10 are detected. IFN-gamma mRNA production is higher than the blood IFN-gamma mRNA in the last week of pregnancy. However, the main difference is found for IL-4 mRNA expression where the placenta levels are 5- to 10-fold higher than the blood mRNA expression. We discuss these results in the context of the placenta as a privileged immune site, where IL-4, being the main cytokine, may play a major regulatory role.

Definition and spatial location of mouse interleukin-2 residues that interact with its heterotrimeric receptor.

The high affinity receptor for interleukin-2 (IL-2) contains three subunits called IL-2R alpha, beta and gamma. A biological and receptor binding analysis based on 1393 different mutant mouse IL-2 (mIL-2) proteins was used to define the function of each of the 149 residues. By this genetic analysis, 44 residues were assigned important functions, 21 of which were structural. The remaining 23 residues consisted of 19 residues, from three separate regions, that were important for IL-2R alpha interaction; three residues, from two separate regions, that were important for IL-2R beta interaction; and a single residue important for IL-2R gamma interaction. We built a model mIL-2 structure based on the homologous human IL-2 (hIL-2) crystal structure. The roles of the 21 residues presumed to be important for structure were consistent with the model. Despite discontinuity in the primary sequence, the residues specific for each IL-2R subunit interaction were clustered and located to three disparate regions of the tertiary mIL-2 structure. The relative spatial locations of these three surfaces are different from the two receptor binding sites known for the structurally related human growth hormone and the significance of this observation is discussed.

High-affinity and intermediate-affinity forms of the human interleukin 2 receptor, expressed in an interleukin 9-dependent murine T cell line, deliver proliferative signals via differences in their transduction pathways

Although several reports have claimed that the p70 IL-2R per se transduces a growth signal in lymphoid and non-lymphoid systems, there is no convincing evidence for this in lymphoid T cell lines. In order to investigate the mechanism of IL-2R-dependent signal transduction pathways via the p70 IL-2R in lymphoid T cells, we have established two IL-9-dependent murine (TS1) cell lines stably expressing the human p70 IL-2R subunit or the human p55-p70 IL-2R complex. Whereas the parental T cell line, TS1, proliferated in response to IL-9 or IL-4 but not IL-2, cell lines stably expressing human p70 IL-2R or p55-p70 IL-2R complex proliferated in response to IL-2. This implies that the murine T cell, TS1, contains all the intracellular components necessary for directing IL-2 signalling. In human p55-p70 IL-2R-transfected cells, the expression of a functional murine p55 IL-2R seemed to be induced and regulated by IL-2. In human p70 IL-2R-transfected cells, in the presence of IL-2 an interaction requiring only intermediate-affinity was sufficient for transduction of a proliferative signal. In addition human p70 IL-2R per se, is biologically functional via a transduction pathway not requiring induction of murine p55 IL-2R and consecutive high-affinity complex reconstitution. Thus, although both transfected cell lines can transduce a proliferative signal in the presence of IL-2, a difference in their transduction pathway is probably involved for the induction of p55 IL-2R.

Molecular area involved in the in-vitro dimerization of the murine p55 IL-2 receptor

In order to study structure-function relationship of the M r 55 000 subunit of the murine IL-2R (p55 IL-2R) by epitope mapping, we have expressed the p55 IL-2R molecule in a cell-free translation system. Under these in vitro conditions, we detected the expected p55 IL-2R polypeptide initiated at Met 1 and, in addition, two internally initiated molecules at Met 64 and Met 105. In this report we describe that from such a mixture, containing three molecular species of p55 IL-2R, mAb 135D5 imunoprecipitated the polypeptide initiated at Met 105 although this N-terminally truncated form of p55 IL-2R does not contain its epitope located between amino acids 72–88. This observation can be taken as a further evidence for the formation of p55 IL-2R dimers. Finally, we identified the region implicated in the formation of p55 IL-2R dimers close to the transmembrane region of the molecule.

Impaired post-transcriptional expression of interleukin-2 receptor in pokeweed mitogen-activated T cells.

The expression and role of interleukin-2/interleukin-2 receptor (IL-2/IL-2R) system in the pokeweed mitogen (PWM)-induced T cell mitogenesis was studied. In the absence of monocytes (Mo), both soluble and Sepharose-bound PWM fail to induce T cell mitogenesis even when exogenous IL-2 or IL-1 or IL-1 + IL-2 or IL-4 are also present. In the presence of Mo, PWM stimulation of T lymphocytes (highly depleted of B lymphocytes) induces as much IL-2 mRNA as phytohemagglutinin (PHA), but results in higher and persistent IL-2 levels in culture supernatants despite the concomitant T cell mitogenesis, suggesting that PWM-activated T cells do not utilize the IL-2 they produce. Confirming this notion, Mo-dependent PWM-preactivated T cells, as compared to PHA-preactivated ones: (a) failed to consume exogenous IL-2 and their mitogenic response did not increase upon exposure to exogenous IL-2; (b) exhibited very low numbers of high-affinity IL-2R; and (c) showed lower expression of IL-2R p55 and undetectable expression of IL-2R p75 on their surface. Moreover, the PWM-induced T cell mitogenesis was not inhibited by anti-IL-2 or CD25 antibodies and only partially (50%-60%) inhibited by cyclosporin A, while these treatments abrogated the PHA-induced one. PWM-activated T cells, as compared to the PHA-activated ones, exhibited as high (p55) or even higher (p75) mRNA expression of both IL-2R p55 and p75 subunits. The possibility that PWM interferes with IL-2R subunits once expressed on the T cell surface was excluded. Thus, intracellular PWM-related events are likely to impair IL-2R expression post-transcriptionally. Possible explanations for this effect and its relation with the capacity of PWM to induce T cell-dependent B cell differentiation are discussed.

Reconstitution of the intermediate-affinity interleukin-2 receptor by cell fusion.

Although IL-2 receptor beta chain (IL-2R beta) expressed in various lymphoid cell lines binds IL-2 with an intermediate affinity, IL-2R beta expressed in fibroblasts is unable to bind IL-2, suggesting that IL-2R beta is on its own not sufficient for generating the intermediate-affinity receptor and that lymphoid-specific regulatory control may be operated to allow IL-2R beta to bind IL-2. In the present study, we observed that human IL-2R beta expressed in a mouse myeloma X63-Ag8.653 (X63) by cDNA transfection did not bind IL-2, while the same IL-2R beta expressed in an IL-6-dependent mouse B cell hybridoma F12-28, which was obtained by cell fusion between X63 and lipopolysaccharide (LPS)-induced lymphoblasts, bound IL-2 with the intermediate affinity. Interestingly, when the human IL-2R beta cDNA-transfected X63 clone, which by itself manifests no IL-2 binding, was fused with LPS-induced lymphoblasts, the resultant hybridomas manifested intermediate-affinity IL-2 binding. The IL-2 binding was specifically inhibited by addition of antihuman IL-2R beta mAb (Mik-beta 1) but not by mAb against mouse IL-2R subunits, indicating that human IL-2R beta was responsible for the IL-2 binding, i.e. non-functional human IL-2R beta in X63 was converted to competent IL-2R beta by complementation with a mouse spleen cell-derived factor(s) through the cell fusion. Cross-linking experiments with [125I]IL-2 revealed the presence of a 61 kDa protein other than IL-2R beta in cells expressing the intermediate-affinity IL-2R.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping.

Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins.

Production of granulocyte/macrophage-colony-stimulating factor by human natural killer cells. Modulation by the p75 subunit of the interleukin 2 receptor and by the CD2 receptor.

Resting natural killer (NK) cells express the p75 chain of the IL-2 receptor (IL-2R beta) and most NK cells express the CD2 (erythrocyte rosette) receptor. The cell adhesion molecule, LFA-3, is a natural co-ligand for CD2. Tac antigen (IL-2R alpha), a p55 IL-2R subunit, can be expressed after NK activation and may play a role in IL-2-induced NK proliferation. Little is known of the molecular mechanisms underlying cytokine production in NK cells. We investigated the roles of IL-2R alpha, IL-2R beta, and CD2/LFA-3 in the molecular regulation of NK cell granulocyte/macrophage-colony-stimulating factor (GM-CSF) production. Enriched populations of peripheral blood NK cells were separated into CD16-positive and CD16-negative fractions by flow cytometry; positively selected cells were greater than 97% positive for CD16 (the FcIII receptor for IgG which is present on almost all NK cells), less than 1% positive for the T cell antigen CD3, and did not demonstrate rearrangement of the T cell receptor beta chain gene by Southern blot. NK cell supernatants were harvested after 3-4 d of incubation with 0-100 U/ml IL-2, or after incubation with anti-CD2 (T11(3] MAb and sheep red blood cells (SRBC are a homologue for LFA-3). Parallel cell aliquots were harvested at 3-16 h for transcriptional run-on assays, S1 nuclease assays, and actinomycin D mRNA t1/2 determinations. IL-2-activated NK supernatants contained large amounts of GM-CSF (178 +/- 35 pg/ml) by ELISA as did supernatants from CD2-activated NK cells (T11(3) MAb + SRBC: 212 +/- 42) vs. less than 20 pg/ml for NK cells incubated alone or with either SRBC or T11(3) MAb alone. Sepharose-linked anti-CD3 MAb did not induce GM-CSF release from NK cells. By S1 analysis, both IL-2 and CD2 stimulation markedly augmented GM-CSF mRNA expression but with very different latencies of onset. IL-2R beta MAb inhibited greater than 85% of GM-CSF release from IL-2-activated NK cells and markedly suppressed IL-2-induced GM-CSF mRNA expression, whereas IL-2R alpha MAb even at 2,000-fold molar excess of IL-2 had little effect (less than 10%) on either GM-CSF release or mRNA expression. Run-on assays showed that GM-CSF is constitutively transcribed in NK cells and that IL-2 and CD2-activated cells had a three- to fourfold increased rate of GM-CSF transcription compared to nonstimulated cells. The t1/2 of GM-CSF mRNA in IL-2-activated NK cells was identical to that of unstimulated NK cells (15 min), whereas GM-CSF mRNA t1/2 in CD2-activated NK cells was increased 2.5-fold. We conclude that GM-CSF production in NK cells is regulated by both the IL-2Rbeta and the CD2 receptor but not by IL-2Ralpha, that both transcriptional and posttranscriptional signals act together to modulate the level of GM-CSF mRNA in NK cells, and that the molecular mechanisms underlying NK cell GM-CSF production are dependent in part on differential surface receptor activation.

IL-2-dependent proliferation of murine T cells requires expression of both the p55 and p70 subunits of the IL-2 receptor.

An IL-4-dependent T cell clone (LD8) was isolated from the murine IL-2-dependent cytotoxic T cell line C30.1. This clone has lost the capacity to proliferate in response to IL-2 after long-term culture in IL-4. LD8 cells express the p70, but not the p55, subunit of the IL-2R on their cell surface. The number of p70 IL-2R molecules on LD8 cells is comparable with the number of high-affinity IL-2R on the parental C30.1 cell line. LD8 cells can efficiently internalize IL-2 through the p70 IL-2R subunit. Following stimulation by IL-2, LD8 cells up-regulate p70 IL-2R mRNA, but do not express p55 IL-2R mRNA. IL-2-dependent proliferation of LD8 cells was reconstituted after introduction and expression of a human p55 IL-2R cDNA. To further investigate the role of p70 IL-2R, we have measured IL-2-induced proliferation of C30.1 cells in the presence of three anti-p55 IL-2R mAb (5A2, PC61, and 7D4) that recognize different epitopes. Under the experimental conditions used, the combination of anti-p55 IL-2R mAb prevents the formation of high-affinity IL-2R, but does not affect the binding of IL-2 to p70 IL-2R or IL-2 internalization. However, these three mAb inhibit proliferation of C30.1 cells even in the presence of IL-2 concentrations sufficient to saturate p70 IL-2R. Together these results demonstrate that p70 IL-2R alone is not sufficient to transmit IL-2-induced growth signals and that formation of p55-p70 IL-2R complex is required for IL-2-dependent proliferation of murine T cells.

CAMP regulation of IL-2 receptor expression. Selective modulation of the p75 subunit.

As previous experiments have shown that IL-2 activity is abrogated by cAMP, its effect on IL-2R expression by normal and leukemic T lymphocytes was evaluated in detail. The exposure of murine or human T cells to dibutyryl cAMP or the cAMP-elevating drug, forskolin, resulted in a decrease in high affinity IL-2 binding. Equilibrium binding analyses revealed that elevation of cAMP for 4 to 5 h produced a 40 to 50% decrease in the number of detectable receptors, whereas the affinity of the IL-2R interaction remained unchanged. The effect of cAMP could be attributed to a selective effect on the 75-kDa chain of the IL-2R (p75) subunit of the 55-kDa chain of the IL-2R/p75 heterodimer. The mechanism for the decreased expression of high affinity IL-2R appears to be due to a dual effect of cAMP, which functions to both increase the rate of IL-2R internalization, and to decrease the rate of expression of new receptors. Moreover, the effect of cAMP on IL-2 binding to p75 subunits is post-transcriptional, because the steady state levels of p75 mRNA expression are not altered within a time interval that produced nearly a 50% reduction in p75 binding.

Expression and role of p75 interleukin 2 receptor on human monocytes.

We investigated the expression of IL-2R subunits in human monocytes using the TU27 mAb, which recognizes the p75 chain, and anti-Tac mAb, which recognizes the p55 moiety of the IL-2R. We found that p75 but not p55 is constitutively expressed in more than 90% of fresh human monocytes. Antibody to p75, but not to p55, inhibited the activation of monocytes to a cytotoxic stage induced by IL-2 but did not block IFN-gamma-induced cytotoxicity. Our data demonstrate that the p75 chain is expressed on human monocytes and is involved in the activation of monocytes by IL-2.

Cell division-associated expression of an epitope, KH17, on early developing thymocytes

A newly established monoclonal antibody, KH17, detects a unique epitope temporarily expressed on early developing CD3-thymocytes confined to a cycling stage. KH17 is detectable on a part of CD4-CD8-,CD4-CD8+, and CD4+CD8+ cells, but not on CD4+CD8- thymocytes. By four-color flow cytometry analysis using KH17, we were able to define the heterogeneity of immature CD4-CD8- thymocytes by the expression of KH17 and IL-2R. In Thy-1-congeneic bone marrow chimeras, the appearance of KH17-IL-2R+ thymocytes preceded the increase of KH17+IL-2R- cells. The antibody could also divide CD3-CD4-CD8+ cells into two subpopulations, KH17+ and KH17-, which showed a continuum. In the fetal thymus there was a rapid and dramatic increase of KH17-CD4+CD8+ thymocytes concomitant with a decrease of KH17+CD4-CD8+ thymocytes in later gestation days. KH17 is not expressed on resting peripheral T cells, but is expressed on a large proportion of Con A-activated blastic spleen cells. The KH17 molecules precipitated from Con A-activated spleen cells were 55 and 75 kd polypeptides, but different from IL-2R subunits.

Differential expression of the IL-2 receptor subunits, p55 and p75 on various populations of primary peripheral blood mononuclear cells.

Abstract Unstimulated PBL were examined for expression of IL-2R subunits, IL-2Rp55 and IL-2Rp75, by two-color flow cytometric analyses using mAb. NKH-1+ non-T non-B cells expressed IL-2Rp75 but not IL-2Rp55, and the IL-2Rp75 sites on purified NKH-1+ cells were determined to be 1630 sites/cell by binding of 125I-labeled TU27 mAb specific for IL-2Rp75. In the CD4+ T cell population, IL-2Rp55+ cells were significantly detected, but little or marginally of the IL-2Rp75+ cells. However, IL-2Rp75+ cells were significantly detected, but little of the IL-2Rp55+ cells in the CD8+ T cell population. The IL-2Rp75 sites on CD8+ T cells were estimated at approximate 180-410 sites/cell. In the CD4+ T cells, expression of IL-2Rp75 as well as IL-2Rp55 was induced by stimulation with PHA. IL-2Rp75+ cells, but not IL-2Rp55+ cells, were also detected in the CD14+ monocyte population. In the CD20+ B cell population, a small number of IL-2Rp55+ cells was detected, but little of the IL-2Rp75+ cells.