IL-2R Beta Chain Research Articles

Role of Interleukin (IL)-2 Receptor β-Chain Subdomains and Shc in p38 Mitogen-activated Protein (MAP) Kinase and p54 MAP Kinase (Stress-activated Protein Kinase/c-Jun N-terminal Kinase) Activation: IL-2-DRIVEN PROLIFERATION IS INDEPENDENT OF p38 AND p54 MAP KINASE ACTIVATION

We have shown recently that interleukin (IL)-2 activates the mitogen-activated protein (MAP) kinase family members p38 (HOG1/stress-activated protein kinase II) and p54 (c-Jun N-terminal kinase/stress-activated protein kinase I). Furthermore, the p38 MAP kinase inhibitor SB203580 inhibited IL-2-driven T cell proliferation, suggesting that p38 MAP kinase might be involved in mediating proliferative signals. In this study, using transfected BA/F3 cell lines, it is shown that both the acidic domain and the membrane-proximal serine-rich region of the IL-2Rbeta chain are required for p38 and p54 MAP kinase activation and that, as for p42/44 MAP kinase, this activation requires the Tyr338 residue of the acidic domain, the binding site for Shc. It is well established that the acidic domain of the IL-2Rbeta chain is dispensable for IL-2-driven proliferation, and thus our observations suggest that neither p38 nor p54 MAP kinase activation is required for IL-2-driven proliferation of BA/F3 cells. In addition, the tetravalent guanylhydrazone inhibitor of proinflammatory cytokine production, CNI-1493, can block the activation of p54 and p38 MAP kinases by IL-2 but has no effect on IL-2-driven proliferation of BA/F3 cells, activated primary T cells, or a cytotoxic T cell line. Furthermore, our observations provide evidence for the existence of an additional, unknown target of the p38 MAP kinase inhibitor SB203580, the activation of which is essential for mitogenic signaling by IL-2.

Defective IL-2 receptor expression in lymphocytes of patients with arsenic-induced Bowen's disease.

The immune function of peripheral mononuclear cells (MNC) in patients with endemic arsenic-induced Bowen's disease (BD) was investigated. Many cytokines and immune-related factors were determined in the present study. Interleukin-1beta and TNF-alpha production was used as an indicator of monocyte/macrophage function. II-2 and sIL-2R production was used as an indicator of lymphocyte activation. The release of sCD4 and sCD8 was used as an indicator of activation of respective T-cell subpopulations. Production of IFN-gamma and IL-2 reflected the cellular effector function of helper T-cells type 1. In vivo cell-mediated immunity was also assessed by estimation of the percentage of T-cells in peripheral blood MNC and the nonspecific delayed-type hypersensitivity (DTH) response to 2,4-dinitrochlorobenzene (DNCB). Both assays revealed depressed cell-mediated immunity in BD. Compared with healthy controls, spontaneous and PHA-induced IFN-gamma and TNF-alpha production was significantly decreased in BD whereas spontaneous release of IL-2, sCD4 and sCD8 was significantly increased. Although PHA stimulation increased IL-2 release, the expression of IL-2R alpha and beta chains and the release of sIL-2R were not proportionately increased in BD. In addition, IL-2-mediated [3H]-thymidine incorporation by MNC in patients with BD was significantly decreased. These findings suggest that the defective cell-mediated immune function in BD is due to impairment of membrane IL-2R expression in lymphocytes after stimulation.

The murine anti-human common gamma chain monoclonal antibody CP.B8 blocks the second step in the formation of the intermediate affinity IL-2 receptor.

A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the human common gamma (gammac) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfected with the cDNA for the full-length IL-2 receptor beta (IL-2Rbeta) and gammac chains, components which together comprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NK cells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, the extracellular portions of the IL-2Rbeta and gammac chains were expressed and purified, and their interactions with each other and with IL-2 were studied by gel filtration and by surface plasmon resonance (SPR). By gel filtration, a stable ternary complex was formed by association of the three proteins, while no stable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2 was shown to associate rapidly with IL-2Rbeta, forming a binary complex with an equilibrium dissociation constant (Kd) of 800 nM, which permitted subsequent association of the gammac chain. Dissociation of the IL-2/IL-2Rbeta/gammac chain complex was significantly slower than dissociation of the IL-2/IL-2Rbeta complex. Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the gammac chain with IL-2 and IL-2Rbeta. By gel filtration, mAb CP.B8 formed a stable complex with the gammac chain, preventing its association with IL-2 and IL-2Rbeta. MAb CP.B8 was also capable of dissociating the gammac chain already complexed with IL-2 and IL-2Rbeta. SPR analysis confirmed these findings and showed, in addition, that the Fab fragment of CP.B8 was also capable of inhibiting the association of the gammac chain with the IL-2/IL-2Rbeta complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediate affinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the gammac chain that is involved in contact with IL-2 and/or IL-2Rbeta.

Molecular analysis of the IL-2 receptor beta chain gene expressed in human tumor cells.

Interleukin-2 (IL-2) is recognized as a T cell growth factor. We have previously reported that human carcinoma cell lines are inhibited in growth by exogenous IL-2, which binds to the IL-2 receptor beta (IL-2Rbeta) chain ubiquitously expressed on the surface of tumor cells. A possibility was considered that IL-2Rbeta on carcinomas responsible for negative signaling was different from that expressed on hematopoietic cells. To investigate this possibility, mRNA for the IL-2Rbeta chain was amplified and compared in carcinoma and lymphoid cells. Using RT-PCR with pairs of sense-antisense oligonucleotide primers specific for the various regions of extracellular, transmembrane and intracellular domains of the IL-2Rbeta chain, we amplified mRNA obtained from three human carcinoma cell lines and human lymphoid cells as controls. The identity of the amplicons was confirmed by Southern analysis with the 32P-labeled cDNA probe coding for the entire span of the IL-2Rbeta chain. In addition, genomic DNA obtained from the tumor cell lines was sequenced to examine the possibility that a mutation is present in the gene coding for the intracellular IL-2Rbeta chain domain. No mutations or deletions were detected. The message for all three domains of the beta chain was identical in tumor cells and in normal lymphoid cells used as controls. Also, by Western blot and northern analyses no differences between IL-2Rbeta chain in tumors vs that expressed in lymphoid cells were demonstrable. The IL-2Rgamma chain, which participates in IL-2/IL-2R signaling pathway, was expressed in tumor cells. Expression of JAK1 transcripts in these cells was comparable to that in lymphocytes. However, RT-PCR analysis identified differences in expression of JAK3 splice variants (B and M) in tumor cells. These differences may be responsible for altered downstream signaling by IL-2. Overall, our data indicate that the same IL-2/IL-2R pathway is operative in human carcinomas and in normal epithelial or lymphoid cells.

Biology of the Interleukin-2 Receptor

Studies of the biology of the IL-2 receptor have played a major part in establishing several of the fundamental principles that govern our current understanding of immunology. Chief among these is the contribution made by lymphokines to regulation of the interactions among vast numbers of lymphocytes, comprising a number of functionally distinct lineages. These soluble mediators likely act locally, within the context of the microanatomic organization of the primary and secondary lymphoid organs, where, in combination with signals generated by direct membrane-membrane interactions, a wide spectrum of cell fate decisions is influenced. The properties of IL-2 as a T-cell growth factor spawned the view that IL-2 worked in vivo to promote clonal T-cell expansion during immune responses. Over time, this singular view has suffered from increasing appreciation that the biologic effects of IL-2R signals are much more complex than simply mediating T-cell growth: depending on the set of conditions, IL-2R signals may also promote cell survival, effector function, and apoptosis. These sometimes contradictory effects underscore the fact that a diversity of intracellular signaling pathways are potentially activated by IL-2R. Furthermore, cell fate decisions are based on the integration of multiple signals received by a lymphocyte from the environment; IL-2R signals can thus be regarded as one input to this integration process. In part because IL-2 was first identified as a T-cell growth factor, the major focus of investigation in IL-R2 signaling has been on the mechanism of mitogenic effects in cultured cell lines. Three critical events have been identified in the generation of the IL-2R signal for cell cycle progression, including heterodimerization of the cytoplasmic domains of the IL-2R beta and gamma(c) chains, activation of the tyrosine kinase Jak3, and phosphorylation of tyrosine residues on the IL-2R beta chain. These proximal events led to the creation of an activated receptor complex, to which various cytoplasmic signaling molecules are recruited and become substrates for regulatory enzymes (especially tyrosine kinases) that are associated with the receptor. One intriguing outcome of the IL-2R signaling studies performed in cell lines is the apparent functional redundancy of the A and H regions of IL-2R beta, and their corresponding downstream pathways, with respect to the proliferative response. Why should the receptor complex induce cell proliferation through more than one mechanism or pathway? One possibility is that this redundancy is an unusual property of cultured cell lines and that primary lymphocytes require signals from both the A and the H regions of IL-2R beta for optimal proliferative responses in vivo. An alternative possibility is that the A and H regions of IL-2R beta are only redundant with respect to proliferation and that each region plays a unique and essential role in regulating other aspects of lymphocyte physiology. As examples, the A or H region could prove to be important for regulating the sensitivity of lymphocytes to AICD or for promoting the development of NK cells. These issues may be resolved by reconstituting IL-2R beta-/-mice with A-and H-deleted forms of the receptor chain and analyzing the effect on lymphocyte development and function in vivo. In addition to the redundant nature of the A and H regions, there remains a large number of biochemical activities mediated by the IL-2R for which no clear physiological role has been identified. Therefore, the circumstances are ripe for discovering new connections between molecular signaling events activated by the IL-2R and the regulation of immune physiology. Translating biochemical studies of Il-2R function into an understanding of how these signals regulate the immune system has been facilitated by the identification of natural mutations in IL-2R components in humans with immunodeficiency and by the generation of mice with targeted mutations in these gen

Dissociation of cytokine signals for proliferation and apoptosis.

Cytokines such as IL-2 or IL-3 prevent cell death through apoptosis, either by preventing apoptosis directly or by sensitizing cells to survival factors present in serum. We demonstrate herein that BAF-B03 cells transfected with the wild-type IL-2R beta-chain undergo apoptosis when stimulated with IL-2 or IL-3 in the absence of serum. IL-2 also induced apoptosis in normal IL-2-responsive human T cell blasts in the absence of serum, and furthermore, epidermal growth factor and fibroblast growth factor induced increased rates of apoptosis in fibroblasts in the absence of serum, suggesting that cytokine-induced apoptosis in the absence of serum survival factors might represent an important biologic phenomenon. In the presence or the absence of serum, IL-2 and IL-3 induced expression of both c-Myc and Bax. In contrast, optimal cytokine-induced expression of Bcl-2 requires serum. Constitutive expression of Bcl-2 prevented cytokine-induced apoptosis. Transferrin mimicked serum by inducing an increase in Bcl-2 expression levels and concurrently prevented apoptosis. These results suggest that the balance between cytokine- and serum-induced Bcl-2 expression and cytokine-induced Bax expression may determine whether a cell undergoes cytokine-induced apoptosis. In BAF/BO3 cells expressing a mutant IL-2Rbeta with a deletion of the acidic domain, IL-2 did not induce either Bax expression or apoptosis. This suggests that the acidic domain of the IL-2R beta-chain plays an essential role in regulating IL-2-mediated Bax expression and apoptosis. Cytokine-induced apoptosis and its counterbalance by survival factors present in serum may play an important role in the regulation of cellular homeostasis during pathophysiologic processes.

TGF-beta inhibits IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 in T lymphocytes.

Signaling through IL-2R, IL-2 induces tyrosine phosphorylation and activation of Jak-1 and Jak-3 kinases and Stat 3 and Stat 5 transcription factors leading to cell cycle progression of activated T cells from G1 to S phase. TGF-beta is an immunosuppressive cytokine, which inhibits T cell proliferation at G1 to S phase transition. We examined the effect of TGF-beta on IL-2R signal transduction pathway in activated T cells. We show here that treatment of activated T cells with TGF-beta inhibited IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 but not Jak-3 and Stat 3. TGF-beta also inhibited IL-2-induced expression of alpha- and beta-chains of IL-2R and induced apoptotic cell death in T cells. These results suggest that TGF-beta-induced growth arrest and apoptosis are associated with the modulation of IL-2-induced activation of Jak-Stat pathway in T cells.

Intermediate TCR cells in mouse lung: their effector function to induce pneumonitis in mice with autoimmune-like graft-versus-host disease.

The lung comprises lymphocytes even under noninflammatory conditions. We investigated the presence of NK cells, extrathymic T cells, and thymus-derived T cells in this organ. As shown previously in mouse liver, two-color staining for CD3 and IL-2R beta-chain (IL-2Rbeta) identifies CD3- IL-2Rbeta+ NK cells, CD3int IL-2Rbeta+ cells (int indicates intermediate; of extrathymic origin), and CD3high IL-2Rbeta- cells (high indicates high; of thymic origin). These populations were present in the lungs of normal mice in a unique manner (i.e., NK cells > CD3high cells > CD3int cells). The proportion of CD3int cells increased in the lung with age. In athymic mice, only CD3int cells were detected in the lung. In contrast to CD3int cells in the liver, the majority of these cells in the lung did not express NK1.1 Ags. Other properties of CD3int cells in the lung were the same as those in the liver, e.g., double-negative CD4- 8- cells and gammadelta T cells. CD3int cells in the lung contained forbidden clones similar to those in other organs. When (B6 x bm12) F1 mice (Ly5.2) were injected with CD3high cells of B6-Ly5.1 origin, F1 mice fell victim to chronic graft-vs-host disease and pneumonitis, showing the expansion of CD3int cells. Almost all of them were of recipient origin (Ly5.2+). More importantly, such CD3int cells induced autoimmune-like pneumonitis when injected into irradiated F1 mice. CD3int cells isolated from the lung in mice with graft-vs-host disease exerted autologous cytotoxicity against thymocytes in vitro. These results suggest that extrathymic T cells exist in the lung and that their expansion may be responsible for inflammatory lung diseases.

Pattern of cytokine receptors expressed by human dendritic cells migrated from dermal explants.

Different reasons account for the lack of information about the expression of cytokine receptors on human dendritic cells (DC): (a) DC are a trace population; (b) the proteolytic treatment used to isolate DC may alter enzyme-sensitive epitopes; and (c) low numbers of receptors per cell. In the present work the expression of cytokine receptors was analysed by flow cytometry on the population of dermal DC (DDC) that spontaneously migrate from short-term culture dermal explants. DDC obtained after dermal culture were CD1alow, CD1b+, CD1c+, human leucocyte antigen (HLA)-DR+, CD11chigh, CD11b+ and CD32+. The DC lineage was confirmed by ultrastructural analysis. DDC expressed interleukin (IL)-1R type 1 (monoclonal antibody (mAb) hIL-1R1-M1; and 6B5); IL-1R type 2 (mAb hIL-1R2-M22); IL-2R alpha chain (mAb anti-Tac; and hIL-2R-M1) and IL-2R gamma chain (mAb 3B5; and AG14C). DDC did not stain for IL-2R beta chain using four mAbs recognizing two different epitopes of IL-2R beta (mAb 2R-B; Mik-beta 1; and CF1; Mik-beta 3, respectively). DDC were also positive for the cytokine binding chains (alpha chains) of IL-3R (mAb 9F5); IL-4R (mAb hIL-4R-M57; and S456C9); and IL-7R (mAb hIL-7R-M20; and R3434). DDC showed low levels of IL-6R alpha chain (mAb B-F19; B-R6; and B-E23) and its signal transducer gp130 (mAb A2; and B1). DDC strongly expressed interferon-gamma receptor (IFN-gamma R) (mAb GIR-208) and were negative for IL-8R (mAb B-G20; and B-F25). All DDC were highly positive for granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha chain (mAb hGM-CSFR-M1; SC06; SC04, and 8G6) and to a lesser extent for the common beta chain of GM-CSFR, IL-3R and IL-5R (mAb 3D7). On the other hand, reactivity was not found for granulocyte colony-stimulating factor receptor (G-CSFR) (mAb hGCSFR-M1) nor macrophage colony-stimulating factor receptor (M-CSFR) (mAb 7-7A3-17) confirming the DC lineage of DDC. As previously reported for lymphoid DC, DDC expressed tumour necrosis factor receptort (TNFR) 75000 MW (mAb utr-1; hTNFR-M1; and MR2-1) but lacked TNFR 55000 MW (mAb htr-9; MR1-1; and MR1-2). In summary, DDC express receptors for a broad panel of cytokines, even receptors for cytokines whose effects on DC are still unknown (i.e. IL-2R alpha gamma; IL-6R alpha/gp 130; IL-7R alpha gamma).

Interleukin-2 induces tyrosine phosphorylation of SHP-2 through IL-2 receptor beta chain.

Coupling of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) induces rapid increase in tyrosine phosphorylation of cellular substrates through activation of non-receptor protein tyrosine kinases. Here, we report that stimulation through the IL-2R induced tyrosine phosphorylation of the SH2-containing protein-tyrosine phosphatase SHP-2 in F7, a hematopoietic BAF-B03 transfectant clone expressing the IL-2Rbeta chain. The tyrosine phosphorylation of SHP-2 was specific since another protein-tyrosine phosphatase SHP-1, which is structurally homologous to SHP-2, was not tyrosine phosphorylated. The IL-2-induced tyrosine phosphorylation of SHP-2 required the acidic region within the IL-2Rbeta chain where Src-family PTKs interact. Though the serine-rich region within IL-2Rbeta chain was also required for the phosphorylation of SHP-2, Jak3 activation was dispensable. In COS-7 cells, co-expression of SHP-2 with Lyn resulted in increased tyrosine phosphorylation levels of SHP-2, whereas co-expression of SHP-2 with Fyn failed to alter the levels significantly. Considering that Lyn and Fyn are major Src-family PTKs expressed in BAF-B03 cells, our data suggest that Lyn may be principally responsible for the tyrosine phosphorylation of SHP-2 in F7 cells. Furthermore, the IL-2 stimulation also induced tyrosine phosphorylation of SHP-2 in the human IL-2-dependent T-cell line ILT-Mat. Taken together, these studies demonstrate an involvement of SHP-2 in the IL-2-mediated signaling events through the activation of specific PTKs.

Requirement for an initial signal from the membrane-proximal region of the interleukin 2 receptor gamma(c) chain for Janus kinase activation leading to T cell proliferation.

The interleukin 2 receptor (IL-2R) generates proliferative signals in T lymphocytes by ligand-induced heterodimerization of two chains, IL-2Rbeta and gamma(c), which associate with the tyrosine kinases Jak1 and Jak3, respectively. Genetic and molecular studies have demonstrated that Jak3 is essential for mitogenic signaling by the gamma(c) chain; because it is also the only molecule known to associate with gamma(c), we speculated that Jak3 might be sufficient for signaling by this chain. Therefore, fusion proteins were constructed in which all or part of the cytoplasmic domain of gamma(c) was replaced by Jak3. Signaling was evaluated in the IL-2-dependent T cell line CTLL-2 using chimeric IL-2Rbeta and gamma(c) chains that bind and are activated by the cytokine granulocyte-macrophage colony-stimulating factor. Chimeric gamma(c) chains containing only Jak3 in the cytoplasmic domain failed to mediate proliferation of CTLL-2 cells, but addition of a conserved membrane-proximal (PROX) domain of gamma(c) in tandem with Jak3 fully reconstituted gamma(c) function. The requirement for the PROX domain reflected an essential role in the activation of Jak3 in vivo. Despite lacking defined catalytic motifs, PROX induced an early Jak-independent signal, including tyrosine phosphorylation of IL-2Rbeta and the tyrosine phosphatase SHP-2. The results define the minimal signaling components of gamma(c) and suggest a new mechanism by which the IL-2R initiates signaling in response to ligand.

IL-2 and TNF receptors as targets of regulatory T-T interactions: isolation and characterization of cytokine receptor-reactive T cell lines in the Lewis rat.

T cells are considered to be of prime importance in immune regulation of both B and T cell functions. The targets of recognition in T-T cell interactions are not clear. Most recent experimental work has focused on the idiotypic regulatory interactions mediated by TCR peptides. There is experimental evidence that regulatory cells exist that do not recognize the TCR. This type of regulation is selectively induced by activated T cells. Therefore, we designed this study to examine the possible role of cytokine receptors as targets of immune regulation. We tested two peptides of IL-2R alpha-chain, 2 of IL-2R beta-chain, and one of TNFR (p60). All peptides were found to be immunogenic at inducing T cell proliferation and four induced Abs in Lewis rats. We generated T cell lines to these five peptides, and tested them both in vitro and in vivo. We found that the T cells exhibited a proliferative response when cultured with activated, irradiated stimulator cells that were augmented upon addition of the cytokine receptor peptide. The cytokine profile of the lines was characterized as well as the Vbeta gene composition. One of the lines significantly protected against active encephalomyelitis. These results point at cytokine receptors as possible targets of immune regulation and T-T cell interactions.

IFN-gamma-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones.

Abstract NK cells, non-T non-B immune effector lymphocytes, are localized in many organs, including liver, as well as in the circulation. To investigate the regulatory mechanism of killing apparatus in hepatic NK cells, we established IL-2-dependent NK cell clones from liver lymphocytes of BALB/c nude mice. To generate the NK cell clones, we incubated liver lymphocytes with a high dose of IL-2 in the presence of irradiated Kupffer cells, as feeder cells and as the source of IL-12, originally identified as NK cell stimulatory factor. Unless liver lymphocytes were incubated with both IL-2 and Kupffer cells, no cell growth was observed. Hepatic NK cell clones were established from this cell line by limiting dilution. The surface phenotypes of cloned NK cells were IL-2R beta-chain+ CD16+ CD3- IgM-. The clones did not express NK2.1, which is expressed by a half of NK-enriched spleen cells of BALB/c mice. Although the cells contained dense granules reactive to mAb against perforin, they exerted no conventional cytolytic activity against YAC-1. They constitutively expressed Fas ligand (FasL) and specifically killed Fas-positive target cells by fragmenting DNA. This Fas-FasL-mediated killing activity was enhanced by IFN-gamma-inducing factor, a recently identified novel cytokine produced by activated Kupffer cells, but was not affected by other Kupffer cell-produced cytokines, such as IL-12, IL-1beta, and TNF-alpha. Taken together, these findings suggest that hepatic NK cells participate in the immune response as effector cells through the Fas-FasL system in collaboration with cytokines from Kupffer cells.

Distribution of IL-15 receptor alpha-chains on human peripheral blood mononuclear cells and effect of immunosuppressive drugs on receptor expression.

IL-15, a newly described cytokine exerting IL-2-like in vitro activities, binds to and induces proliferation of cells co-expressing IL-15R alpha, IL-2R beta, and IL-2R gamma chains. To study the expression of human IL-15R alpha chains, we have utilized tagged human IL-15 protein and FACS analysis. In contrast to resting cells, mitogen-activated macrophages, NK cells, and CD4+ and CD8+ T cells express IL-15R alpha chains. Neither IL-2R alpha nor IL-2R beta chains are required for IL-15 binding. Dexamethasone, but not cyclosporine or rapamycin, blocks mitogen-induced IL-15R alpha expression. Dexamethasone-pretreated cells respond to IL-15 poorly, while the response to IL-2 is not affected. Thus, despite structural and functional similarities between IL-2R alpha and IL-15R alpha chains, the activation-triggered mechanisms of induction are different. Since IL-15R alpha chain is necessary and sufficient for IL-15 binding, regulation of IL-15R alpha expression may represent a new target for T cell-directed pharmacologic intervention.

Activation of naive and memory T cells by interleukin-15

Abstract Interleukin-15 (IL-15), a product of monocytes and other cells, has biological activities similar to those of IL-2, including growth stimulation of activated T cells, induction of cytolytic effector cells, and B-cell costimulation for proliferation and lg production. We report that IL-15 at optimal concentrations rapidly induced memory (CD45RO+) CD4+ and CD8+ T cells and naive (CD45RO-) CD8+ T cells to express the CD69 activation marker followed by proliferation. By contrast, IL-15 failed to induce naive (CD45RO-) CD4+ T cells to express CD69 or to proliferate. Similar findings were obtained with IL- 2. Unlike the other T-cell subsets, CD4+ T cells with a naive phenotype expressed little or no IL-2R beta chain, a shared component of the IL-2 and IL-15 receptors required for receptor function. A monoclonal antibody to the IL-2R beta chain, Mik beta 1, reduced CD69 expression and proliferation in CD4+ memory, CD8+ memory, and CD8+ naive T cells activated by IL-15. These results confirm the biological similarities of IL-2 and IL-15. They further document that the pool of naive CD4+ cells, unlike the pool of memory CD4+, memory CD8+, and naive CD8+ cells, is not regulated directly by the T-cell growth factors IL-2 or IL-15.

Activation of naive and memory T cells by interleukin-15

Interleukin-15 (IL-15), a product of monocytes and other cells, has biological activities similar to those of IL-2, including growth stimulation of activated T cells, induction of cytolytic effector cells, and B-cell costimulation for proliferation and lg production. We report that IL-15 at optimal concentrations rapidly induced memory (CD45RO+) CD4+ and CD8+ T cells and naive (CD45RO-) CD8+ T cells to express the CD69 activation marker followed by proliferation. By contrast, IL-15 failed to induce naive (CD45RO-) CD4+ T cells to express CD69 or to proliferate. Similar findings were obtained with IL-2. Unlike the other T-cell subsets, CD4+ T cells with a naive phenotype expressed little or no IL-2R beta chain, a shared component of the IL-2 and IL-15 receptors required for receptor function. A monoclonal antibody to the IL-2R beta chain, Mik beta 1, reduced CD69 expression and proliferation in CD4+ memory, CD8+ memory, and CD8+ naive T cells activated by IL-15. These results confirm the biological similarities of IL-2 and IL-15. They further document that the pool of naive CD4+ cells, unlike the pool of memory CD4+, memory CD8+, and naive CD8+ cells, is not regulated directly by the T-cell growth factors IL-2 or IL-15.

Circulating interleukin-2 receptors are a group of multimeric proteins with immunoreactivity for interleukin-2 receptor alpha, beta, and gamma chains.

Soluble interleukin-2 receptor (sIL-2R) alpha chain serum levels as determined by enzyme-linked immunosorbant assay (ELISA) are commonly used to monitor various inflammatory and neoplastic disorders associated with lymphocytic proliferation and activation. The in vivo structure of this soluble receptor species, however, is not characterized. We investigated sera with elevated sIL-2R serum levels of patients with histologically proven cutaneous T cell lymphoma (CTCL) and high-dose IL-2-treated melanoma patients and healthy donors. Purified recombinant IL-2R alpha and beta chain molecules, produced by transfection of NIH/3T3 fibroblasts and CHO cells, served as positive controls for purification and detection procedures. For selective enrichment of IL-2R molecules from supernatants and sera, affinity columns were prepared by coupling recombinant IL-2 or monoclonal antibodies against the alpha and the beta chain of the IL-2R complex to cyanogen bromide-activated Sepharose. Western blotting with affinity-purified fractions under reducing and nonreducing conditions revealed proteins that showed immunoreactivity for IL-2R alpha, beta, and gamma chain using several detection antibodies against these molecules. We conclude that the composition of sIL-2R in vivo is more complex than that of recombinant sIL-2R and can include all three IL-2R chains.

Lack of intermediate‐affinity interleukin‐2 receptor in mice leads to dependence on interleukin‐2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependent mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor beta (IL-2R beta) and gamma (IL-2R gamma) chains, but has lost expression of IL-2 receptor alpha chain (IL-2R alpha). To investigate the properties of the mouse IL-2R beta gamma complex and the role of IL-2R alpha gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind 125I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TM beta 1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TM beta 1 to C30-1 cells expressing the IL-2 alpha beta gamma complex. Since TM beta 1 recognizes an epitope related to the IL-2 binding site of IL-2R beta, these results can be taken as a demonstration that mouse IL-2R beta gamma does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2R beta and mouse IL-2R gamma chains bind 125I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2R beta chains. Transfection of T cell clone 8.2 with human IL-2R alpha genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2R alpha chain. When grown in IL-4, the endogeneous mouse IL-2R alpha gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2R alpha gene is therefore required for the formation of the functional IL-2R in mice.

Analysis of IL-2, IL-4 and their receptors in clonally-related cell lines derived from a patient with a progressive cutaneous T-cell lymphoproliferative disorder.

Three clonally related T-cell lymphoma lines (PB-1, 2A, and 2B) were examined for expression of IL-2, IL-4, and their receptors. All three lines were derived from a single patient who had an atypical, progressive T-cell lymphoproliferative disorder involving primarily skin (Davis, T.H. et al. 1992, N. Engl. J. Med. 326:1115). The PB-1 cell line was obtained from a relatively early, clinically indolent stage of the cutaneous lymphoma, whereas the 2A and 2B lines were established from a late, aggressive stage of the lymphoma. Reverse-transcriptase PCR performed with primer pairs specific for IL-2 and IL-4 showed that no mRNA coding for these cytokines was present in any of the lines with the exception of IL-4 mRNA in the 2A line. No IL-4 protein, however, was found in any of the cell lines including 2A by immunocytochemical staining with anti-IL-4 mAb. Accordingly, no bioactive IL-4 was present in the supernatants of these lines. In contrast, all three T-cell lymphoma lines contained mRNA for IL-2R alpha, IL-2R beta, IL-4R and common gamma chain. Immunocytochemical analysis revealed that only the PB-1 line stained strongly with mAbs specific for IL-2R alpha, IL-2R beta, and IL-4R whereas the 2A and 2B lines showed only limited staining with these mAbs. In contrast to expression of IL-2R alpha and IL-4R primarily on the cell surface, IL-2R beta was localized mainly in the cell cytoplasm. Testing supernatants of the cell lines by ELISA for the presence of soluble alpha chain of the IL-2R (sIL-2R) has shown that only PB-1 secreted a large amount of sIL-2R, whereas the 2A and 2B lines secreted lesser amounts. Furthermore, the PB-1 cells expressed a relatively large number of IL-4R as determined by IL-4 binding studies using an IL-4-alkaline phosphatase fusion protein. The remaining two lines displayed only limited binding of IL-4. Addition of IL-2 and/or IL-4 to the culture medium did not modulate growth of PB-1 and the other two lines. These findings may indicate that at least some types of T-cell lymphoma evolve from cells which lose the capacity to synthesize T-cell autocrine growth factors such as IL-2 and IL-4, and show progressive loss of receptors for these cytokines.

Analysis of Interleukin-2-dependent Signal Transduction through the Shc/Grb2 Adapter Pathway: INTERLEUKIN-2-DEPENDENT MITOGENESIS DOES NOT REQUIRE Shc PHOSPHORYLATION OR RECEPTOR ASSOCIATION

The interleukin (IL)-2 receptor system has previously been shown to signal through the association and tyrosine phosphorylation of Shc. This study demonstrates that the IL-2 receptor beta (IL-2R beta) chain is the critical receptor component required to mediate this effect. The use of IL-2R beta chain deletion mutants transfected into a Ba/F3 murine cell model describes a requirement for the IL-2R beta "acid-rich" domain between amino acids 315 and 384 for Shc tyrosine phosphorylation and receptor association. COS cell co-transfection studies of IL-2R beta chain constructs containing point mutations of tyrosine to phenylalanine along with the tyrosine kinase Jak-1 and a hemagglutinin-tagged Shc revealed that the motif surrounding phosphorylated tyrosine 338 within the acid-rich domain of the IL-2R beta is a binding site for Shc. Deletion of this domain has previously been shown to abrogate the ability of IL-2 to activate Ras but does not affect IL-2-dependent mitogenesis in the presence of serum. Proliferation assays of Ba/F3 cells containing IL-2R beta chain deletion mutants in serum-free medium with or without insulin shows that deletion of the acid-rich domain does not affect IL-2-driven mitogenesis regardless of the culture conditions. This study thus defines the critical domain within the IL-2R beta chain required to mediate Shc binding and Shc tyrosine phosphorylation and further shows that Shc binding and phosphorylation are not required for IL-2-dependent mitogenesis. Neither serum nor insulin is required to supplement the loss of induction of the Shc adapter or Ras pathways, which therefore suggests a novel mechanism for mitogenic signal transduction mediated by this hematopoietin receptor.