Proliferation Response to Interleukin-2 and Jak/Stat Activation of T Cells Immortalized by Human T-Cell Lymphotropic Virus Type 1 Is Independent of Open Reading Frame I Expression

Human T-cell lymphotropic virus type 1 (HTLV-1), a complex retrovirus, encodes a hydrophobic 12-kD protein from pX open reading frame (ORF) I that localizes to cellular endomembranes and contains four minimal SH3 binding motifs (PXXP). We have demonstrated the importance of ORF I expression in the establishment of infection and hypothesize that p12(I) has a role in T-cell activation. In this study, we tested interleukin-2 (IL-2) receptor expression, IL-2-mediated proliferation, and Jak/Stat activation in T-cell lines immortalized with either wild-type or ORF I mutant clones of HTLV-1. All cell lines exhibited typical patterns of T-cell markers and maintained mutation fidelity. No significant differences between cell lines were observed in IL-2 receptor chain (alpha, beta, or gamma(c)) expression, in IL-2-mediated proliferation, or in IL-2-induced phosphorylated forms of Stat3, Stat5, Jak1, or Jak3. The expression of ORF I is more likely to play a role in early HTLV-1 infection, such as in the activation of quiescent T cells in vivo.     

A membrane-proximal region of the interleukin-2 receptor gamma c chain sufficient for Jak kinase activation and induction of proliferation in T cells.

The interleukin-2 (IL-2) receptor (IL-2R) consists of three distinct subunits (alpha, beta, and gamma c) and regulates proliferation of T lymphocytes. Intracellular signalling results from ligand-mediated heterodimerization of the cytoplasmic domains of the beta and gamma c chains. To identify the residues of gamma c critical to this process, mutations were introduced into the cytoplasmic domain, and the effects on signalling were analyzed in the IL-2-dependent T-cell line CTLL2 and T-helper clone D10, using chimeric IL-2R chains that bind and are activated by granulocyte-macrophage colony-stimulating factor. Whereas previous studies of fibroblasts and transformed T cells have suggested that signalling by gamma c requires both membrane-proximal and C-terminal subdomains, our results for IL-2-dependent T cells demonstrate that the membrane-proximal 52 amino acids are sufficient to mediate a normal proliferative response, including induction of the proto-oncogenes c-myc and c-fos. Although gamma c is phosphorylated on tyrosine upon receptor activation and could potentially interact with downstream molecules containing SH2 domains, cytoplasmic tyrosine residues were dispensable for mitogenic signalling. However, deletion of a membrane-proximal region conserved among other cytokine receptors (cytoplasmic residues 5 to 37) or an adjacent region unique to gamma c (residues 40 to 52) abrogated functional interaction of the receptor chain with the tyrosine kinase Jak3. This correlated with a loss of all signalling events analyzed, including phosphorylation of the IL-2R beta-associated kinase Jak1, expression of c-myc and c-fos, and induction of the proliferative response. Thus, it appears in T cells that Jak3 is a critical mediator of mitogenic signaling by the gamma c chain.     

Identification of a novel receptor/signal transduction pathway for IL-15/T in mast cells.

Interleukin-15/T(IL-15) is a growth factor that utilizes IL-2 receptor (IL-2R) components in addition to its private binding protein IL-15R(alpha) in T-cells. Here, we report that IL-15 induces mast cell proliferation in the absence of IL-2R alpha and beta. Using transfectants of these cells with a cytoplasmic-truncated mutant of gamma(c), we demonstrated that IL-15 signaling in mast cells does not involve gamma(c). Cross-linking of mast cells with [(125)I]IL-15 revealed a 60-65 kDa IL-15 binding protein that is distinct from known components of T-cell IL-15 receptors. Mast cell IL-15 receptors recruit JAK-2 and STAT-5, instead of JAK1/3 and STAT3/5 that are activated in T-cells. Thus IL-15 is a mast cell growth factor that utilizes a novel receptor and distinct signaling pathway.     

Three residues in the common beta chain of the human GM-CSF, IL-3 and IL-5 receptors are essential for GM-CSF and IL-5 but not IL-3 high affinity binding and interact with Glu21 of GM-CSF.

The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is essential for high affinity ligand-binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM-CSF, IL-3 and IL-5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL-2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site-directed mutagenesis and binding assays with radiolabelled GM-CSF, IL-3 and IL-5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365-Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM-CSF and IL-5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL-3 binding to high affinity by beta c. To identify likely contact points in GM-CSF involved in binding to the 365-368 beta c region we used the GM-CSF mutant eco E21R which is unable to interact with wild-type beta c whilst retaining full GM-CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild-type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM-CSF and IL-5 can be dissociated from IL-3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM-CSF.     

Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor.

Interleukin-15 (IL-15) is a novel cytokine of the four-helix bundle family which shares many biological activities with IL-2, probably due to its interaction with the IL-2 receptor beta and gamma (IL-2R beta and gamma c) chains. We report here the characterization and molecular cloning of a distinct murine IL-15R alpha chain. IL-15R alpha alone displays an affinity of binding for IL-15 equivalent to that of the heterotrimeric IL-2R for IL-2. A biologically functional heteromeric IL-15 receptor complex capable of mediating IL-15 responses was generated through reconstruction experiments in a murine myeloid cell line. IL-15R alpha is structurally similar to IL-2R alpha; together they define a new cytokine receptor family. The distribution of IL-15 and IL-15R alpha mRNA suggests that IL-15 may have biological activities distinct from IL-2.     

A Lysine-to-Arginine Change Found in Natural Alleles of the Human T-Cell Lymphotropic/Leukemia Virus Type 1 p12I Protein Greatly Influences Its Stability

The HTLV-1 singly spliced open reading frame I protein, p12(I), is highly unstable and appears to be necessary for persistent infection in rabbits. Here we demonstrate that p12(I) forms dimers through two putative leucine zipper domains and that its stability is augmented by specific proteasome inhibitors. p12(I) is ubiquitylated, and mutations of its unique carboxy-terminus lysine residue to an arginine greatly enhance its stability. Interestingly, analysis of 53 independent HTLV-1 strains revealed that the natural p12(I) alleles found in ex vivo samples of tropical spastic paraparesis-HTLV-1-associated myelopathy patients contain a Lys at position 88 in some cases, whereas arginine is consistently found at position 88 in HTLV-1 strains from all adult T-cell leukemia-lymphoma (ATLL) cases and healthy carriers studied. This apparent segregation of different alleles in tropical spastic paraparesis-HTLV-associated myelopathy and ATLL or healthy carriers may be relevant in vivo, since p12(I) binds the interleukin-2 receptor beta and gammac chains, raising the possibility that the two natural alleles might affect differently the regulation of these molecules.     

Compensatory energetic mechanisms mediating the assembly of signaling complexes between interleukin-2 and its alpha, beta, and gamma(c) receptors

Interleukin-2 is a key immuno-regulatory cytokine whose actions are mediated by three different cell surface receptors: the alpha, beta and the "common gamma" (gamma(c)) chains. We have undertaken a complete thermodynamic characterization of the stepwise assembly cycle for multiple possible combinations of the receptor-ligand, and receptor-receptor interactions that are necessary for formation of the high-affinity IL-2/alphabetagamma(c) signaling complex. We find an entropically favorable high affinity interaction between IL-2 and its alpha receptor, a moderately entropically favorable low affinity interaction between IL-2 and its beta receptor, and no interaction between IL-2 and the shared receptor, gamma(c). Formation of the stable intermediate trimolecular complexes of IL-2 with alpha and beta receptors, as well as IL-2 with beta and gamma(c) receptors proceeds through enthalpy-entropy compensation mechanisms. Surprisingly, we see a moderate affinity interaction between the unliganded receptor alpha and beta chains, suggesting that a preformed alphabeta complex may serve as the initial interaction complex for IL-2. Reconstitution of the IL-2/Ralphabetagamma(c) high-affinity quaternary signaling complex shows it to be assembled through cooperative energetics to form a 1:1:1:1 assembly. Collectively, the favorable entropy of the bimolecular interactions appears to be offset by the loss in rigid body entropy of the receptor components in the higher-order complexes, but overcome by the formation of increasingly enthalpically favorable composite interfaces. This enthalpic mechanism utilized by gamma(c) contrasts with the favorable entropic mechanism utilized by gp130 for degenerate cytokine interaction. In conclusion, we find that several energetically redundant pathways exist for formation of IL-2 receptor signaling complexes, suggesting a more complex equilibrium on the cell surface than has been previously appreciated.     

Time course and cytokine dependence of human T-cell lymphotropic virus type 1 T-lymphocyte transformation as revealed by a microtiter infectivity assay.

Human T-cell lymphotropic virus type 1 (HTLV-1) enhances the growth of T lymphocytes, allowing the generation of T-lymphocyte cell lines. This report describes a limiting-dilution assay system which uses low input numbers of HTLV-1-producing cells for generation of T-lymphocyte cultures. The HTLV-1 transformants generated with this assay system produced high levels of HTLV-1 p24 antigen and required exogenous cytokines for maintenance. Clonal populations of CD4- or CD8-positive HTLV-1 transformants were generated with transformation efficiency rates as high as 78%. An exogenous cytokine is necessary for HTLV-1 T-lymphocyte transformation, and cytokine dependence is the most likely outcome of infection and transformation. HTLV-1 T-lymphocyte transformation can occur in the presence of cytokines other than interleukin-2 (IL-2), such as IL-4 or IL-7. IL-4- or IL-7-dependent HTLV-1 transformants underwent T-lymphocyte mitogenesis in response to their homologous cytokines but proliferated best in the presence of IL-2. Since the receptors for IL-2, IL-4, and IL-7 share the IL-2 gamma chain, this component may be the common element in the signaling pathway for HTLV-1-associated transformation.     

Activation of the interleukin 2 receptor: a possible role for tyrosine phosphatases

Engagement of interleukin-2 (IL-2) mediates the heterodimeridation of the common beta chain (beta(c)) and common gamma chain (gamma(c)) of the IL-2 receptor (IL-2R). This is sufficient and necessary for receptor activation and signal transduction. It is generally held that the IL-2R is activated by the trans-activity of the protein tyrosine kinases (PTKs) Jak1 and Jak3 associated with beta(c) and gamma(c) respectively. Transduction of proliferative signals requires Jak3 activity. A Jak3 independent signalling pathway involving p56(lck), generating anti-apoptotic signals, can be observed and requires the PROX domain of gamma(c). p56(lck) can be activated by dephosphorylation of an inhibitory carboxyl terminal phosphorylated tyrosine residue (Y505). We propose that this is mediated by a PROX domain associated protein tyrosine phosphatase (PTP). Activation of p56(lck) alone is insufficient for transduction of proliferative signals and thus works in concert with Jak3 mediated receptor activation. This indicates that both gamma(c) domains are vital for signal transduction.     

Mendelian predisposition to mycobacterial infections in humans

Selective susceptibility to poorly pathogenic mycobacteria, such as bacille Calmette-Guérin (BCG) vaccine and environmental non-tuberculous mycobacteria (NTM), bas long been suspected to be a mendelian disorder but its molecular basis has remained elusive. Recently, recessive mutations in the interferon gamma receptor ligand-binding chain (IFN gamma R1), interferon gamma receptor signalling chain (IFN gamma R2), interleukin 12 p40 subunit (IL-12 p40), and interleukin 12 receptor beta 1 chain (IL-12R beta 1) genes have been identified in a number of patients with disseminated BCG or NTM infection. Although genetically distinct, these conditions are immunologically related and highlight the essential role of interferon gamma-mediated immunity in the control of mycobacteria in man.     

Human T-cell leukemia virus type 1 (HTLV-1) p12I down-modulates ICAM-1 and -2 and reduces adherence of natural killer cells, thereby protecting HTLV-1-infected primary CD4+ T cells from autologous natural killer cell-mediated cytotoxicity despite the reduction of major histocompatibility complex class I molecules on infected cells

Although natural killer (NK) cell-mediated control of viral infections is well documented, very little is known about the ability of NK cells to restrain human T-cell leukemia virus type 1 (HTLV-1) infection. In the current study we show that NK cells are unable to kill HTLV-1-infected primary CD4+ T cells. Exposure of NK cells to interleukin-2 (IL-2) resulted in only a marginal increase in their ability to kill HTLV-1-infected primary CD4+ T cells. This inability of NK cells to kill HTLV-1-infected CD4+ T cells occurred despite the down-modulation of major histocompatibility complex (MHC) class I molecules, one of the ligands for the major NK cell inhibitory receptor, by HTLV-1 p12(I) on CD4+ T cells. One reason for this diminished ability of NK cells to kill HTLV-1-infected cells was the decreased ability of NK cells to adhere to HTLV-1-infected cells because of HTLV-1 p12(I)-mediated down-modulation of intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. We also found that HTLV-1-infected CD4+ T cells did not express ligands for NK cell activating receptors, NCR and NKG2D, although they did express ligands for NK cell coactivating receptors, NTB-A and 2B4. Thus, despite HTLV-1-mediated down-modulation of MHC-I molecules, HTLV-1-infected primary CD4+ T cells avoids NK cell destruction by modulating ICAM expression and shunning the expression of ligands for activating receptors.     

IL-2-dependent in vivo and in vitro tyrosine phosphorylation of IL-2 receptor gamma chain.

We previously reported a molecule, p64, which was tentatively named the gamma chain, coprecipitable with the beta chain of human interleukin-2 receptor (IL-2R). The present study demonstrated that the gamma chain, as well as the beta chain expressed on IL-2-responsive cells, is phosphorylated on tyrosine residues in an IL-2-dependent manner in vivo and in vitro. The in vivo tyrosine phosphorylation of both chains was similarly induced within 1 min after IL-2 stimulation, and their in vitro tyrosine phosphorylation with the anti-IL-2R beta antibody-directed immunocomplex was also increased by treatment of cells with IL-2. These results suggest that a tyrosine kinase is associated with the beta gamma subunit complex, of which activation by IL-2 may result in transduction of intracellular signals.     

Free major histocompatibility complex class I heavy chain is preferentially targeted for degradation by human T-cell leukemia/lymphotropic virus type 1 p12(I) protein

Human T-cell leukemia virus type 1 (HTLV-1) establishes a persistent infection in the host despite a vigorous virus-specific immune response. Here we demonstrate that an HTLV-1-encoded protein, p12(I), resides in the endoplasmic reticulum (ER) and Golgi and physically binds to the free human major histocompatibility complex class I heavy chains (MHC-I-Hc) encoded by the HLA-A2, -B7, and -Cw4 alleles. As a result of this interaction, the newly synthesized MHC-I-Hc fails to associate with beta(2)-microglobulin and is retrotranslocated to the cytosol, where it is degraded by the proteasome complex. Targeting of the free MHC-I-Hc, and not the MHC-I-Hc-beta(2)-microglobulin complex, by p12(I) represents a novel mechanism of viral interference and disrupts the intracellular trafficking of MHC-I, which results in a significant decrease in surface levels of MHC-I on human T-cells. These findings suggest that the interaction of p12(I) with MHC-1-Hc may interfere with antigen presentation in vivo and facilitate escape of HTLV-1-infected cells from immune recognition.     

Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.