Interleukin-7 induces the association of phosphatidylinositol 3-kinase with the α chain of the interleukin-7 receptor

The recently characterized receptor for interleukin (IL)-7 (IL-7R) includes a unique α chain as well as a common γ chain shared with the receptors for IL-2 and IL-4. Engagement of the IL-7R activates the intracellular enzyme phosphatidylinositol (PtdIns) 3-kinase but the mechanism of PtdIns 3-kinase activation and the molecular basis of its interaction with IL-7R are not known. Here we show that IL-7 causes the 85-kDa regulatory subunit of PtdIns 3-kinase (p85), and PtdIns 3-kinase activity, to associate with the IL-7R. This interaction can be ascribed to ligand-induced phosphorylation of a single Tyr residue in the receptor's unique α chain. Herbimycin A, a specific protein tyrosine kinase inhibitor, suppresses not only tyrosine phosphorylation of the IL-7R but also its association with p85. A phosphopeptide corresponding to the sequence surrounding Tyr⁴⁴⁹ in the cytoplasmic tail of the IL-7R α chain, but not its non-phosphorylated analogue or phosphopeptides coincident with the sequences surrounding other α chain Tyr residues, efficiently competes out p85 binding. Replacement of Tyr⁴⁴⁹ with Phe results in a loss of p85 binding. Finally, soluble forms of the src homology 2 domains of p85, which bind directly to phosphotyrosyl peptides, specifically inhibit the association of p85 with the IL-7R. Thus, PtdIns 3-kinase recruitment occurs through a single, phosphotyrosine dependent recognition motif surrounding Tyr⁴⁴⁹ in the IL-7R α chain. This motif corresponds to a canonical sequence for p85 binding, Tyr(P)-X-X-Met. Since the closely related IL-2R and IL-4R also activate PtdIns 3-kinase but are devoid of such canonical motifs, our results suggest that the mechanism by which IL-7R recruits and activates PtdIns 3-kinase differs fundamentally from that used by the other receptors. PtdIns 3-kinase may, therefore, play a unique and important role in the biological response to IL-7.     

A transgenic T cell receptor restores thymocyte differentiation in interleukin-7 receptor α chain-deficient mice

Interleukin-7 (IL-7) receptor α chain-deficient (IL-7Rα^-/-) mice have severely depleted lymphocyte populations and thymocyte development is arrested at the double-negative (DN) stage. We show that thymocyte development in these mice can be reconstituted by the introduction of a transgenic T cell receptor (TCR), implying that one function of the IL-7Rα chain is to initiate TCR gene rearrangement. Expression of the recombinase-activating genes RAG1 and RAG2 was greatly reduced in the IL-7Rα^-/- thymuses, and in DN thymocytes from the TCR transgenic IL-7Rα^-/- mice, but was restored in double-positive thymocytes from the TCR transgenic IL-7Rα^-/- mice. These data suggest that the IL-7Rα chain controls RAG expression and initiation of TCRβ chain VDJ rearrangement in DN cells. In contrast, once cells have progressed beyond the DN stage of development the IL-7Rα chain becomes no longer essential for RAG expression.     

Differences in the interleukin-2 (IL-2) receptor system in human and mouse: α chain is required for formation of the functional mouse IL-2 receptor

Reconstitution with mouse interleukin-2 (IL-2) receptor subunits demonstrated that the mouse IL-2 receptor complex was different from the human complex in the α chain requirement for the functional mouse receptor complex. The heterotrimeric complex of the mouse exogenous α and β chains and the endogenous γ chain on mouse lymphoid BW5147 cells showed the ability to bind IL-2 with high affinity, resulting in IL-2-induced tyrosine phosphorylation of a cytosolic tyrosine kinase, JAK3, which is involved in IL-2-dependent signals. Exogenous introduction of the β chain with the endogenous γ chain, however, could neither confer appreciable IL-2 binding nor IL-2-induced signal transduction on BW5147 cells, unlike the human βγ heterodimer. Mouse spleen CD8⁺ cells, not having the α chain initially, showed IL-2-dependent cell proliferation only when expression of the α chain was induced. Collectively, these results illustrate that the functional mouse IL-2 receptor complex necessarily includes the α chain, and that the regulation of CD8⁺ T cell growth during immune reaction depends upon α chain expression.     

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

To gain an insight into roles of the interleukin-2 (IL-2) receptor γ 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 α, β and γ genes. The association rate constant (k) with the αβγ complex on L cells was fourfold larger than that with the αβ complex on L cells, and the dissociation rate constant (k') was one fifth of that with the αβ complex. These results indicate that the γ 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 α and β chains. During the course of this study, we found that the IL-2 dissociation from αβγ complex on lymphoid cells was a lot slower than that from the αβγ complex on fibroblast cells. A similar difference was observed with the βγ complex. These observations may indicate functional and constitutional differences of the high- and intermediate-affinity IL-2 receptor complexes between lymphoid and fibroblastoid cells.     

Dispensability of Jak1 tyrosine kinase for interleukin-2-induced cell growth signaling in a human T cell line

The tyrosine kinases Jak1 and Jak3 are known to be associated with the β and γ chains of interleukin-2 receptor (IL-2R). They are activated by stimulation with IL-2, IL-4, IL-7, IL-9, or IL-15, receptors of which share the γ chain of the IL-2R. We have obtained direct evidence of Jak1 association with the α chains of receptors for IL-4, IL-7 and IL-9 and with the β chain of IL-2R, which is also common to the IL-15R. Furthermore, we have prepared mutant IL-2R β chains with a mutation in the box 1 region, which is conserved among the IL-2R β chain and the α chains of the other cytokine receptors sharing the IL-2R γ chain. Using MOLT-4 transfectants with the mutant β chains, we found that two conserved proline residues within the box 1 region are essentially involved in association with Jak1. The MOLT-4 transfectants with the mutant β chains lacking Jak1 association showed IL-2 responsiveness, in terms of activation of Jak3 and Stat5 and induction of cell growth, indicating that Jak1 is dispensable for IL-2-mediated cell growth signaling and that Jak1 activation is not required for activation of Jak3 and Stat5 in the MOLT-4 transfectants.     

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 dependant 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 β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα 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 TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ 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-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα 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-2Rα gene is therefore required for the formation of the functional IL-2R in mice.     

X-SCID B cell responses to interleukin-4 and interleukin-13 are mediated by a receptor complex that includes the interleukin-4 receptor α chain (p140) but not the γ chain

This study investigates the effect of interleukin (IL)-4 mutant proteins and a monoclonal antibody to the IL-4 receptor α chain on IL-4 and IL-13 response by B cells from X-linked severe combined immunodeficiency (X-SCID) patients in which the common γ chain (γc chain) gene mutations have been fully characterized and no γc chain expression was detected. In this γc chain gene knockout model, it was confirmed that the γc chain is essential for B cell responses to IL-2 but not for IL-4 or IL-13. Dose-response curves for X-SCID and normal B cell responses to IL-4 were indistinguishable, showing that the loss of the γc chain did not diminish the sensitivity of B cells to IL-4. The mutant protein IL-4_Y124D and an antibody to the IL-4R α chain both inhibited responses of X-SCID B cells to IL-4 and IL-13, showing that X-SCID B cell responses to these cytokines are mediated by a receptor complex that includes the IL-4R α chain but not the γc chain. Another mutant protein, IL-4_R88D, which has greatly reduced affinity for IL-4Rα, was found to inhibit responses by normal B cells to IL-4 but not to IL-13. IL-4_R88D did not, however, inhibit X-SCID B cell responses to IL-4. This result is consistent with IL-4_R88D inhibition of responses mediated by receptor complexes that include the γc chain. We propose that X-SCID B cells responses to IL-4 are mediated by an IL-13 receptor complex comprised of the IL-4R α chain associated with the recently cloned IL-13R binding protein. This model has major implications for understanding normal B cell responses to IL-4.     

Expression of the mouse interleukin-2 receptor γ chain in various cell populations of the thymus and spleen

Expression of the γ chain, which is shared among functional receptor complexes for interleukin (IL)-2, IL-4 and IL-7, was examined with hematopoietic cells in mouse thymus and spleen by flow cytometry. The γ chain was expressed in cell populations from the spleen. Stimulation with concanavalin A and lipopolysaccharide caused fluctuation in expression of the γ chain in T and B cells, respectively. T lineage cells developing in the adult thymus expressed the γ chain. Fetal thymus at day 15 contained mostly immature thymocytes, which also expressed the γ chain. These results demonstrate that the γ chains is widely expressed in T lineage cells, probably indicating that the γ chain plays a role not only in the proliferation of mature hematopoietic cells but also in the development of immature cells through signal transduction as a common receptor subunit for multiple cytokines.     

Interleukin-15 up-regulates interleukin-2 receptor α chain but down-regulates its own high-affinity binding sites on human T and B cells

The cytokines interleukin (IL)-2 and IL-15 share many biological activities as a consequence of their utilization of the β and γ chains of the IL-2 receptor. However, each cytokine binds to a specific receptor α chain; IL-2 with low affinity and IL-15 with high affinity. Here, we demonstrate that IL-15, like IL-2, up-regulates expression of IL-2Rα on human T and B cells, but rapidly down-regulates IL-15 high-affinity binding sites, which represent IL-15Rα. This leads to a decreased responsiveness to IL-15 as measured by induction of Jak3 tyrosine phosphorylation. These results suggest a mechanism by which IL-15, a product of activated macrophages, may cooperate with IL-2 at the initiation of an immune response and enhance subsequent IL-2 responsiveness during T cell expansion.     

Characterization of critical residues in the cytoplasmic domain of the human interleukin-5 receptor α chain required for growth signal transduction

Interleukin (IL)-5 binds to a cell surface receptor composed of two polypeptide chains, α and β, both belonging to the hemopoietic cytokine receptor family. Mouse cells expressing common mouse β chain (AIC2B) that were transfected with human IL-5 receptor (R)α cDNA proliferated in response to picomolar concentrations of human IL-5, indicating that a functional receptor was reconstituted. We show that in these cells, human (h)IL-5 as well as mouse (m)IL-3 induce tyrosine phosphorylation of β chain and JAK 2 kinase. Phosphorylated β receptor was co-precipitated with anti-JAK 2 antibodies, suggesting that both molecules were physically associated. IL-5 and IL-3 also induce cytosolic DNA binding activity as measured by an electrophoretic mobility shift assay using the interferon-γ responsive region of human Fc γ1 gene DNA element. A deletion mutant of hIL-5Rα lacking the cytoplasmic part could bind hIL-5 normally in association with the β chain, but was unable to transmit a biological signal. The cytoplasmic domain was also indispensable for tyrosine phosphorylation and activation of DNA binding proteins. A membrane-proximal proline-rich element of the hIL-5Rα cytoplasmic domain that is conserved among different members of the hemopoietic cytokine receptor family was essential for biological activity. Point mutations in this motif also knocked out IL-5-inducible JAK 2 phosphorylation.     

IL-7 receptor is present on human microvascular endothelial cells

Interleukin-7 (IL-7) is a pleiotropic, non-redundant cytokine crucial for development of B and T lymphocytes. The cellular response to IL-7 is triggered by binding of the cytokine to its receptor, IL-7R. Until now the expression of the receptor was evidenced only in lymphoid and myeloid cell lineages. The receptor consists of two chains: IL-7 specific alpha chain (CD127) and the common gamma(c) chain (CD132) which is a component of several other cytokine receptors: IL-2, IL-4, IL-9 and IL-15. The former observation that exogenous IL-7 is biologically active towards murine endothelial cell lines from secondary lymphoid organs was the starting point of our studies. This observation has prompted us to search for the presence of IL-7 receptor in human microvascular endothelial cells. We used in our studies a set of human endothelial cell lines established from various organs. Our results demonstrate the presence of IL-7R in human microvascular endothelial cells, mainly in the mesenteric but also in the peripheral and to a lesser extent, in the mucosa-associated lymph node endothelial cells. On the basis of the RT-PCR reaction, molecular weight estimated in Western blot and IL-7 binding activity the identified endothelial IL-7 receptor was identical to the lymphocyte-type IL7-R.     

Dysregulation of interleukin-7 receptor may generate loss of cytotoxic T cell response in human immunodeficiency virus type 1 infection

Virus-specific cytotoxic T lymphocytes (CTL) play a crucial role in modulating an immune response against human immunodeficiency type 1 (HIV-1) infection. The generation of effector cytotoxic cells from CTL precursors involves intricate interactions with antigen via T cell receptors (TcR) and soluble cytokines. Interleukin (IL)-7 can affect T cell maturation and differentiation. Here we report on a group of five HIV-1-positive individuals who tested negative for env-and gag-specific CTL activity. When exogenous recombinant human IL-7 was added as a stimulus to the cultures, none (0/5) of the CTL-negative individuals exhibited a CTL response. Individuals that were negative for HIV-1-specific CTL activity were found to lack IL-7 receptor (IL-7R) on CD8⁺ cells with a comparable reduction on CD4⁺ cells. Increased shedding of IL-7R in the culture supernatant was observed. A significant reduction in receptor number was detected by binding of ¹²⁵I-labeled IL-7 and Scatchard analysis. The lack of IL-7R is probably not due to endogenous IL-7, since it was not detectable in the culture supernatants of the patients studied. HIV-1 proteins may cause down-modulation of IL-7R expression, either by producing an insufficient number of molecules or by rapid decay of IL-7R on T cells. These changes may alter the cells' capability to respond to the IL-7 growth signal, resulting in CTL failure and subsequent mishandling of the virus.