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.     

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.     

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.     

Regulation of interleukin-12 receptor β1 chain expression and interleukin-12 binding by human peripheral blood mononuclear cells

The interleukin-12 receptor (IL-12R)β1 chain is an essential component of the functional IL-12R on both human T and natural killer cells. In this report it is shown that activation of human peripheral blood mononuclear cells (PBMC) with anti-CD3 monoclonal antibody (mAb) or phytohemagglutinin resulted in the up-regulation of IL-12Rβ1 expression and IL-12 binding. Kinetic studies revealed that maximum expression of IL-12Rβ1 and IL-12 binding occurred on days 3–4. Anti-CD3-induced expression of IL-12Rβ1 chain and IL-12 binding by PBMC was augmented by anti-CD28 mAb, indicating that the potentiating effect of anti-CD28 on T cell responses to IL-12 could be mediated, at least in part, by the enhancement of IL-12R expression. Among 16 cytokines tested, IL-2, IL-7 and IL-15 markedly induced IL-12Rβ1 expression and IL-12 binding on resting PBMC, whereas IL-1α and tumor necrosis factor-α had a minimal enhancing effect. In contrast, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-α, IFN-γ, granulocyte/macrophage colony-stimulating factor and transforming growth factor (TGF)-β2 had no detectable enhancing effect. Anti-CD3-induced expression of IL-12Rβ1 and of low-affinity IL-12 binding sites was partially inhibited by TGF-β2, IL-10 and IL-4; however, TGF-β2 and IL-10 completely abolished anti-CD3-induced expression of high-affinity IL-12 binding sites. Consistent with the reduction of high affinity IL-12 binding sites, PBMC activated with anti-CD3 mAb in the presence of TGF-β2 or IL-10 failed to produce IFN-γ or to proliferate in response to IL-12. These results suggest that Th2 cell-derived cytokines can inhibit IL-12-induced biological functions by inhibiting IL-12R expression and that expression of a second subunit of the IL-12R (IL-12Rβ2), required for the formation of high-affinity IL-12 binding sites, may be more highly regulated by TGF-β2 and IL-10 than is expression of IL-12Rβ1.     

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.     

Lineage relationships of the fetal thymocyte subset that expresses the β chain of the interleukin-2 receptor

The β chain (p75) of the interleukin-2 (IL-2) receptor (IL-2R) is expressed on up to 5-7% of fetal thymocytes on day 16 of gestation, declining thereafter to a minute proportion of less than 1% around birth, and of 1-2% of adult thymocytes. A significant part of fetal IL-2R β⁺ thymocytes are γδ cells. The precursor-progeny relationships of fetal DL-2Rβ⁺ thymocytes to the αδ T cell lineage have not been previously studied, nor has their position within the developmental sequence been determined. Here we show that IL-2Rβ is expressed on a subset of very immature cells, along with high amounts of Pgp1 and FCγRII/III, partially preceding the expression of intracellular CD3?. IL-2-Rβ disappears before expression of IL-2Rα. IL-2Rβ⁺ cells, purified by sorting on day 15 of gestation, efficiently reconstituted fetal thymic lobes depleted of lymphoid cells by treatment with desoxyguanosine. They developed into T cell receptor (TCR)αβ⁺, TCRγδ⁺, and CD4/CD8 double- and single-positive cells in similar proportions as did sorted IL-2Rα⁺ day 15 fetal thymocytes. These data suggest that IL-2Rβ expression marks a short period of very early thymocyte development, perhaps immediately after entry into the thymus.     

Identification and cellular distribution of the rat interleukin-2 receptor β chain: induction of the IL-2Rα−β+ phenotype by major histocompatibility complex class I recognition during T cell development in vivo and by T cell receptor stimulation of CD4+8+ immature thymocytes in vitro

A mouse monoclonal antibody (mAb) to the rat interleukin-2 receptor β (IL-2Rβ) chain was generated using IL-2Rβ cDNA-transfected mouse L929 cells for immunization and differential screening. This antibody, called L316, detects a cell surface protein with an apparent molecular mass of about 80 kDa. In peripheral lymphoid organs of young adult rats, IL-2Rβ expression is restricted to T and natural killer (NK) cells, and less than 10% of IL-2Rβ⁺ cells co-express the IL-2Rα chain. IL-2Rβ was detected on all NKRP-1^hi (NK⁻) and NKRP-1^lo cells (T-lineage cells of unknown function), most peripheral γδ T cells and on 30–40% of CD8 and 10% of CD4 αβ T cells. In the adult rat thymus, mAb L316 detects a small subset (about 1%) of predominantly IL-2Rα⁻ cells which express cell surface markers characteristic of mature T lymphocytes and contain a high proportion of CD4⁻8⁻ and CD4⁻8⁺ αβ T cell receptor (TCR)⁺ thymocytes. TCR-V usage suggests that major histocompatibility complex (MHC) class I plays a more important role than MHC class II in the selection of these cells. On immature CD4⁺8⁺ rat thymocytes, IL-2Rβ cell surface expression is readily induced by TCR stimulation in vitro, supporting the idea that in vivo, the IL-2Rβ⁺ phenotype is the result of TCR engagement during thymic selection.     

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.     

Selective activation of resting human γδ T lymphocytes by interleukin-2

In rheumatoid arthritis and other inflammatory diseases we and others have found that γδ T cells express activation antigens, suggesting that they are involved in the pathogenesis of these disorders. In this study we have stimulated peripheral blood mononuclear cells from normal donors with recombinant interleukin-2 (rIL-2) to see whether such a stimulus alone could activate γδ T cells. Short-term exposure (24-96 h) to rIL-2 selectively stimulated the γδ but not the αβ T cells to express activation antigens (CD69, CD25 and HLA-DR). Long-term culture (2 weeks) in rIL-2-containing medium caused a selective increase in the proportion of the γδ T cells and a corresponding reduction of the fraction of αβ T cells. Limiting dilution analysis revealed that approximately 1/60 of the γδ T cells responded to IL-2 in contrast to only 1/250 of the αβ T cells. Comparison of the expression of the IL-2 receptor (IL-2R) a and P chains showed that there was a similar expression of the α chain on γδ and αβ T cells whereas the relative density of the β chain was more than twice as high on γδ T cells. Both the IL-2-induced proliferation of γδ T cells and the expression of activation antigens on these cells could be inhibited by an anti-IL-2Rβ monoclonal antibody (mAb) but not by an anti-IL-2Rα mAb. Expression of CD69 on γδ T cells was dependent neither on the presence of B cells, monocytes, nor αβ T cells. Finally, we found that the IL-2-induced expression of CD69 was inhibited by activation of cAMP-dependent protein kinase and by inhibition of the Src-family of the tyrosine protein kinase, but not by inhibition of protein kinase C or by activation of the CD45 associated tyrosine phosphatase. The ability of γδ T cells to be activated by IL-2 is a feature which they have in common with natural killer cells. Moreover, it may be possible that the expression of activation antigens on γδ T cells in inflammatory diseases is an epiphenomenon secondary to IL-2 produced by activated αβ T cells.     

Selection of internalization-deficient cells by interleukin-2-Pseudomonas exotoxin chimeric protein: the cytoplasmic domain of the interleukin-2 receptor β chain does not contribute to internalization of interleukin-2

To study the structural basis of ligand-induced receptor-mediated internalization of interleukin-2 (IL-2), a strategy has been developed to generate variant T cells that are deficient in internalization of this cytokine. IL-2 receptor (IL-2R) α- and β-bearing EL4 cells, that express high-affinity IL-2R and internalize IL-2, were treated with low doses of IL-2-Pseudomonas exotoxin chimeric protein (IL-2-PE40). This treatment resulted in isolation of a variant (CX1) that was unable to express high-affinity IL-2R or internalize IL-2. Transfection of CX1 with the IL-2Rβ cDNA led to surface expression of IL-2Rβ and high-affinity IL-2R as well as the ability to internalize IL-2. This finding indicates that the absence of the p subunit was the sole defect in CX1 responsible for its failure to internalize IL-2. By transfecting CX1 with mutated β cDNA, several CX1 transfectants were produced that expressed a β-subunit that lacked all amino acids of the intracytoplasmic region. These transfectants expressed high-affinity IL-2R and internalized IL-2 at a rate comparable to cells expressing wild-type β-chain. These results demonstrate that internalization of IL-2 is independent of any signals contained in the intracytoplasmic tail of the β subunit and raise the possibility that such signals may be entirely contained within the γ subunit.     

Enhanced production of biologically active interleukin-1α and interleukin-1β by psoriatic epidermal cells ex vivo: evidence of increased cytosolic interleukin-1β levels and facilitated interleukin-1 release

The expression of interleukin (IL)-1 is altered in psoriatic lesions. However, little is known about the actual production of IL-1α and IL-1β by psoriatic epidermal cells (EC). We monitored IL-1 in the extracellular, the membrane and the intracellular compartment of freshly isolated EC from untreated lesional psoriatic (PP) and normal healthy (NN) skin during non-stimulated short-term cultures, representing a psoriasis model ex vivo. Cytokines were measured using bioassays combined with neutralizing antibodies and enzyme-linked immunosorbent assay in parallel. PP EC released significantly increased amounts of biologically active IL-1α and IL-1β in a ratio of 3:1, whereas NN EC only released IL-1α. Also, the release of IL-6, but not of TNF-α, by PP EC was significantly increased. Membrane-associated IL-1 activity, analyzed using glutaraldehydefixed EC, was low and not unique to PP EC. The cytosol of PP EC contained significantly increased levels of immunoreactive IL-1β. Furthermore, PP EC displayed loss of membrane integrity, as determined by trypan blue exclusion and release of cytosolic lactate dehydrogenase. This facilitated release of intracellular IL-1. Depletion of CD45⁺ cells showed that intraepidermal leukocytes did not contribute to the production of IL-1. Our observations show that resident PP EC express enhanced IL-1 production ex vivo, which is due to an increased cytosolic IL-1β content and facilitated IL-1 release. This study provides the first evidence that PP EC can produce bioactive IL-1β.     

Distinct roles of the interleukin-7 receptor α chain in fetal and adult thymocyte development revealed by analysis of interleukin-7 receptor α-deficient mice

Mouse mutants lacking expression of the IL-7 receptor (IL-7R) α chain are defective in thymopoiesis. The adult thymus has multiple defects, including reduced cell numbers and proportions of the more mature thymocyte subsets, a complete absence of CD25⁺ cells and a reduced level of RAG1 and RAG2 expression. We show here that, in contrast to the profound developmental arrest observed in the adult thymus, fetal thymocytes from IL-7Rα^−/− mice have normal proportions of all of the major thymocyte subpopulations, including CD25⁺ thymocytes and the most mature single-positive subsets. Moreover, normal levels of RAG1 and RAG2 were observed. Total thymocyte numbers, however, remained reduced. These data suggest that the IL-7Rα chain is a key regulator of both survival and proliferation during thymocyte development but that it is not essential for the production of T cells during fetal thymopoiesis.     

Endocytosis of the β chain of interleukin-2 receptor requires neither interleukin-2 nor the γ chain

Interleukin-2 (IL-2) and IL-2 receptors (IL-2R) critically regulate the magnitude and duration of T cell expansion required in an immune response. Modulation occurs at the level of receptor number and affinity. IL-2R is a multisubunit receptor which contains at least three chains, IL-2Rα (p55), IL-2Rβ (p70) and IL-2Rγ (p64). Some components of high-affinity receptors (αβγ) are continuously internalized in the absence as well as in the presence of IL-2. From studies on other receptors, it is known that endocytosis of ligand-receptor complexes is due to an intrinsic property of the receptor. However, the specific chains responsible for endocytosis of high-affinity IL-2 receptors have not been fully elucidated. IL-2Rγ has been reported to be necessary for IL-2 internalization, based on the fact that fibroblasts transfected with IL-2Rα and -β do not internalize IL-2. However, IL-2 dissociates too rapidly from IL-2Rαβ receptors to allow for its internalization. From the reported results on IL-2 internalization in transfected fibroblasts, it cannot be concluded as to the respective roles of IL-2Rβ and/or IL-2Rγ in endocytosis. As modulation of receptor number is important for biological activity, we have attempted to define the chains responsible for receptor internalization. In this work, we have studied the endocytic properties of IL-2Rβ. We demonstrate that IL-2Rβ is constitutively endocytosed in a B cell line, derived from a X-linked severe combined immunodeficiency patient, which lacks expression of IL-2Rγ. IL-2Rβ was also constitutively internalized in T and natural killer cell lines independently of IL-2Rγ. These results suggest that IL-2Rβ is endowed with endocytic capacity and carries internalization signals.     

The role of the interleukin-2 (IL-2)/IL-2 receptor pathway in MRL/lpr lymphadenopathy: The expanded CD4−8− T cell subset completely lacks functional IL-2 receptors

Autoimmune MRL/MP-lpr/lpr (MRL/lpr) mice spontaneously develop a systemic lupus erythematosus-like disease accompanied by a profound lymphadenopathy that consists of CD4^?8^?B220⁺ a P T cells. By the use of cross-linking experiments with radiolabeled interleukin-2 (IL-2), these abnormal T cells have been reported to constitutively express the IL-2 receptor β chain (IL-2Rα), a signal transducing component of IL-2R, in the absence of the a chain (IL-2Rα).To critically reevaluate the role of the IL-2/IL-2R pathway in the pathogenesis of lymphadenophathy we examined expression of the IL-2Rα and IL-2Rβ in MRL/lpr mice by ¹²⁵I-IL-2 binding analysis and also by flow cytometric analysis using monoclonal antibodies against each component of the receptor. We found that, contrary to the previous report, the CD4^?8^?B220⁺ α β T cells in lymph node (LN) of MRL/lpr mice were negative for both IL-2Rα and IL-2Rβ expression. The lpr liver CD4^?8^?B220⁺ a P T cells that had been implicated in the genesis of these abnormal LN T cells were also negative for IL-2Rβ expression. Therefore, our results indicate that the IL-2/IL-2R system plays little role, if any, in the expansion of abnormal CD4^?8^? B220⁺ α β T cells in MRL/lpr mice.