Reconstitution of a functional interleukin (IL)-7 receptor demonstrates that the IL-2 receptor γ chain is required for IL-7 signal transduction

The interleukin (IL)-2 receptor γ chain has recently been shown to be a component of the IL-7 and IL-4 receptors. Using a transient transfection assay and the trans-activation of reporter gene constructs which are under the control of cytokine-responsive promoter elements, we have studied signal transduction through the IL-7 receptor (IL-7R). The reporter gene expression was not stimulated by receptors that contained the cytoplasmic domain of the IL-7R, either as intact IL-7R or as part of a chimeric receptor. However, co-expression of the IL-7R with the IL-2 receptor γ chain was able to stimulate gene activation. For maximal stimulation the intact cytoplasmic domains of each chain was required.     

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.     

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.     

Lack of association of Vβ8+ T cells with lupus-like syndrome in MRL-lpr/lpr mice

To evaluate the role of Vβ8⁺ T cells in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice, we treated them with the F23.1 anti-Vβ8 monoclonal antibody (mAb) from birth to 4 months of age. Here we report that almost complete depletion of Vβ8⁺ T cells by the F23.1 mAb treatment neither inhibited nor delayed the development of hypergammaglobulinemia, autoantibody production and autoimmune glomerulonephritis in MRL-lpr/lpr mice. In addition, the F23.1 mAb treatment did not prevent the development of lymphadenopathy and the generation of a CD4^?CD8^? double-negative T cell subset, characteristically accumulating in lpr lymph nodes. Our results strongly argue against the idea that the Vβ8⁺ T cells play a critical role in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice.     

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.     

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.     

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.     

Interleukin-2 receptor β and γ chain dysregulation during the inhibition of CD4 T cell activation by human immunodeficiency virus-1 gp120

We have observed that CD4 T lymphocytes from human immunodeficiency virus (HIV)-infected patients marginally express interleukin-2 receptor (IL-2R)β and IL-2Rγ chains which are essential for IL-2 signal transduction. To analyze this observation further, we studied the influence of gp120 on the cell surface expression of IL-2Rβ and IL-2Rγ by purified CD4 lymphocytes in vitro. Cross-linking of the T cell receptors of these lymphocytes initiates entry into the cell cycle as measured by CD69 and CD71 cell surface expression and [³H]thymidine incorporation. It also induces the cell surface expression of IL-2Rβ and IL-2Rγ. We have shown that treatment of the CD4 T lymphocytes with HIV-1 gp120 before anti-CD3 stimulation impedes cell cycle progression as measured by reduced CD71 expression and inhibition of [³H]thymidine incorporation. Furthermore, cell surface expression of IL-2Rβ and IL-2Rγ subunits, which form the functional intermediate-affinity IL-2R, are significantly inhibited. More importantly, addition of exogenous IL-2 does not restore the proliferation of the CD4 T cells treated with gp120, suggesting that cells are anergic and/or that the remaining IL-2R are not functional. This is the first study of IL-2Rβ and IL-2Rγ dysregulation in the context of HIV infection and shows that CD4 is also involved in IL-2R expression.     

Self-reactive T cell clones in a restricted population of interleukin-2 receptor β+ cells expressing intermediate levels of the T cell receptor in the liver and other immune organs

T cells expressing high levels of the T cell receptor (TCR^high) differentiate in the major intrathymic pathway and then distribute to the peripheral immune organs, whereas T cells expressing intermediate levels of the TCR (TCR^int) differentiate in both extrathymic pathways and an alternative intrathymic pathway and localize in unique sites, including the liver and thymic medulla. Since TCR^int cells constitutively express interleukin-2 receptor β-chain (IL-2R), two-color staining for CD3 (or TCR) and IL-2Rβ clearly distinguished IL-2RßT⁺ CD3^int (or TCR^int) cells from IL-2Rβ^?, CD3^high cells. CD3^int cells may be considered to be primordial T cells based on their phenotype, morphology and other functional properties. In this study, using anti-Vβ mAb in conjunction with the endogenous superantigen Mls, the distribution of self-reactive clones among T cells generated in all of the above pathways was investigated in mice. Self-reactive T cell clones were confined to IL-2Rβ⁺, CD^int cells, in all of the organs tested. A significant proportion of self-reactive clones was never identified among CD3^high cells in the thymus and peripheral immune organs in either young (8 week old) or old (50 week old) mice. Possibly reflecting their self-reactivity, CD3^int cells, but neither NK cells nor CD3^high cells had a potent cytotoxic effect against a syngeneic hepatoma in the presence of anti-CD3 mAb. These results raise the possibility that CD3^int cells seen in the liver and thymus might belong to a similar primordial lineage of T cells, and that self-reactive clones are not generated through the major intrathymic pathway, but only through extrathymic pathways and an alternative intrathymic pathway.     

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.     

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.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

Interleukin-1β converting enzyme in murine Langerhans cells and epidermal-derived dendritic cell lines

Interleukin (IL)-1β plays an essential role in the induction of T cell-mediated immune responses in skin. Langerhans cells (LC), which constitutively express IL-1β mRNA, have been assumed to be the primary source of IL-1β in murine epidermis. The purpose of this study was to determine whether LC express mRNA for the IL-1β converting enzyme (ICE), a protease that is required for processing pro-IL-1β into an active form. Here, we report that both IL-1β and ICE mRNA are expressed by the Ia⁺ population (i.e. LC) in murine epidermis. Moreover, murine epidermal-derived DC lines (XS series) also express both IL-1β and ICE mRNA, and they secrete relatively large amounts of IL-1β following lipopolysaccharide (LPS) stimulation. Finally, LPS-triggered IL-1β secretion by XS cells is blocked almost completely by the ICE inhibitor acetyl-Tyr-Val-Ala-Asp-CH₂OC(O)-[2,6-(CF₃)₂]Ph. These results demonstrate that LC are the primary source of IL-1β within the epidermis, and suggest that the proinflammatory role of IL-1β may be regulated pharmacologically by ICE inhibitors in vivo.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Interleukin-15 preferentially promotes the growth of intestinal intraepithelial lymphocytes bearing γδ T cell receptor in mice

Several cytokines including stem cell factor (SCF) and interleukin (IL)-7 are known to be required for development of γδ T cell receptor (TCR) intestinal intraepithelial lymphocytes (i-IEL) in mice. We show here the effects of IL-15 on the proliferation and maintenance of murine γδ i-IEL in vitro. γδ i-IEL constitutively expressed a high level of IL-15 receptor α mRNA and proliferated in response to IL-15 more vigorously than αβ i-IEL. Vγ/δ repertoire analysis revealed that IL-15, like IL-2, induced polyclonal expansion of γδ i-IEL, whereas γδ i-IEL responding to IL-7 showed a Vγ/δ repertoire skewed towards Vγ1/Vδ4, Vδ5. IL-15 efficiently prevented γδ i-IEL from apoptosis induced by growth factor deprivation. This rescue was accompanied by up-regulation of Bcl-2 expression. These results suggest that IL-15 plays important roles in proliferation and maintenance of γδ i-IEL.