Reconstitution of functional receptor for human interleukin-2 in mouse cells

Interleukin-2 (IL-2) has a key role in the antigen-specific clonal growth of T lymphocytes, by virtue of its interaction with a specific cell-surface receptor (IL-2R). The growth signal seems to be delivered by IL-2 bound to the high-affinity, but not the low-affinity, receptor. Genes encoding IL-2 and its receptor (that is, Tac-antigen) have been cloned and analysed in detail. We have now achieved cell-type-specific reconstitution of the high-affinity human IL-2R by expressing the complementary DNA cloned from normal lymphocytes. A mouse T-lymphocytic line, EL-4, expressed human IL-2R with high (dissociation constant (Kd) = 160-220 pM) and low (Kd = 2.1-2.2 nM) affinity for recombinant human IL-2, while mouse L929 cells expressed only a single class of the IL-2R with lower affinity (Kd = 34.5 nM) for the ligand. We also show that the human IL-2R expressed in EL-4 cells responds to IL-2 and mediates reversed signal transduction: growth of the EL-4 cells harbouring the IL-2R is inhibited specifically by human recombinant IL-2. The approach described here may provide a general experimental framework for elucidating the molecular basis of signal transduction mediated by specific receptor-ligand interaction.     

Similarities between interleukin-2 receptor number and affinity on activated B and T lymphocytes

Interleukin-2 (IL-2) is a T-cell-derived polypeptide hormone of 133 amino acids which exerts its growth-promoting activity via a surface receptor. Originally, IL-2 was believed to be a unique growth factor for activated T cells; more recent studies, however, have demonstrated that certain B-cell tumours as well as normal activated B lymphocytes express a surface molecule which is recognized by monoclonal antibodies directed against the IL-2 receptor. Furthermore, we and others have shown recently that activated B cells proliferate in response to either immunoaffinity-purified or recombinant IL-2. These controversial findings prompted us to undertake a detailed quantitative comparison of IL-2 receptor expression on activated B and T cells. We show here, using biosynthetically labelled IL-2(3H-IL-2) and anti-IL-2 receptor antibody (3H-PC61) that activated B and T cells express both high-affinity (apparent dissociation constant, Kd approximately 20 pM) and low-affinity (Kd approximately 1,000 pM) IL-2 receptors. Binding of IL-2 to both classes of receptor is inhibited by the monoclonal anti-IL-2 receptor antibody PC61. B blasts express half as many total IL-2 binding sites or PC61 binding sites as T blasts, and the ratio of the number of low- to high-affinity receptors for each cell type is approximately 10:1. Immunoprecipitation analysis of surface-labelled blasts indicates that B and T cells have IL-2 receptors of similar relative molecular mass. Taken together, these data suggest strongly that IL-2 can act as a growth hormone for both B and T lymphocytes.     

Interleukins 4 and 5 control expression of IL-2 receptor on murine B cells through independent induction of its two chains

Resting B cells express few, if any, receptors for interleukin-2 (IL-2), whereas activated B cells can express receptors for and respond to IL-2. IL-2 receptors can exist on the cell surface in three different forms; the complete high-affinity receptor, a heterodimer consisting of a chain of relative molecular mass (Mr) 70-75,000 (70-75K) and a chain of Mr 55K; the 70-75K chain alone, with intermediate affinity for IL-2; or the 55K chain alone, with low affinity for IL-2. We have previously reported that IL-5-stimulated B cells are induced to express the 55K chain. We report here evidence for the differential regulation of the expression of the two chains, namely that IL-4 and IL-5 can independently induce expression of the 70-75K and 55K chains respectively on murine B cells. As expected, cells stimulated to express the 55K chain alone are unresponsive to IL-2, whereas cells stimulated to express either the 70-75K chain or the 70-75/55K heterodimer respond to IL-2, at a high and low ligand concentration respectively, with a marked increase in proliferation. This orchestration of receptor expression and factor responsiveness may represent a novel activation pathway for B cells, where the two chains of a compound receptor are shown to be independently regulated.     

Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.     

Self-tolerance to transgenic gamma delta T cells by intrathymic inactivation

During their intrathymic differentiation, T lymphocytes expressing alpha beta T-cell receptors (TCR) are negatively and positively selected. This selection contributes to the establishment of self-tolerance and ensures that mature CD4+ and CD8+ cell populations are restricted by the self major histocompatibility complex. Little is known, however, about gamma delta T-cell development. To investigate whether selection operates in the establishment of the gamma delta T-cell class, we have generated transgenic mice using gamma- and delta-transgenes encoding a TCR that is specific for a product of a gene in the TL-region of the TLb haplotype. Similar numbers of thymocytes expressing the transgenic TCR were generated in mice of TLb and TLd haplotypes. But gamma delta thymocytes from TLb and TLd transgenic mice differed in cell size, TCR density and in their capacity to respond to TLb stimulator cells or interleukin-2 (IL-2). In contrast to gamma delta T cells from TLd transgenic mice, gamma delta T cells from TLb transgenic mice did not produce IL-2 and did not proliferate in response to TLb stimulator cells, but they did proliferate in the presence of exogenous IL-2. These results indicate that functional inactivation of self-antigen-specific T cells could contribute to the establishment of self-tolerance to thymic determinants.     

The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3

CD2 (known also as T11 (ref. 1), LFA-2 (ref. 2) and the erythrocyte rosette receptor (ref. 3] is a functionally important T lymphocyte surface glycoprotein of relative molecular mass 50,000 to 58,000 (Mr 50-58 K) which appears early in thymocyte ontogeny and is present on all mature T cells. Monoclonal antibodies to CD2 inhibit cytotoxic T-lymphocyte (CTL)-mediated killing by binding to the T lymphocyte and blocking adhesion to the target cell. Such antibodies also inhibit T helper cell responses including antigen-stimulated proliferation, interleukin-2 (IL-2) secretion, and IL-2 receptor expression. Certain combinations of monoclonal antibodies to CD2 epitopes trigger proliferation of peripheral blood T lymphocytes, cytotoxic effector function and expression of IL-2 receptors by thymocytes, resulting in thymocyte proliferation in the presence of exogenous IL-2 (ref. 11). These findings suggest that CD2 can function in signalling as well as being an adhesion molecule. To understand the role of CD2 in T-cell adhesion and activation, it is essential to define its natural ligand. Our previous observation that purified CD2 inhibits rosetting of T lymphocytes with sheep erythrocytes and can be absorbed by sheep erythrocytes suggested it also might bind with detectable affinity to human cells. We now report that CD2 binds to a cell-surface antigen known as lymphocyte function-associated antigen-3 (LFA-3) with high affinity, and can mediate adhesion of lymphoid cells via interaction with LFA-3.     

Participation of CD4 coreceptor molecules in T-cell repertoire selection.

During thymocyte development, progenitor cells bearing both CD4 and CD8 coreceptor molecules mature into functional T lymphocytes that express these proteins in a mutually exclusive way. Although T-cell specificity is determined primarily by the structure of the T-cell antigen receptor (TCR) heterodimer, a developmentally regulated process acts to ensure that cells bearing class II-restricted TCRs are CD4+ and those bearing class I-restricted TCRs express only CD8. To investigate this maturation process, we have engineered transgenic mice in which CD4 is expressed in all thymocyte subsets and in all peripheral T cells. Peripheral CD4+8+ T lymphocytes from these mice react with both class I and class II alloantigens. Moreover, expression of the CD4 transgene disrupts the positive selection of doubly transgenic thymocytes bearing a class I-restricted TCR specific for the male (H-Y) antigen. Hence the CD4 coreceptor participates directly in T-cell repertoire selection.     

Phenotypic differences between alpha beta versus beta T-cell receptor transgenic mice undergoing negative selection

T-cell differentiation in the thymus is thought to involve a progression from the CD4-CD8- phenotype through CD4+CD8+ intermediates to mature CD4+ or CD8+ cells. There is evidence that during this process T cells bearing receptors potentially reactive to 'self' are deleted by a process termed 'negative selection' One example of this process occurs in mice carrying polymorphic Mls antigens, against which a detectable proportion of T cells are autoreactive. These mice show clonal deletion of thymic and peripheral T-cell subsets that express the autoreactive V beta 3 segment of the T-cell antigen receptor, but at most a two-fold depletion of thymic cells at the CD4+CD8+ stage. By contrast, transgenic mice bearing both alpha and beta chain genes encoding autoreactive receptors recognizing other ligands, show severe depletion of CD4+CD8+ thymocytes as well, suggesting that negative selection occurs much earlier. We report here the Mls 2a/3a mediated elimination of T cells expressing a transgene encoded V beta 3-segment, in T-cell receptor alpha/beta and beta-transgenic mice. Severe depletion of CD4+CD8+ thymocytes is seen only in the alpha/beta chain transgenic mice, whereas both strains delete mature V beta 3 bearing CD4+ and CD8+ T cells efficiently. We conclude that severe CD4+CD8+ thymocyte deletion in alpha/beta transgenic mice results from the premature expression of both receptor chains, and does not reflect a difference in the timing or mechanism of negative selection for Mls antigens as against the allo- and MHC class 1-restricted antigens used in the other studies.     

A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors

Although activated human T and B lymphocytes express both high-affinity and low-affinity membrane receptors for interleukin-2 (IL-2), the structural features that distinguish these receptors have remained unresolved. The high-affinity receptors appear to mediate IL-2 induced T cell growth and internalization of IL-2, whereas no function has yet been ascribed to the low-affinity receptors. The Tac antigen is an IL-2 binding protein of relative molecular mass 55,000 (Mr 55K) that participates in the formation of both high- and low-affinity receptors. But Tac complementary DNA transfection and membrane fusion studies have suggested that additional T-cell components are required to produce high-affinity IL-2 receptors. In this study, we report the identification of a second human IL-2 binding protein that (1) has an Mr of approximately 70K, (2) lacks reactivity with the anti-Tac antibody, (3) binds IL-2 with intermediate affinity and (4) is present on the surface of resting T cells, large granular lymphocytes (natural killer cells), and certain T and B cell lines in the absence of the Tac antigen. Chemical crosslinking of 125I-labelled IL-2 bound to high-affinity IL-2 receptors produces labelling of both the p70 protein and the Tac antigen and the anti-Tac antibody blocks the crosslink detection of both of these proteins. Expression of Tac cDNA in a T cell line expressing the p70 protein, but lacking both Tac and high-affinity receptors, results in the reconstitution of high-affinity IL-2 receptors in these cells. Together, these findings suggest that the high-affinity human IL-2 receptor may be a membrane complex composed of at least the p70 protein and Tac antigen.     

Expression and functional characterization of artificial mutants of interleukin-2 receptor

The physiological proliferation of T lymphocytes (T cells) requires interaction between the humoral growth factor, interleukin 2 (IL-2) and its cell-surface receptor. Studies of IL-2 binding to the IL-2 receptor (IL-2R) on T cells have revealed that there are two distinct species of IL-2R, one with high and one with low affinity. Isolation and characterization of cDNA for the human IL-2R made it possible to deduce the complete primary sequence (251 residues) of the receptor protein. However, expression of IL-2R alone is not sufficient for either growth signal transduction or high-affinity site formation: another lymphocyte-specific molecule called converter seems to be required for the biological activity of IL-2R. We found that the converter did not form a stable complex with IL-2R unless the receptor bound the ligand (the 'affinity conversion' model). To discover which are the functionally important parts of the human IL-2R we have constructed artificial mutant cDNAs encoding the receptor. The mutant receptors produced from them had deletions or substitutions in the cytoplasmic region (13 residues), the transmembrane region (19 residues) or the carboxy-terminal portion of the extracellular region (219 residues). All were active in growth signal transduction, efficient internalization and high-affinity site formation in two mouse T-cell lines, suggesting that the extracellular region of IL-2R and the converter may be responsible for growth signal transduction.     

Absence of growth by most receptor-expressing fetal thymocytes in the presence of interleukin-2

The growth of mature T cells is regulated by receptors for interleukin-2 (IL-2) and by IL-2 itself. Binding of antigen to T-cell antigen receptors induces the expression of IL-2 receptors, and binding of IL-2 to these receptors induces transferrin receptor expression and is sufficient to promote the growth of T cells for several days. However, nothing is known about the growth requirements of pre-T cells. We have therefore studied the dividing population of T-cell precursors which carry the Thy-1 surface antigen, but lack surface antigens Ly2 and L3T4; these cells are present in 14-day-old embryonic thymus. If the thymus is removed at this stage and placed in organ culture, all lymphocyte subpopulations normally present in thymuses of adult mice develop in vitro, that is, the nonfunctional Ly2+, L3T4+ population and the functional Ly2+, L3T4- and Ly2-, L3T4+ populations. We now report that, in contrast to their progeny, the early Ly2-, L3T4- cells express large amounts of IL-2 receptors, but most of them do not grow in IL-2-containing media outside the thymus. In contrast to dividing mature T cells, most fetal thymocytes express low amounts of transferrin receptors.     

Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain

Interleukin-12 (IL-12) is a heterodimeric molecule composed of p35 and p40 subunits. Analyses in vitro have defined IL-12 as an important factor for the differentiation of naive T cells into T-helper type 1 CD4+ lymphocytes secreting interferon-gamma (refs 1, 2). Similarly, numerous studies have concluded that IL-12 is essential for T-cell-dependent immune and inflammatory responses in vivo, primarily through the use of IL-12 p40 gene-targeted mice and neutralizing antibodies against p40. The cytokine IL-23, which comprises the p40 subunit of IL-12 but a different p19 subunit, is produced predominantly by macrophages and dendritic cells, and shows activity on memory T cells. Evidence from studies of IL-23 receptor expression and IL-23 overexpression in transgenic mice suggest, however, that IL-23 may also affect macrophage function directly. Here we show, by using gene-targeted mice lacking only IL-23 and cytokine replacement studies, that the perceived central role for IL-12 in autoimmune inflammation, specifically in the brain, has been misinterpreted and that IL-23, and not IL-12, is the critical factor in this response. In addition, we show that IL-23, unlike IL-12, acts more broadly as an end-stage effector cytokine through direct actions on macrophages.     

Abnormal tyrosine phosphorylation on T-cell receptor in lymphoproliferative disorders

The study of human autoimmune diseases has benefited greatly from analysis of animal models. Mice that are homozygous for either the lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) mutant genes develop a disease characterized by massive lymphadenopathy and autoantibody formation. With age, the lymphoid organs in these mice are replaced with a greatly expanded population of abnormal lymphocytes. Recent work has shown that these cells are likely to be in the T-cell lineage. They rearrange and transcribe the genes for the alpha and beta subunits of the T-cell receptor (TCR) and a third, T-cell receptor-like gene, T gamma. As determined by immunofluorescence with anti-receptor antibodies the cells also express TCR on the cell surface. The murine T-cell receptor consists of the alpha and beta chains, derived from the rearranged alpha and beta genes, in non-covalent association with seven other chains; the delta chain, of relative molecular mass (Mr) 26,000 (26K), the epsilon chain (25K), a glycosylated 21K chain (gp21) which is probably the homologue of the gamma chain of T3 (CD3), a 16K homodimer (zeta) and a 21K dimer (p21). This multichain complex is thought to be the murine analogue of the human T3 complex. After activation of normal T cells by antigen or lectin, p21 is phosphorylated on tyrosine residues and gp21 is phosphorylated on serine residues. In contrast, in the gld and lpr cells, p21 is phosphorylated even in the absence of antigen or lectin, whereas gp21 is not phosphorylated.     

Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis

Antigen receptor stimulation of mature alpha beta T lymphocytes can lead either to proliferation or death. Programmed cell death, termed apoptosis, leads to the clonal deletion of both thymocytes and mature T cells that establishes tolerance. How a mature T cell selects between proliferation and death is not understood. Here I show that interleukin-2 (IL-2) is a critical determinant of the choice between these two fates. Both CD4+ and CD8+ T cells previously exposed to IL-2 undergo apoptosis after antigen-receptor stimulation. Antibody blockade of IL-2 but not IL-4 reverses the marked reduction of lymph node V beta 8+ T cells caused in mice by the bacterial superantigen Staphylococcus aureus enterotoxin B. IL-2 may thus participate in a feedback regulatory mechanism by predisposing mature T lymphocytes to apoptosis.     

Positive and negative selection of an antigen receptor on T cells in transgenic mice

The T-cell repertoire found in the periphery is thought to be shaped by two developmental events in the thymus that involve the antigen receptors of T lymphocytes. First, interactions between T cells and major histocompatibility complex (MHC) molecules select a T-cell repertoire skewed towards recognition of antigens in the context of self-MHC molecules. In addition, T cells that react strongly to self-MHC molecules are eliminated by a process called self-tolerance. We have recently described transgenic mice expressing the alpha beta T-cell receptor from the cytotoxic T lymphocyte 2C (ref. 11). The clone 2C was derived from a BALB.B (H-2b) anti-BALB/c (H-2d) mixed lymphocyte culture and is specific for the Ld class I MHC antigen. In transgenic H-2b mice, a large fraction of T cells in the periphery expressed the 2C T-cell receptor. These T cells were predominantly CD4-CD8+ and were able to specifically lyse target cells bearing Ld. We now report that in the periphery of transgenic mice expressing Ld, functional T cells bearing the 2C T-cell receptor were deleted. This elimination of autoreactive T cells appears to take place at or before the CD4+CD8+ stage in thymocyte development. In addition, we report that in H-2s mice, a non-autoreactive target haplotype, large numbers of CD8+ T cells bearing the 2C T-cell receptor were not found, providing strong evidence for the positive selection of the 2C T-cell receptor specificity by H-2b molecules.     

Peptide sequences of T-cell receptor delta and gamma chains are identical to predicted X and gamma proteins

Although most mature peripheral T lymphocytes express a major histocompatibility complex restricted, CD3-associated, antigen receptor (TCR) which has been well characterized, some T cells carry a different CD3-associated heterodimer on their surface. One of the two disulphide-linked chains of this putative second receptor, which in mice has relative molecular mass (Mr) 35,000 (35K), has been identified as a product of the group of gamma genes. The other chain, termed delta (Mr 45K in mice), is not as well characterized. Although gamma/delta-bearing cells are a minor subset among peripheral T lymphocytes, they are the only CD3+ cells in the thymus early in ontogeny. Taking advantage of these kinetics, we have generated gamma/delta-bearing hybridomas, using a new TCR alpha chain-negative variant of the AKR thymoma BW5147 as tumour parent, fetal thymocytes as normal cell partners, and an anti-CD3 monoclonal antibody (mAb) as screening reagent. Gamma and delta chains from one of these hybrids have been purified and partially sequenced. The sequences obtained indicate that delta is indeed identical to the polypeptide encoded by the recently described gene X, as suggested by Chien et al.     

Molecular cloning of cDNA encoding human interleukin-2 receptor

The human interleukin-2 (IL-2) receptor was purified by affinity chromatography using the anti-Tac monoclonal antibody, and its N-terminal amino acid sequence was determined. Complementary DNA clones were isolated and sequenced to reveal the primary structure of the IL-2 receptor precursor, which has 272 amino acid residues. The receptor is separated into two domains by a putative 19-residue transmembrane region. Two mRNAs (1.4 and 3.5 kilobases) hybridizing to the cDNA clone were found in human T cells bearing the IL-2 receptor. The cDNA directed synthesis of the IL-2 receptor in COS cells.     

Expression of functional human interleukin-2 receptor in mouse T cells by cDNA transfection

Interleukin-2 (IL-2) in combination with the IL-2 receptor has an essential role in antigen-stimulated proliferation of T lymphocytes. It has been proposed that the constitutive expression of the IL-2 receptor on adult T-cell leukaemia (ATL) cells may be associated with transformation of T cells. Although we and others have isolated complementary DNA clones encoding a protein that binds IL-2, formal proof that this protein is the IL-2 receptor requires demonstration of IL-2-dependent growth stimulation of cells expressing the protein. In addition, a functional assay system other than binding of IL-2 is required to investigate the molecular mechanism of signal transmission through the IL-2 receptor using artificially mutated cDNA. The IL-2 receptor expressed in non-lymphoid cells by cDNA transfection did not mediate a growth signal, implying that lymphoid cells expressing the functional receptor might have specific accessory molecule(s) for signal transmission by the receptor. Therefore, we established a line of IL-2-dependent mouse cells (CT/hR) expressing both murine (endogenous) and human IL-2 receptors. Here, by blocking the endogenous mouse IL-2 receptors with monoclonal antibodies, we show that the human IL-2 receptor of CT/hR cells is functionally active. Although CT/hR expressed the human IL-2 receptor constitutively, growth of these cells was strictly dependent on IL-2, indicating that uncontrolled over-expression of the IL-2 receptor was not by itself sufficient for T-cell transformation.     

Transient expression of IL-2 receptor precedes the differentiation of immature thymocytes

The growth of mature T lymphocytes after activation by antigen is regulated by the binding and endocytosis of interleukin-2 (IL-2). In the thymus, approximately 50% of adult thymocytes that carry neither the CD4 nor the CD8 antigen and day 14-15 fetal CD4-8- thymocytes express receptors for IL-2(IL-2R). The CD4-8- (double-negative) subpopulation of thymocytes contains the precursors of cells that can differentiate along an unknown pathway into thymocytes bearing either CD8 or CD4, with the characteristics of mature T lymphocytes. The basis for IL-2R expression by double-negative thymocytes is unclear as they appear to lack a functional T-cell receptor/CD3 complex through which activation of peripheral T cells is mediated. The argument for a role for IL-2 in thymocyte differentiation has also been complicated by conflicting reports on the inability or capability of double-negative thymocytes to respond to IL-2 in vitro. At present, both the nature of the stimuli within the thymic micro-environment which induce IL-2R expression and its relevance to thymocyte differentiation are not known. We show here that the IL-2R-bearing subset has a greater potential to differentiate into phenotypically mature T lymphocytes than do IL-2R-negative thymocytes. In addition, progeny of IL-2R-negative donor cells transiently express IL-2R in the thymuses of adoptive hosts before generating CD8 and/or CD4-positive thymocytes. These results identify the IL-2R-positive cells as a more differentiated double-negative thymocyte subset on the pathway to mature T lymphocytes.