IL-2-dependent proliferation of murine T cells requires expression of both the p55 and p70 subunits of the IL-2 receptor

An IL-4-dependent T cell clone (LD8) was isolated from the murine IL-2-dependent cytotoxic T cell line C30.1. This clone has lost the capacity to proliferate in response to IL-2 after long-term culture in IL-4. LD8 cells express the p70, but not the p55, subunit of the IL-2R on their cell surface. The number of p70 IL-2R molecules on LD8 cells is comparable with the number of high-affinity IL-2R on the parental C30.1 cell line. LD8 cells can efficiently internalize IL-2 through the p70 IL-2R subunit. Following stimulation by IL-2, LD8 cells up-regulate p70 IL-2R mRNA, but do not express p55 IL-2R mRNA. IL-2-dependent proliferation of LD8 cells was reconstituted after introduction and expression of a human p55 IL-2R cDNA. To further investigate the role of p70 IL-2R, we have measured IL-2-induced proliferation of C30.1 cells in the presence of three anti-p55 IL-2R mAb (5A2, PC61, and 7D4) that recognize different epitopes. Under the experimental conditions used, the combination of anti-p55 IL-2R mAb prevents the formation of high-affinity IL-2R, but does not affect the binding of IL-2 to p70 IL-2R or IL-2 internalization. However, these three mAb inhibit proliferation of C30.1 cells even in the presence of IL-2 concentrations sufficient to saturate p70 IL-2R. Together these results demonstrate that p70 IL-2R alone is not sufficient to transmit IL-2-induced growth signals and that formation of p55-p70 IL-2R complex is required for IL-2-dependent proliferation of murine T cells.     

IL-4 is a mediator of IL-12p70 induction by human Th2 cells: reversal of polarized Th2 phenotype by dendritic cells

IL-12 is a key inducer of Th1-associated inflammatory responses, protective against intracellular infections and cancer, but also involved in autoimmune tissue destruction. We report that human Th2 cells interacting with monocyte-derived dendritic cells (DC) effectively induce bioactive IL-12p70 and revert to Th0/Th1 phenotype. In contrast, the interaction with B cells preserves polarized Th2 phenotype. The induction of IL-12p70 in Th2 cell-DC cocultures is prevented by IL-4-neutralizing mAb, indicating that IL-4 acts as a Th2 cell-specific cofactor of IL-12p70 induction. Like IFN-gamma, IL-4 strongly enhances the production of bioactive IL-12p70 heterodimer in CD40 ligand-stimulated DC and macrophages and synergizes with IFN-gamma at low concentrations of both cytokines. However, in contrast to IFN-gamma, IL-4 inhibits the CD40 ligand-induced production of inactive IL-12p40 and the production of either form of IL-12 induced by LPS, which may explain the view of IL-4 as an IL-12 inhibitor. The presently described ability of IL-4 to act as a cofactor of Th cell-mediated IL-12p70 induction may allow Th2 cells to support cell-mediated immunity in chronic inflammatory states, including cancer, autoimmunity, and atopic dermatitis.     

Interleukin-4 downregulates the p70 chain of the IL-2 receptor on peripheral blood mononuclear cells

Data that support a differential regulation of the interleukin-2 receptor (IL-2R) alpha-(p55 or Tac) and beta-chain (p70) expression by IL-4 are presented. Cytofluorometric analysis performed on peripheral blood mononuclear cells, some of which had been nylon wool passed (enriched for T-cells), in the presence or absence of phytohemagglutinin (PHA) or OKT3, demonstrated that IL-4 has a dose-dependent capacity to inhibit beta-chain IL-2R expression, whereas the alpha-chain is nearly unaffected. We could also, as a consequence of the decreased p70 expression, detect a slight increase in the amounts of IL-2 obtained from PHA-stimulated cultures, when IL-4 was present. Further, the proliferative response, especially to IL-2, but also to PHA alone, was depressed in the presence of IL-4. These data thus give further support to the idea that not only the IL-2R complex as such, but also the two individual IL-2R chains, can be independently regulated.     

IL-2 suppression of IL-12p70 by a recombinant HSV-1 expressing IL-2 induces T-cell auto-reactivity and CNS demyelination

To evaluate the role of cellular infiltrates in CNS demyelination in immunocompetent mice, we have used a model of multiple sclerosis (MS) in which different strains of mice are infected with a recombinant HSV-1 expressing IL-2. Histologic examination of the mice infected with HSV-IL-2 demonstrates that natural killer cells, dendritic cells, B cells, and CD25 (IL-2rα) do not play any role in the HSV-IL-2-induced demyelination. T cell depletion, T cell knockout and T cell adoptive transfer experiments suggest that both CD8(+) and CD4(+) T cells contribute to HSV-IL-2-induced CNS demyelination with CD8(+) T cells being the primary inducers. In the adoptive transfer studies, all of the transferred T cells irrespective of their CD25 status at the time of transfer were positive for expression of FoxP3 and depletion of FoxP3 blocked CNS demyelination by HSV-IL-2. The expression levels of IL-12p35 relative to IL-12p40 differed in BM-derived macrophages infected with HSV-IL-2 from those infected with wild-type HSV-1. HSV-IL-2-induced demyelination was blocked by injecting HSV-IL-2-infected mice with IL-12p70 DNA. This study demonstrates that suppression of the IL-12p70 function of macrophages by IL-2 causes T cells to become auto-aggressive. Interruption of this immunoregulatory axis results in demyelination of the optic nerve, the spinal cord and the brain by autoreactive T cells in the HSV-IL-2 mouse model of MS.     

The beta-chain of the IL-2 receptor (p70) is tyrosine-phosphorylated on YT and HUT-102B2 cells

IL-2 has previously been shown to rapidly induce activity of a tyrosine kinase. High-affinity IL-2 receptors that mediate the major mitogenic signals of IL-2 contain both p70 and p55 chains. p55 has no potential tyrosine phosphorylation sites and lacks consensus sequences found in protein tyrosine kinases. Inasmuch as the phosphorylation of hormone receptors is generally an important mechanism for regulating receptor function, we have now investigated the phosphorylation status of p70. By using anti-phosphotyrosine antibodies to immunoprecipitate affinity-labeled IL-2 receptors and to probe Western blots, we provide data suggesting that p70, but not p55, is constitutively tyrosine-phosphorylated on the leukemic cell lines studied.     

Detection of avian hematopoietic cell surface antigens with monoclonal antibodies to myeloid cells : Their distribution on normal and leukemic cells of various lineages

The isolation and characterization of monoclonal antibodies reacting with cell surface antigenic determinants of normal and leukemic avian hematopoietic cells is described. The antibodies were produced by immunizing mice with normal macrophages, as well as with myeloid cells transformed with the avian acute leukemia viruses MC29, AMV and E26. Eleven antibodies were characterized for their reactivity with a variety of normal and leukemic cells of the myeloid, B- and T-lymphoid and of the erythroid cell lineage. Using several methods, they could be subdivided into five distinct types: I. Four antibodies were specific for the myeloid lineage, predominantly reacting with immature myeloid cells. II. One antibody reacted with mature and immature myeloid cells as well as with T-lymphoid cells. III. Four antibodies reacted with myeloid, erythroid and T-lymphoid cells. IV. One antibody reacted with myeloid as well as with T- and B-lymphoid cells. V. One antibody reacted with all kinds of chicken hematopoietic cells except erythrocytes. The first type of antibodies detected glycoproteins with MWs of 170 and 130 kD. The pattern of antigens precipitated varied with the different monoclonal antibodies of this group. The antibody of the fourth type precipitated a 30 kD polypeptide from extracts of myeloid and lymphoid cells. None of the other antibodies precipitated any detectable proteins.     

Expression and the functional role of a p70/75 interleukin 2-binding molecule in human B cell

Expression of p70/75 IL-2-binding molecules and their functional roles in induction of Ig secretion by IL-2 were examined in human B cells. IL-2, at high concentrations induced higher levels of Ig secretion in Staphylococcus aureus strain Cowan I (SAC)-activated B cells than at low concentrations. About 50% of SAC-activated B cells, lacking Tac antigen, were also responsive to Ig secretion by IL-2, although the required dose of IL-2 was higher than that for Tac-positive B cells. H-31 antibody which recognizes Tac antigen did not inhibit the induction of Ig secretion by high concentrations of IL-2 in both Tac-negative and Tac-positive B cells, suggesting that IL-2 might induce Ig secretion through a receptor distinct from Tac antigens. In contrast, IL-2 was ineffective in the absence of SAC stimulation even at high concentrations. Upon analysis by SDS-PAGE, p70/75 IL-2-binding molecules were detected on Tac-negative SAC-activated B cells. Similar IL-2-binding molecules distinct from Tac antigen (p55) were detected in both Tac-positive B and T cells. However, neither p55 nor p70/75 IL-2-binding molecules could be detected in the absence of SAC stimulation. These observations suggest that p70/75 IL-2 binding molecules are induced in human B-cells in the presence or absence of Tac antigen by SAC stimulation and these determinants play an important function in the transduction of IL-2 associated signal for B cell differentiation.     

Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells

A glycoprotein that stimulates the proliferation of multiple hemopoietic stem and progenitor cell types was purified to apparent homogeneity. The factor, termed P cell-stimulating factor (PSF), was assayed by its ability to support the growth of murine factor-dependent hemopoietic cell lines operationally termed persisting cells (P cells). PSF was purified 50,000-fold from serum-free medium conditioned by the myelomonocytic cell line WEHI-3B by sequential ammonium sulfate precipitation, phenyl boronate chromatography, gel filtration on Sephadex G-100, neuraminidase treatment, Mono Q anion exchange chromatography, reverse phase high performance liquid chromatography on a C18 silica column, and two steps of high performance gel permeation chromatography on a TSK 3000 SW column operated under first neutral and then acidic solvent conditions. Although purified PSF could not be detected on sodium dodecyl sulfate-polyacrylamide gels stained with silver, following electrophoresis of purified PSF labeled with iodine-125, autoradiography showed only a single broad band of Mr = 30,000. This labeled band corresponded to the profile of PSF activity eluted from polyacrylamide gel slices. After reduction, labeled PSF had a slightly higher Mr of 32,000, although reduction resulted in loss of 98% of PSF activity, thus suggesting that the integrity of internal disulfide bond(s) was required for activity. When purified PSF was chromatographed on a TSK 3000 SW column under denaturing conditions in 0.1% sodium dodecyl sulfate, the single peak of absorbance at 280 nm coincided with a sharp peak of biological activity. The following unique NH2-terminal amino acid sequence of the purified PSF was obtained: NH2ALA -SER-Ile-Ser-X-X-Asp-Thr-His-Arg-Leu-Thr-Arg-. The concentration of PSF required for half-maximal stimulation of P cell growth was estimated as 1.3 X 10(-13) M or 4 pg/ml. The availability of purified PSF will allow rigorous examination of the hypothesis that a single molecule acts on multiple hemopoietic cell lineages.     

Differential expression of the IL-2 receptor subunits, p55 and p75 on various populations of primary peripheral blood mononuclear cells

Unstimulated PBL were examined for expression of IL-2R subunits, IL-2Rp55 and IL-2Rp75, by two-color flow cytometric analyses using mAb. NKH-1+ non-T non-B cells expressed IL-2Rp75 but not IL-2Rp55, and the IL-2Rp75 sites on purified NKH-1+ cells were determined to be 1630 sites/cell by binding of 125I-labeled TU27 mAb specific for IL-2Rp75. In the CD4+ T cell population, IL-2Rp55+ cells were significantly detected, but little or marginally of the IL-2Rp75+ cells. However, IL-2Rp75+ cells were significantly detected, but little of the IL-2Rp55+ cells in the CD8+ T cell population. The IL-2Rp75 sites on CD8+ T cells were estimated at approximate 180-410 sites/cell. In the CD4+ T cells, expression of IL-2Rp75 as well as IL-2Rp55 was induced by stimulation with PHA. IL-2Rp75+ cells, but not IL-2Rp55+ cells, were also detected in the CD14+ monocyte population. In the CD20+ B cell population, a small number of IL-2Rp55+ cells was detected, but little of the IL-2Rp75+ cells.     

Selection of CC chemokine receptor 5-binding peptide from a phage display peptide library

Human CC chemokine receptor (CCR) 5 is a G protein-coupled receptor involved in a broad range of human diseases that mediates HIV-1 viral entry into cells. Certain small molecule receptor antagonists to CCR5 have been useful in therapy for these diseases. In this study, CCR5-expressing CHO cells (CHO/CCR5 cells) were used to select CCR5-binding peptides from a phage-displayed 12-mers peptide library. All of the 30 clones selected from the library showed specific binding to CHO/CCR5 cells by enzyme linked immunosorbent assay (ELISA). Seventeen out of the 30 clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages and synthetic peptide AFDWTFVPSLIL blocked the binding of mAb 2D7 to CHO/CCR5 cells and competitively inhibited the ability of chemokine regulated on activation normal T cell expressed and secreted (RANTES) binding to CHO/CCR5 cells. Furthermore, the peptide AFDWTFVPSLIL also inhibited RANTES induced increase in the intracellular Ca2+ level in CHO/CCR5 cells. These results suggest that the peptide AFDWTFVPSLIL was specific for CCR5 and that it might become a CCR5 antagonist.     

CTL recognition of a novel HLA-A*0201-binding peptide derived from glioblastoma multiforme tumor cells

Genetic instability of tumor cells can result in translation of proteins that are out of frame, resulting in expression of neopeptides. These neopeptides are not self-proteins and therefore should be immunogenic. By eluting peptides from human glioblastoma multiforme (GBM) tumor cell surfaces and subjecting them to tandem mass spectrometry, we identified a novel peptide (KLWGLTPKVTPS) corresponding to a frameshift in the 3′ beta-hydroxysteroid dehydrogenase type 7 (HSD3B7) gene. HLA-binding algorithms predicted that a 9-amino acid sequence embedded in this peptide would bind to HLA-A*0201. We confirmed this prediction using an HLA-A*0201 refolding assay followed by live cell relative affinity assays, but also showed that the 12-mer binds to HLA-A*0201. Based on the 9-mer sequence, optimized peptide ligands (OPL) were designed and tested for their affinities to HLA-A*0201 and their abilities to elicit anti-peptide and CTL capable of killing GBM in vitro. Wild-type peptides as well as OPL induced anti-peptide CTL as measured by IFN-γ ELISPOTS. These CTL also killed GBM tumor cells in chromium-51 release assays. This study reports a new CTL target in GBM and further substantiates the concept that rational design and testing of multiple peptides for the same T-cell epitope elicits a broader response among different individuals than single peptide immunization.     

Failure to detect IL-3-binding sites on human mast cells

IL-3, a pleiotropic lymphokine, has been termed mast cell growth factor because it promotes growth and differentiation of murine mast cells. Murine mast cells, in turn, express cell surface receptors for IL-3. Human rIL-3 has been shown to induce proliferation and differentiation of human basophils and to activate basophils via high affinity binding sites. To investigate whether human mast cells express IL-3R, binding studies with 125I-radiolabeled human rIL-3 were performed on HMC-1, a novel human mast cell line, and on pure populations (i.e., 93 to 99% purity) of human tissue mast cells obtained with mAb and C from dispersed lung (n = 2). Unexpectedly, neither enriched human lung mast cells nor HMC-1 cells bound radiolabeled human rIL-3 specifically. Moreover, human rIL-3 failed to promote uptake of [3H]thymidine, synthesis of histamine, histamine releasability, or changes in expression of mast cell differentiation Ag (YB5B8, CD54/ICAM-1, CD9/p24, CD33/gp67) on either human lung mast cells or HMC-1 cells. It is hypothesized that the fundamental difference in the biologic response to IL-3 between human and murine mast cells is due to a loss during evolution of mast cell high affinity IL-3 binding sites.     

Inhibition of IL-2 receptor induction and IL-2 production in the human leukemic cell line Jurkat by a novel peptide inhibitor of protein kinase C

We recently reported that the myristoylated peptide N-myristoyl-Lys-Arg-Thr-Leu-Arg (N-m-KRTLR) is a novel protein kinase C inhibitor. In this study, we investigated the biological effects of N-m-KRTLR using as an in vitro model the induction of the IL-2 receptor and IL-2 secretion by Jurkat cells in response to stimulation with 12-O tetradecanoylphorbol-13-acetate (TPA) plus phytohemagglutinin (PHA) and TPA plus OKT3 mAb. N-m-KRTLR significantly suppressed induction of the IL-2 receptor on the surface of the Jurkat cells by TPA plus either PHA or OKT3 mAb. Furthermore, N-m-KRTLR inhibited the production and release of IL-2 from cultured Jurkat cells stimulated with TPA plus either PHA or OKT3 mAb. Similarly, this peptide significantly inhibited the IL-2 production in normal human peripheral blood mononuclear cells in response to stimulation by TPA and PHA. In contrast, this peptide did not affect expression of the CD3 complex on the surface of the Jurkat cells either alone or in the presence of TPA or PHA. Furthermore, N-m-KRTLR did not interfere with the spontaneous proliferation of the Jurkat cells, and its effects on IL-2 secretion and IL-2 receptor expression in the Jurkat cells were evident without loss of cell viability. These results suggest that the novel protein kinase C inhibitor N-m-KRTLR may selectively inhibit certain activation pathways of Jurkat cells and indicate the usefulness of N-m-KRTLR in the analysis of discrete events in T cell activation.     

Perlecan: a major IL-2-binding proteoglycan in murine spleen

Although interleukin-2 (IL-2) is typically considered a soluble cytokine, our laboratory has shown that the availability of IL-2 in lymphoid tissues is regulated, in part, by an association with heparan sulfate glycosaminoglycan. Heparan sulfate is usually found in proteoglycan form, in which the heparan sulfate chains are covalently linked to a specific core protein. We now show that perlecan is one of the major IL-2-binding heparan sulfate proteoglycans in murine spleen. IL-2 binds perlecan via heparan sulfate chains, as enzymatic removal of heparan sulfate from splenic perlecan abolishes its ability to bind IL-2. Furthermore, we demonstrate that perlecan-bound IL-2 supports the proliferation of an IL-2-dependent cell line. Identification of perlecan as a major heparan sulfate proteoglycan that binds IL-2 has implications for both the localization and regulation of IL-2 in vivo.     

Following direct CD40 activation, human primary microglial cells produce IL-12 p40 but not bioactive IL-12 p70

There is accumulating evidence that interleukin 12 (IL-12) is involved in the pathogenesis of multiple sclerosis. In the periphery, this cytokine is produced by antigen-presenting cells (APCs) following interaction with activated T cells. CD40 ligation plays a crucial role in this production. Microglial cells are thought to play a major role in antigen presentation in the central nervous system. In this work, we examined IL-12 production by human primary microglial cells after CD40 ligation. These cells expressed CD40 and MHC class II following interferon-gamma activation. IL-12 p40 mRNA and protein, but not bioactive IL-12 p70, were detected in response to direct CD40 activation. Microglial cells co-cultured with activated allogenic CD4+ T lymphocytes also produced IL-12 p40 but not IL-12 p70. This IL-12 p40 production was inhibited by anti-CD40 ligand. Altogether, these results suggest that CD40-CD40-ligand interaction provides a signal that triggers IL-12 p40 expression. However, other interaction(s) may be required during antigen presentation for bioactive heterodimeric IL-12 p70 to be produced by microglial cells.