Genomic structure and chromosomal localization of the human interleukin 15 gene (IL-15).

Interleukin 15 (IL-15) is a novel cytokine whose biological activities are similar to those of interleukin 2 (IL-2). The genomic sequence of human IL-15 has been isolated based on its sequence homology with a cDNA clone encoding human IL-15. The human sequence is 14968 bp in length and includes all six protein-coding exons and five introns. The location of the introns in the human sequence is identical to their positions in the murine IL-15 gene. The same is true for the overall size of the gene, which was estimated to be at least 32 kb. Using Polymerase Chain Reaction (PCR) with gene-specific primers on a panel of human/rodent hybrid cell DNAs, as well as by fluorescence in situ hybridization the human IL-15 gene was mapped to chromosome 4 region q25-35.

Temporary anti-CD25/CsA therapy induces a CD4+ T-cell-mediated tolerance in BB/OK rats.

Pancreatic islets obtained from the congenic LEW.1BB/OK rat strain (MHC identical, different in genetic background from BB/OK rats) were grated into diabetic BB/OK rats. The recipients were treated for 10 d with 1.0 mg/kg bw anti-IL2 receptor monoclonal antibody (ART-18) in combination with 1.5 mg/kg bw cyclosporin A, which resulted in indefinite graft survival in the majority of animals. The successfully treated recipients (normoglycaemia for > 120 days) relapsed immediately into hyperglycaemia after graft removal. A second donor-identical graft was accepted without any further immunotherapy, whereas MHC-different islet grafts (obtained from LEW.1A or DA rats) were rejected, demonstrating the induction of donor-specific tolerance. Splenocytes or thoracic duct lymphocytes (TDL) obtained from successfully treated recipients were transfused into naive diabetic BB/OK rats grafted with LEW.1BB/OK islets. Independent of the origin of the transfused cells, 64% of recipients maintained normoglycaemia for more than 120 days. To characterize the cell population(s) responsible for transfer of tolerance, B-lymphocytes were removed from the TDLs using the monoclonal antibody OX33 and magnetic beads. When OX33-depleted TDLs were transfused into naive diabetic BB/OK with a LEW.1BB/OK islet graft, all recipients maintained normoglycaemia. The OX33-depleted TDLs consisted only of CD4+ T-lymphocytes, which were either negative for CD45RC or coexpressed the CD45RC at low levels. We conclude that the cell-dependent tolerance transfer is mediated by a TH2-like suppressor cell.

Effects of depletion of cells bearing the interleukin-2 receptor on immunoglobulin production and allergic airway responses in the rat.

Lymphocytes, key cells in chronic inflammation, are increased in the airways of asthmatics and have increased expression of the interleukin-2 (IL-2) receptor, a sign of activation. We determined the effects of depleting cells bearing IL-2 receptors on immunoglobulin (Ig) production, airway inflammation, and airway responses after antigen challenge of Brown Norway rats that were sensitized to ovalbumin (OA). Both control and ART-18 (antirat IL-2 receptor) antibodies inhibited plasma specific IgE and the early (ER) and late (LR) airway responses to antigen when given from zero to 14 d after sensitization. When ART-18 was administered from 4 to 14 d after sensitization and compared with control animals, it inhibited OA specific IgE production from Day 21 onward, but it increased total IgE and specific IgG. These changes followed a significant increase in blood CD4+ lymphocytes (%) in ART-18-treated animals 14 d after sensitization. The same protocol of administration did not affect Ig levels at 14 d, but it decreased neutrophil influx into the lungs 8 h after antigen challenge without any effects on the ER and LR. Administration of ART-18 at the time of antigen challenge did not affect the subsequent airway inflammation or the increased responsiveness to methacholine that occurs 32 h after antigen challenge. In summary, depletion of IL-2-receptor-bearing cells affects lymphocyte subsets and immunoglobulin production and it decreases the influx of neutrophils into the lungs 8 h after OA challenge, but it does not significantly inhibit the ER, LR, or increased airway responsiveness after antigen challenge.

Suppression of cell-mediated and humoral immune responses by an interleukin-2-immunoglobulin fusion protein in mice.

Interleukin-2 (IL-2) plays a pivotal role in the cellular and humoral immune responses directed against foreign antigens. We characterized the in vitro and in vivo properties of a chimeric protein consisting of mouse IL-2 fused to the mouse IgG2b Fc domains. This fusion protein binds to IL-2 and Fc receptors and supports IL-2-dependent cell proliferation but does not mediate lysis of IL-2 receptor-positive cells in the presence of murine complement in vitro. However, in vivo the IL2-IgG2b fusion protein suppresses both cellular and humoral immune responses after immunization with sheep erythrocytes. Surprisingly, delayed hypersensitivity is inhibited despite a dramatic increase of splenic CD3+ and NK1.1+ lymphocytes, indicating that altered homing of IL2-IgG2b-activated lymphocytes rather than cytolysis prevents these cells from accumulating in areas of inflammation. Although in vitro the IL2-IgG2b fusion protein does not alter proliferation of B cells in response to mitogenic stimulation, IgM production in response to sheep erythrocytes is profoundly inhibited in mice treated with the IL2-IgG2b fusion protein. Since no side effects are observed, the IL2-IgG2b fusion protein may expand the therapeutic repertoire of reagents used for the treatment of allograft rejection and autoimmune diseases.

The rat interleukin 4 receptor: coevolution of ligand and receptor.

A rat interleukin 4 receptor (IL-4R) cDNA was cloned by polymerase chain reaction (PCR) using RNA of Con A activated T cells and primers deduced from mouse and human IL-4R sequences. Sequence analysis revealed an open reading frame for a putative membrane protein of 800 amino acids in length. It comprises an overall identity of 52 and 78% to its human and mouse homologues, respectively. The extracellular part of the rat IL-4R contains a number of residues including cysteines and a WSXWS motif typical for the cytokine receptor superfamily. Analysis of amino acid exchanges between rat and mouse IL-4 receptors deciphered for replacement (R) or silent (S) mutations suggested different types of selective pressure acting on the extracellular and intracellular domains. A high R/S value that indicates selective pressure for amino acid exchanges was found for the extracellular domain and a low R/S value for the intracellular part of the IL-4R. Since we previously found a similar high R/S value in the rat IL-4 gene encoding the ligand for the IL-4R, the high amino acid exchange rate can best be explained by coevolution between IL-4 and the ligand binding domain of the IL-4R to improve or retain affinity.

Production of cytokines by human melanoma cells and melanocytes.

Experimental animal models have shown that various cytokines, depending of their specific properties, may support growth and metastasis of tumor cells or even lead to tumor rejection. The analysis of expression of cytokine genes by melanoma cell lines indicated that melanoma cells constitutively produce both autostimulatory and inhibitory cytokines. Using reverse transcriptase polymerase chain reaction analysis, simultaneous expression of several cytokines, including interleukin-1 beta (IL-1 beta), IL-6, IL-8, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor, by melanoma cells was found. The same cytokine transcripts were detected in melanocytes, suggesting that cells of the melanocytic lineage express a specific pattern of cytokines in vitro. All these cytokines are known to be able to stimulate effector cells of the host. Additionally, production of mRNA for IL-10, a cytokine with potential immunosuppressive properties, was detected in melanoma cells and melanocytes. These and other cytokines are likely to be involved in the immune response to cancer and at this time it is unknown what the net effects of multiple cytokines are on the outcome of the host response to tumor.

Expression of interleukin 10 in human melanoma.

The expression of interleukin 10 (IL-10) mRNA in human malignant melanoma was investigated by reverse transcriptase polymerase chain reaction analysis. Selective expression of IL-10 mRNA in tissues of primary melanomas and melanoma metastases was found in comparison with normal skin. In addition, strong expression of IL-10 mRNA and of biologically active IL-10 was detected in 3 out of 13 melanoma cell lines. Normal melanocytes consistently expressed low levels of IL-10 mRNA but did not produce detectable IL-10 protein, nor did keratinocytes or fibroblasts. The production of biologically active IL-10 by melanoma cell lines suggests that IL-10 mRNA in melanoma lesions may derive at least in part from the tumour cells themselves. Tumour-infiltrating cells, however, could also be a source of IL-10 in melanoma tissues. The presence of IL-10 in melanoma lesions may contribute to the postulated 'paralysis' of an anti-melanoma immune response.

Mechanisms of dichotomous action of IL-2-Pseudomonas exotoxin 40 (IL-2-PE40) on cell-mediated and humoral immune response.

IL-2-PE40 is a chimeric protein composed of human IL-2 genetically fused to the amino terminus of a modified form of Pseudomonas exotoxin lacking its cell recognition domain. The immunosuppressive efficacy of IL-2-PE40 was demonstrated in several experimental murine transplant and autoimmune models. However, some observations suggested that IL-2-PE40 could not inhibit the humoral response. In this report, we describe the dichotomous effects of IL-2-PE40 on humoral and cell-mediated immune response in a simple, well characterized in vivo model. Although IL-2-PE40 inhibited the cell-mediated delayed type hypersensitivity reaction to SRBC, it increased the humoral immune response to the same Ag. To understand the mechanism of dichotomous action of IL-2-PE40 on the immune response, IL-2R-bearing T cells were treated with IL-2-PE40 in vitro and the cytokine expression was studied at mRNA and protein level. Similar to IL-2, IL-2-PE40 promoted the expression of T helper 1-like (IFN-gamma) as well as T helper 2-like (IL-4, IL-10) cytokines. These in vitro studies show that IL-2-PE40 can induce signal transduction in activated T cells through the IL-2R before exerting its cytotoxic effect. In contrast to DTH reaction, humoral immune response requires T cell help only for a limited period. Therefore, the short-term stimulation of T helper cells by IL-2-PE40 may be sufficient in vivo to mediate a B cell response in the local environment, whereas the DTH reaction and other cell-mediated immune responses are inhibited by the toxin moiety of the chimeric protein.

Gene transfer of the interleukin (IL)-2 receptor beta chain into an IL-7-dependent pre-B cell line permits IL-2-driven proliferation: tyrosine phosphorylation of Shc is induced by IL-2 but not IL-7.

Expression of the interleukin (IL)-2 receptor beta chain in the IL-7-dependent pre-B cell line IxN/2B permitted growth in presence of either IL-2 or IL-7, allowing for a direct comparison of intracellular signaling events. Protein tyrosine phosphorylation was essential for IL-2 and IL-7-induced signal transduction since the tyrosine kinase inhibitor herbimycin A blocked proliferation in response to both factors. Western blot analysis of tyrosine-phosphorylated proteins revealed that both IL-2 and IL-7 stimulation led to enhanced phosphorylation of proteins of 170-, 145-, 115- and 99-kDa, as well as induction of phosphorylation of a 96-kDa protein. However, a 55- and a 155-kDa protein were only phosphorylated after IL-2 stimulation. The 55-kDa protein specifically phosphorylated by IL-2 could be identified as p52shc which has recently been shown to be critically involved in Ras activation. Shc tyrosine phosphorylation as a result of IL-2 stimulation was consistently found in CTLL-2 cells and human T lymphoblasts. Taken together our results indicate that the IL-2- and IL-7-stimulated intracellular pathways are partially different and that Shc is a target of IL-2-, but not IL-7-, stimulated tyrosine phosphorylation.

Tyrosine phosphorylation of Shc is induced by IL-3, IL-5 and GM-CSF.

The receptors for IL-3, IL-5 and GM-CSF belong to the hematopoietic receptor superfamily and have no intrinsic tyrosine kinase activity but nevertheless indirectly induce protein tyrosine phosphorylation. In order to directly compare the effects of IL-3, IL-5 and GM-CSF on protein tyrosine phosphorylation we analyzed the murine cell line FDCP-1 which proliferates equally well in response to IL-3 and GM-CSF and the cell line B13 which responds to both IL-3 and IL-5. The protein tyrosine phosphorylation pattern induced by IL-3, IL-5 and GM-CSF in these cell lines was shown to be remarkably similar and all three cytokines induced tyrosine phosphorylation of Shc, a src homology domain-2 containing protein which has been shown to be involved in Ras activation by tyrosine kinase receptors.

Tumor-cell-targeted cytokine gene transfer in experimental models for cancer therapy.

The genetic manipulation of tumor cells to express immunostimulatory molecules provides a current approach for the analysis of immune reactions against tumor cells in vivo. Experiments with multiple cytokines have demonstrated that an array of different host effector cells can be recruited by different cytokines in vivo, but more studies are necessary to distinguish cytokine-specific effects from as yet uncharacterized influences of different tumor models. A technically feasible clinical application of this approach is to be seen in the generation of vaccines by introducing such immunostimulatory genes into cancer cells and boosting systemic immune reactions against the unmodified cells. The experimental basis of these vaccination studies is critically discussed.

A sialyl-Le(x)-negative melanoma cell line binds to E-selectin but not to P-selectin.

The adhesion molecules E-selectin (ELAM-1) and P-selectin (GMP-140/CD62) recognize the carbohydrate motives sialyl-Le(x), sialyl-diLe(x), or sialyl-Lea, though with different affinity. We found that the melanoma cell line NKI-4 bound to E-selectin, but not to P-selectin. This melanoma cell line did not express sialyl-Le(x), but was positive for sialyl-diLe(x) and sialyl-Le(a). In contrast, 2 other melanoma cell lines, MeWo and SK-MEL-28, expressing either sialyl-diLe(x) or sialyl-Le(a) on the cell surface, bound neither E-selectin nor P-selectin. Transfection of the fucosyltransferases Fuc-TIII, Fuc-TIV, and Fuc-TV mediates cell surface expression of sialyl-Le(x) in many cell lines. We detected transcripts of the fucosyltransferases Fuc-TIII and Fuc-TV in 4 melanoma cell lines despite the absence of cell surface sialyl-Le(x). Our observations indicate that expression of fucosyltransferases (Fuc-TIII and -TV) and generation of cell-surface sialyl-diLe(x) are not sufficient to permit adherence to E-selectin or P-selectin. Furthermore, it seems possible that a yet undefined ligand different from sialyl-Le(x), sialyl-diLe(x), or sialyl-Le(a) enables melanoma cells to adhere to E-selectin.

T cells bind to the endothelial adhesion molecule GMP-140 (P-selectin).

A crucial step in an effective immune response is the adhesion of circulating lymphocytes. Lymphocytes must attach to endothelial cells before they can migrate into the graft. It has been shown that T cells bind to ICAM-1 and VCAM-1. Additionally, certain T cell subsets bind to ELAM-1. We now report that resting CD4+ and CD8+ T cells as well as individual CD4+ T cell clones and CD8+ T cell lines bind to GMP-140 in an adhesion assay using protein chimeras consisting of the extracellular domain of GMP-140 linked to the hinge domain of human IgG1. Whereas resting T cells bound similarly to ELAM-1 IgG and GMP-140 IgG, activated T cells represented by CD4+ T cell clones and CD8+ T cell lines bound to GMP-140 IgG, but not to ELAM-1 IgG. Neither the binding to immobilized GMP-140 IgG, nor to immobilized ELAM-1 IgG could provide T cells with costimulatory signals for proliferation in the presence of submitogenic concentrations of anti-CD3 antibodies. The binding of T cells to the endothelial adhesion receptor GMP-140 might be important during the initial adhesion process of lymphocytes in rejecting grafts.

Interleukin 10 transfected into Chinese hamster ovary cells prevents tumor growth and macrophage infiltration.

Expression of cytokines in tumor cells provides a sensitive modality to analyze the consequences of local cytokines in vivo on tumor infiltrating cells and tumorigenicity. We have transfected Chinese hamster ovary (CHO) cells with an interleukin 10 (IL-10) expression vector. CHO-IL10 cells although unaltered with respect to their in vitro growth lost tumorigenicity, both in nude and in SCID mice and in an IL-10 dose dependent manner. In addition, CHO-IL10 cells suppressed the growth of equal numbers of coinjected but not of contralaterally injected CHO cells. Immunohistology with anti-CR3/Mac-1 and anti-Mac-3 monoclonal antibodies revealed that CHO tumors were substantially infiltrated by macrophages. However, in CHO-IL10 tumors macrophages were virtually absent within the tumor tissue. Our results suggest that IL-10 indirectly suppresses tumor growth of certain tumors by inhibiting infiltration of macrophages which may provide tumor growth promoting activity.

An examination of the immune system of the duck (Anas platyrhynchos) for factors resembling some defined mammalian cytokines.

Duck lymphoblasts generated by phytohaemagglutinin (PHA) did not respond to recombinant or Jurkat cell line human interleukin (IL)-2 or possess surface antigens resembling mammalian IL-2 receptors or IL-1 beta. Supernatant fluids from normal and PHA-stimulated duck lymphocyte cultures, and normal and lipopolysaccharide (LPS)-stimulated monocytes, gave negative results in a range of assays for biological activity and immunochemical presence of factors resembling mammalian IL-1 and IL-2. However, supernatant fluids from LPS-stimulated duck monocytes contained IL-6-like activity (up to 35 units/mL) assessed on the 7TD-1 murine cell line. We were unable to demonstrate mRNA that would hybridize to cDNA probes for human IL-1 beta, IL-6, and tumour necrosis factor (TNF) in extracts of blood and lymphoid organs from normal and antigen-stimulated ducks. Because homologous serum or plasma is essential for duck lymphocytes and macrophages to respond to mitogens in vitro, we asked whether this growth-factor-like activity might be caused by substances resembling mammalian cytokines. Serum and plasma were examined for activity consistent with IL-1 and IL-6 on mammalian target cells. None was detected. Instead, both serum and plasma contained inhibitors of human IL-1 beta and IL-6, detected at dilutions up to 1:100. Inhibition by serum was heat (56 degrees C, 30 min) labile but inhibition by plasma was heat stable. The identities and biological functions of these inhibitors remain to be defined.

Expression of tumor necrosis factor by different tumor cell lines results either in tumor suppression or augmented metastasis.

Tumor necrosis factor (TNF) produced by tumor cells after gene transfer can effectively suppress the growth of locally growing tumors. We wanted to test the effects of "local" TNF on the growth of a highly metastatic cell line. Therefore, a recombinant retrovirus allowing expression of the TNF gene by the beta-actin promotor has been constructed and used to infect the two tumor cell lines EB and ESB, which grow as solid tumor or metastasize, respectively. Expression of TNF by EB cells resulted in their rapid and dose-dependent rejection. In sharp contrast, mice injected with ESB cells producing similar amounts of TNF showed no signs of tumor suppression, but rather had reduced survival rates that correlated with enhanced hepatic metastases. The accelerated formation of liver metastases by ESB TNF cells could be reversed by an anti-TNF mAb. These results demonstrate the opposite effects TNF may have on tumor growth: suppression of a locally growing tumor and promotion of metastasis formation.

Mechanisms of rejection induced by tumor cell-targeted gene transfer of interleukin 2, interleukin 4, interleukin 7, tumor necrosis factor, or interferon gamma.

Interleukin (IL)-2, IL-4, IL-7, tumor necrosis factor (TNF), or interferon-gamma (IFN-gamma) has been shown to be able to induce tumor rejection if produced locally by the tumor cells after gene transfer. To analyze whether the cellular rejection mechanisms are different or redundant we have expressed the cytokines in the same tumor cell line (J558L). Cell depletion experiments revealed that all cytokines required CD8+ T cells for complete long-term tumor eradication, although effective but transient host-dependent tumor suppression was also observed in the complete absence of CD8+ T cells. The transient tumor suppression induced by IL-2, IL-4, TNF, or IFN-gamma was also operative in nude and severe combined immunodeficient mice, whereas only tumor suppression induced by IL-7 was dependent on the presence of CD4+ T cells and was not evident in nude mice. The T-cell-independent effector arm of IL-2 and IFN-gamma but not IL-4 and TNF was mediated in part by natural killer cells. The transience of tumor suppression in the absence of T cells reflected loss of cytokine production in the case of TNF, IL-2, and IL-4 but not IFN-gamma. Immunohistologic analysis revealed all cytokine-producing tumors to be heavily infiltrated by macrophages. IL-4 and IL-7 tumors additionally contained eosinophils. The infiltration by T cells did not necessarily reflect their contribution to tumor rejection. Thus, the different cytokines activate heterogeneous transient tumor-suppressive mechanisms but always require CD8+ T cells for complete tumor rejection.

Eosinophils infiltrating interleukin-5 gene-transfected tumors do not suppress tumor growth.

Tumor-associated eosinophils have been observed in human tumors and in experimental tumor models, but their function is poorly understood. To study the role of eosinophils during tumor growth, the plasmacytoma J558L and the mammary adenocarcinoma TS/A were transfected with an expression vector encoding the murine gene for interleukin-5 (IL-5), a cytokine inducing proliferation and activation of eosinophils. Injection of parental cells, mock-transfectants and IL-5-producing cells into syngeneic mice showed that local IL-5 secretion induced rapid tumor infiltration by eosinophils, as evidenced by immunohistochemical staining, but nevertheless did not alter the tumor growth kinetics of IL-5 transfectants. Therefore, the mere presence of IL-5 and eosinophils was not sufficient to induce a protective host immune response.

Vaccinations with tumor cells genetically engineered to produce different cytokines: effectivity not superior to a classical adjuvant.

The potential of tumor cells (J558L) engineered to produce one of 5 different cytokines (interleukin 2, interleukin 4, interleukin 7, tumor necrosis factor, or gamma-interferon) to give rise to systemic immunity protective against a contralateral challenge with the parental cells was analyzed. The rejection of all cytokine-producing cells appeared to induce some systemic response capable of mediating the rejection of low numbers of subsequently contralaterally injected cells, but the effect was much less obvious with higher cell numbers. The injection of any possible combination of two of the cytokine producers did not reveal any synergistic effects. The cytokine gene-transfected tumor cells were not superior to the parental cells admixed with the adjuvant Corynebacterium parvum with respect to their potential as immunogens to induce immunity.