Horst Dürkop

Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin's disease

In man, Hodgkins disease (HD) represents the most frequent lymphoma entity whose pathogenesis is still unknown. In order to contribute to the characterization of the molecular mechanisms of this disease, cDNAs coding for the HD characteristic antigen CD30 were cloned from expression libraries of the human HUT-102 cell line using the monoclonal antibodies Ki-1 and Ber-H2. The open reading frame of the cDNA that can be translated from two mRNA species of 2.6 kb, and 3.8 kb, respectively, predicts a 595 amino acid protein with leader, extracellular, single transmembrane, and intracellular domains. When expressed in COS-1 cells, the cDNA presented properties comparable to native CD30 antigen. The CD30 extracellular domain proved to be homologous to members of the nerve growth factor receptor superfamily. Six cysteine-rich motifs could be recognized within the putative ligand-binding domain.

Expression of the oncofetal ED-B containing fibronectin isoform in hematologic tumors enables ED-B targeted 131I-L19SIP radioimmunotherapy in Hodgkin lymphoma patients

Current treatment of hematologic malignancies involves rather unspecific chemotherapy, frequently resulting in severe adverse events. Thus, modern clinical research focuses on compounds able to discriminate malignant from normal tissues. Being expressed in newly formed blood vessels of solid cancers but not in normal mature tissues, the extradomain B of fibronectin (ED-B FN) is a promising target for selective cancer therapies. Using immunohistology with a new epitope retrieval technique for paraffin-embedded tissues, ED-B FN expression was found in biopsies from more than 200 Hodgkin and non-Hodgkin lymphoma patients of nearly all entities, and in patients with myeloproliferative diseases. ED-B FN expression was nearly absent in normal lymph nodes (n = 10) and bone marrow biopsies (n = 9). The extent of vascular ED-B FN expression in lymphoma tissues was positively correlated with grade of malignancy. ED-B FN expression was enhanced in lymph nodes with severe lymphadenopathy and in some hyperplastic tonsils. The in vivo accessibility of ED-B FN was confirmed in 3 lymphoma patients, in whom the lymphoma lesions were visualized on scintigraphy with (131)I-labeled L19 small immunoprotein ((131)I-L19SIP). In 2 relapsed Hodgkin lymphoma patients(131)I-L19SIP radioimmunotherapy induced a sustained partial response, qualifying ED-B FN as a promising target for antibody-based lymphoma therapies.

The IL-15Rα Chain Signals Through Association with Syk in Human B Cells

The α-chain of the IL-15R (IL-15Rα) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Rα-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Rα and IL-2Rγ chains, but lacks the IL-2Rβ chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Rα chain. Upon association, the activated Syk kinase phosphorylates the IL-15Rα chain as well as phospholipase Cγ, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Rα and phospholipase Cγ phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Rα (Tyr227) abrogates both the IL-15Rα/Syk association and IL-15Rα phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Rα chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.

Expression of the CD30 antigen in non-lymphoid tissues and cells.

Originally, expression of the CD30 antigen was shown to be typical of the tumour cells of Hodgkins disease (HD) and of anaplastic large cell lymphomas (ALCLs). In reactive lymphoid tissue, CD30 is expressed only in a small population of activated lymphoid blasts. Since then, several reports have been published describing CD30 expression in non‐lymphoid tissues and malignancies, such as embryonal carcinomas (ECs), seminomas, cultivated macrophages, two histiocytic neoplasms, decidual cells, and mesotheliomas. As CD30 detection is important in the differential diagnosis of HD and ALCL, the expression of CD30 in different non‐lymphoid tissues was re‐evaluated by immunohistology and in situ hybridization. Extra‐lymphoid CD30 expression was found in 48/51 cases of EC or EC components of germ cell tumours, in decidual cells of 1/10 cases, in activated mesothelium in 16/28 pleural and peritoneal effusions, and in small foci of tumour cells in 2/8 mesotheliomas. CD30 expression was not confirmed in 27 germ cell tumours of the testis without an EC component nor in cultivated macrophages and 17 histiocytic malignancies. The knowledge of these CD30 expression patterns is important for the immunohistological differential diagnosis of anaplastic tumours. The absence of CD30 expression in reactive and neoplastic macrophages does not favour the concept that HD and ALCL are derived from these cells. Copyright

Blocking IL-15 Prevents the Induction of Allergen-Specific T Cells and Allergic Inflammation In Vivo

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Rα (sIL-15Rα) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-α, IL-1β, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Rα treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Rα-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4+/CD25+ T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.

Rescue of Impaired NK Cell Activity in Hodgkin Lymphoma With Bispecific Antibodies In Vitro and in Patients

Natural killer (NK) cells represent a key component of the innate immune system against cancer. Nevertheless, malignant diseases arise in immunocompetent individuals despite tumor immunosurveillance. Hodgkin lymphoma (HL) is characterized by CD30+ tumor cells and a massive infiltration of immune effector cells in affected lymph nodes. The latter obviously fail to eliminate the malignant cell population. Here, we tested for functional NK cell defects in HL and suggest an improvement of NK function by therapeutic means. We demonstrate that peripheral NK cells (pNK) from patients with HL fail to eliminate HL cell lines in ex vivo killing assays. Impaired NK cell function correlated with elevated serum levels of soluble ligands for NK cell receptors NKp30 (BAG6/BAT3) and NKG2D (MICA), factors known to constrict NK cell function. In vitro, NK cell cytotoxicity could be restored by an NKG2D/NKp30-independent bispecific antibody construct (CD30xCD16A). It artificially links the tumor receptor CD30 with the cytotoxicity NK cell receptor CD16A. Moreover, we observed that NK cells from patients treated with this construct were generally activated and displayed a restored cytotoxicity against HL target cells. These data suggest that reversible suppression of NK cell activity contributes to immune evasion in HL and can be antagonized therapeutically.

Expression of the human OX40 (hOX40) antigen in normal and neoplastic tissues

Summary. The Ber‐ACT35 mAb was raised against the human T‐cell lymphotrophic virus (HTLV) 1‐transformed, CD4+ HUT 102 cell line and recognizes the human homologue of the 0X40 (hOX40) antigen. The analysis of the expression of hOX40 by immunohistochernical techniques in malignant lymphomas, carcinomas and non‐malignant tissues of different organs shows that hOX40 expression was almost completely restricted to T lymphocytes. Besides T cells only a small subpopulation of macrophages in Langerhans’cell histiocytosis and a few blasts in B‐cell non‐Hodgkins lymphoma (B‐NHL) revealed a faint immunostaining with the Ber‐ACT35 mAb. Furthermore, most of the hOX40+ T‐cells are CD4+.

Differential expression and function of A20 and TRAF1 in Hodgkin lymphoma and anaplastic large cell lymphoma and their induction by CD30 stimulation

A20 and TRAF1 are two anti‐apoptotic components of the intracellular signalling pathway of the tumour necrosis factor receptor (TNFR) family. Induction of apoptosis seems to be a main function of these receptors. It is astonishing that a member of this family, CD30, is overexpressed by highly proliferating tumours such as Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). It is known that CD30 stimulation leads to the apoptosis of ALCL tumour cells but not of Hodgkin–Reed–Sternberg (HRS) cells. We have already established the overexpression of TRAF1 in HRS cells. In this study we demonstrate that A20 is highly expressed in the HRS cells in 20/22 of cases of classical HL, in 4/4 cases of nodular lymphocyte‐predominant HL (NLPHL), and in 2/2 cases of the anaplastic variant of diffuse large B cell lymphoma. In contrast, all other non‐Hodgkin lymphomas, including ALCL, revealed either no A20 reactivity, or reactivity in less than 1% of all tumour cells. CD30 stimulation induced A20 and TRAF1 expression. This effect was most prominent in HL and ALCL cell lines with low basal expression levels of these molecules. Immunohistological studies of reactive lymphoid blasts in tonsillar tissue demonstrated that co‐expression of CD30, A20, and TRAF1 also occurs in vivo. Cell lines with high basal A20 and TRAF1 expression were resistant to CD30‐mediated apoptosis. The sensitivity to CD30‐induced apoptosis was increased by inhibition of protein synthesis. TRAF1 transfection decreased CD30‐induced apoptosis. Our data suggest that A20 and TRAF1 contribute to apoptosis resistance and, therefore, play an important role in the pathogenesis of classical HL. Copyright

Inhibition of Tumor Growth by Targeted Toxins in Mice is Dramatically Improved by Saponinum Album in a Synergistic Way

The application of targeted toxins in cancer therapy remains a challenge due to the severe side effects as a consequence of the high systemic doses required. Here, we describe the combined application of a glycosylated triterpenoid (Spn) and epidermal growth factor receptor (EGFR)-targeted chimeric toxins (SA2E). The cytotoxicity of SA2E on murine TSA tumor cells transfected with human EGFR was enhanced 20,000-fold by low nonpermeabilizing Spn concentrations in a synergistic manner. Subcutaneous application of Spn and SA2E in BALB/c mice bearing a solid TSA cells transfected with epidermal growth factor receptor tumor resulted in 94% tumor volume reduction with a 50-fold lower chimeric toxin concentration compared with pure SA2E treatment. Side effects as monitored by observable complications, body weight, blood parameters; histologic analyses and antibody responses were only moderate and usually reversible.

The HTLV-I Tax Protein Transcriptionally Modulates OX40 Antigen Expression

OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5′-flanking region. By reporter gene analysis with progressive 5′ deletions from nucleotides −1259 to −64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40+ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-κB sites, which are not active in the constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-κB site demonstrates that cooperative NF-κB binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 ligand system in the proliferation of HTLV-I+ T cells.