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To analyze the impact of TNFα or IL2 on human lymphocytes in vitro and the anti-tumor and immune-modifying effects of L19-IL2 and L19-TNFα on subcutaneously growing J558L myeloma in immunocompetent mice. PBMCs from three healthy volunteers were incubated with IL2, TNFα, or with IL2 plus addition of TNFα (final 20 h). BALB/c J558L mice with subcutaneous tumors were treated with intravenous L19-TNFα plus L19-IL2, or controls. Tumor growth and intra- and peri-tumoral tissues were analyzed for micro-vessel density, necrosis, immune cell composition, and PD1 or PD-L1 expressing cells. Exposure of PBMC in vitro to IL2, TNFα, or to IL2 over 3 and 5 days plus TNFα for the final 20 h resulted in an approximately 50 and 75% reduction of the CD25low effector cell/CD25high Treg cell ratio, respectively, compared to medium control. IL2 or TNFα increased the proportion of CD4− CD25low effector lymphocytes while reducing the proportion of CD4+ CD25low Teff cells. In the J558L myeloma model, tumor eradication was observed in 58, 42, 25, and 0% of mice treated with L19-TNFα plus L19-IL2, L19-TNFα, L19-IL2, and PBS, respectively. L19-TNFα/L19-IL2 combination caused tumor necrosis, capillary density doubling, peri-tumoral T cell and PD1+ T cell reduction (− 50%), and an increase in PD-L1+ myeloma cells. IL2, TNFα, or IL2 plus TNFα (final 20 h) increased the proportion of CD4− CD25low effector lymphocytes possibly indicating immune activation. L19-TNFα/L19-IL2 combination therapy eradicated tumors in J558L myeloma BALB/c mice likely via TNFα-induced tumor necrosis and L19-TNFα/L19-IL2-mediated local cellular immune reactions.

Targeted cancer therapy provides the basis for the arrest of tumor growth in aggressive pancreatic carcinoma, however, a number of protein-based targeted toxins lack efficacy due to insufficient endosomal escape after being endocytosed. Therefore, we tested a fusion protein of the ribosome-inactivating protein dianthin and human epidermal growth factor in combination with a glycosylated triterpene (SO1861) that serves as an endosomal escape enhancer. In vitro investigations with the pancreatic carcinoma cell lines BxPC-3 and MIA PaCa-2 revealed no significant differences to off-target cells in the half maximal inhibitory concentration (IC50) for the fusion protein. In contrast, combination with SO1861 decreased the IC50 for BxPC-3 cells from 100 nM to 0.17 nM, whereas control cells remained unaffected. Monotherapy of BxPC-3 xenografts in CD-1 nude mice led to a 51.7% average reduction in tumor size (40.8 mm3) when compared to placebo, however, combined treatment with SO1861 resulted in a more than 13-fold better efficacy (3.0 mm3 average tumor size) with complete regression in 80% of cases. Immunohistochemical analyses showed that tumor cells with lower target receptor expression are, in contrast to the combination therapy, able to escape from the monotherapy, which finally results in tumor growth. At the effective concentration, we did not observe liver toxicity and saw no other side effects with the exception of a reversible skin hardening at the SO1861 injection site, alongside an increase in platelet counts, plateletcrit and platelet distribution width. In conclusion, combining a targeted toxin with SO1861 is proven to be a very promising approach for pancreatic cancer treatment. This article is protected by copyright. All rights reserved.

// Hinrich P. Hansen 1 , Ahmad Trad 2 , Maria Dams 1 , Paola Zigrino 3 , Marcia Moss 4 , Maximilian Tator 1 , Gisela Schon 1 , Patricia C Grenzi 1 , Daniel Bachurski 1 , Bruno Aquino 5 , Horst Durkop 6 , Katrin S Reiners 1 , Michael von Bergwelt-Baildon 1 , Michael Hallek 1 , Joachim Grotzinger 2 , Andreas Engert 1 , Adriana F Paes Leme 5 , Elke Pogge von Strandmann 1 1 Department of Internal Medicine I, University of Cologne, Cologne, Germany 2 Department of Biochemistry, University Kiel, Kiel, Germany 3 Department of Dermatology, University of Cologne, Cologne, Germany 4 BioZyme Inc., Apex, North Carolina, USA 5 Brazilian Biosciences National Laboratory, LNBio, CNPEM, Campinas, Brazil 6 Pathodiagnostik Berlin, Berlin, Germany Correspondence to: Hinrich P. Hansen, e-mail: h.hansen@uni-koeln.de Keywords: Hodgkin lymphoma, extracellular vesicle, CD30, ADAM10, Brentuximab-Vedotin Received: January 11, 2016 Accepted: March 31, 2016 Published: April 20, 2016 ABSTRACT The goal of targeted immunotherapy in cancer is to damage both malignant and tumor-supporting cells of the microenvironment but spare unaffected tissue. The malignant cells in classical Hodgkin lymphoma (cHL) selectively express CD30. They release this receptor on extracellular vesicles (EVs) for the tumor-supporting communication with CD30 ligand (CD30L)-positive bystander cells. Here, we investigated how CD30-positive EVs influence the efficacy of the CD30 antibody drug conjugate (ADC) Brentuximab Vedotin (SGN-35). The malignant cells and the EVs expressed the active sheddase ADAM10. ADAM10 cleaved and released the CD30 ectodomain (sCD30), causing a gradual depletion of SGN-35 binding sites on EVs and creating a soluble competitor of the ADC therapy. In a 3D semi-solid tumor microenvironment model, the EVs were retained in the matrix whereas sCD30 penetrated readily into the surrounding culture medium. This resulted in a lowered ratio of EV-associated CD30 (CD30EV) to sCD30 in the surrounding medium in comparison to non-embedded cultures. A low percentage of CD30EV was also detected in the plasma of cHL patients, supporting the clinical relevance of the model. The adherence of CD30EV but not sCD30 to CD30 – /CD30L + mast cells and eosinophils allowed the indirect binding of SGN-35. Moreover, SGN-35 damaged CD30-negative cells, provided they were loaded with CD30 + EVs.

Recent advances in the diagnostic of myeloproliferative neoplasms (MPNs) discovered CALRETICULIN (CALR) mutations as a major driver in these disorders. In contrast to JAK2 mutations being mainly associated with polycythaemia vera, CALR mutations are only associated with primary myelofibrosis (PMF) and essential thrombocythaemia (ET). CALR mutations are present in the majority of PMF and ET patients lacking JAK2 and MPL mutations. As these CALR mutations are absent from reactive bone marrow (BM) lesions their presence indicates ET or PMF. So far these mutations are detectable only by molecular assays. Their molecular detection is cumbersome because of the great CALR mutation heterogeneity. Therefore, the availability of a simple assay would be of great help. All CALR mutations reported lead to a frameshift generating a new 36 amino-acid C-terminus. We generated a monoclonal antibody (CAL2) to this C-neoterminus by immunizing mice with a representative peptide and compared its performance with Sanger sequencing data in 173 MPNs and other BM diseases. There was a 100% correlation between the molecular and the CAL2 immunohistochemical (IHC) assays. Thus, the detection of CALR mutations by the CAL2 IHC is a specific, sensitive, rapid, simple and low-cost method.

ABSTRACT Aims: The intention of this work was to lift saponin supported tumor targeted therapies onto the next level by using targeted toxins in nude mice xenotransplant models. Materials & methods: Combined application of dianthin coupled to EGF and saponin SO-1861 was tested in a xenograft model of colon carcinoma. In vitro cytotoxicity was tested in real-time in NIH3T3 cells (no human EGF receptor expression), HER14 and human colon carcinoma HCT116 (both EGF receptor overexpressing) cells. A xenograft model was established using HCT116 cells and tumor-bearing animals were treated with SO-1861 (30 µg/treatment) and dianthin coupled to EGF (0.35 µg/treatment). Tumor progression was monitored, using 18F-2-fluor-2-desoxy-d-glucose, by small animal PET and by x-ray computed tomography. Results: In vitro results demonstrated a high-receptor specificity and the in vivo experiment showed a progressive reduction of the tumor volume and glycolytic activity in the treated group (>95% reduction; p < 0.05). Conclusion: This therapy has great advantage because of high specificity, low side effects and great effectiveness for future development in the treatment of colon cancer.

The extradomain B of fibronectin (ED-B) is a promising vascular target for selective pharmacodelivery in cancer patients. We analyzed a large series of prostatectomies from patients with prostate cancer, hyperplastic prostate disease, and normal prostates to study extent and tumor-selectivity of ED-B expression.Using immunohistology, 68 adenocarcinomas of the prostate or prostate cancer-inflicted lymph nodes, 4 samples of benign prostatic hyperplasia, and 6 normal prostate glands were studied for ED-B expressing newly formed blood vessels. Further, we treated an advanced prostate cancer patient with the anti-ED-B antibody (131)I-L19SIP to study in vivo target accessibility.ED-B-positive blood vessels were found significantly more frequent in prostate cancers as compared with peritumoral prostate tissues or normal prostate glands, independent of tumor differentiation. The ED-B-positive blood vessels' density was 97 (±23), 65 (±9), and 59 (±9)/mm(2) in G3, G2, and G1 prostate cancers, respectively, and 7 (±5)/mm(2) in normal prostate glands. In high-grade (G3) prostate cancers, also the peritumoral tissue showed a higher density of ED-B vessels than normal prostate glands. Similar results were obtained when ED-B-positive vessel density was expressed as a fraction of CD34-positive vessel density. Finally, selective uptake of ED-B-binding (131)I-L19SIP to tumor lesions was found in an advanced prostate cancer patient by whole-body planar scintigraphy.ED-B-positive blood vessels were found to a large extent in prostate cancer tissues, but only rarely in normal prostates or benign prostatic hyperplasia. Whole-body planar scintigraphy in a prostate cancer patient confirmed selective uptake of (131)I-L19SIP in the prostate cancer tissues, qualifying ED-B as a promising target for selective pharmacodelivery of anticancer agents in prostate cancer.

Hirsch B, Grunbaum M, Wagner F, Bi Y, Lucka L, Du M-Q, Stein H & Durkop H (2012) Histopathology 60, E19–E27 A novel A20 (TNFAIP3) antibody (Ber-A20) can be used to detect unmutated A20 by immunohistology Aims: A20 (TNFAIP3) is a nuclear factor-κB (NF-κB)-inducible component of tumour necrosis factor and Toll-like receptor intracellular signal transduction. It negatively regulates NF-κB, and has been identified as a tumour suppressor. Several studies have described A20 inactivation by deletion of the A20 locus at 6q23, inactivating mutations, and/or methylation of the A20 promoter in various lymphoma entities. Methods and results: We generated a monoclonal antibody against the C-terminus of A20 (Ber-A20) and investigated full-length A20 expression of normal lymphoid tissue and lymphomas for the first time. We identified loss of A20 expression in tumour cells of 24% of classical Hodgkin lymphoma, 27% of diffuse large B-cell lymphoma, 20% of chronic lymphocytic leukaemia, 19% of follicular lymphoma, 13% of mantle cell lymphoma and 8% of primary mediastinal B-cell lymphoma cases by immunohistology. Loss of A20 expression rarely occurred in T-cell non-Hodgkin lymphoma. Conclusions: Our data are in agreement with cytogenetic and molecular analyses. Among 21 cases of ocular adnexal marginal zone lymphomas with known A20 mutation status, we detected complete absence of A20 expression, whereas cases with wild-type A20 were weakly A20-positive. We demonstrate that A20 loss can be detected by immunohistology with a sensitivity similar to that of complex molecular and genetic methods.

Stimulation of the TNF receptors CD30 and CD95 exerts opposite effects. Crosstalk of both receptors is unknown. We aimed to reveal regulatory mechanisms of CD30-induced effects on CD95 signaling of cALCL cell lines. "CD30/CD95 crosstalk analysis" was performed in cALCL cell lines by comparison of CD30 or CD95 stimulation and CD30/CD95 costimulation. Receptor expression and induction of apoptosis was investigated by flow cytometry. mRNA expression of CD30-inducible genes (cFLIP, TRAF1, cIAP2, and A20) was compared by semiquantitative reverse transcription (RT-RQ-) PCR in stimulated and unstimulated cells. Protein expression of IκBα, p100/p52, caspase-8, caspase-3, and cFLIP was analyzed by immunoblotting. A lentiviral-based shRNA-mediated approach was used to inhibit cFLIP expression. CD30/CD95 crosstalk experiments revealed that CD30 ligation leads to NFκB-mediated cFLIP upregulation in cALCL cells, which in turn enhanced resistance to CD95-mediated apoptosis. This effect is based on the CD30-induced upregulation of cFLIP. Knockdown of cFLIP restores sensitivity to CD95-mediated apoptosis. We conclude that the anti-apoptotic function of CD30 antibodies should be kept in mind if CD30 antibody-based therapeutic concepts for ALCL lymphoma are considered.

Death ligands, including TNF-alpha, CD95L/FasL, and TRAIL, mediate safeguard mechanisms against tumor growth and critically contribute to lymphocyte homeostasis. We investigated death receptor-mediated apoptosis and CD30/CD95 crosstalk in four CD30-positive cell lines of cutaneous anaplastic large-cell lymphoma (cALCL). Whereas CD95 stimulation strongly induced apoptosis in cALCL cells, the pro-apoptotic pathways of TNF-alpha and TRAIL were completely blocked at an early step. Expression of TNF receptor 1 was lost in three of four cell lines, providing an explanation for TNF-alpha unresponsiveness. TRAIL resistance may be explained by the consistent overexpression of cellular flice inhibitory protein (c-FLIP) (four of four cell lines) and frequent loss of the pro-apoptotic Bcl-2 protein Bid (three of four cell lines). Changes at the receptor-expression level were largely ruled out. CD30/CD95 crosstalk experiments showed that CD30 ligation leads to NF-kappaB-mediated c-FLIP upregulation in cALCL cells, which in turn conferred enhanced resistance to CD95-mediated apoptosis. Knockdown of c-FLIP by a lentiviral approach enhanced basic apoptosis rates in cALCL cells and diminished the CD30-mediated suppression of apoptosis, thus proving the significance of c-FLIP in this context. These in vitro findings may be indicative of the clinical situation of cALCL. Further clarifying the defects in apoptosis pathways in cutaneous lymphomas may lead to improved therapies for these disorders.

Lymphomatoid papulosis (LyP), primary cutaneous anaplastic large T-cell lymphoma (cALCL), and cutaneous infiltrates of systemic anaplastic large cell lymphoma (sALCL) are CD30-positive lymphoproliferative disorders of the skin that overlap clinically, histopathologically, immunophenotypically, and genetically but differ considerably in their prognosis. In particular, lesions of LyP regress spontaneously, whereas those of cALCL and sALCL persist and may progress and spread to extracutaneous sites. In contrast to patients with cALCL, LyP patients do not benefit from an aggressive radio- and/or chemotherapeutic approach. We generated a novel tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) antibody that recognizes a formalin-resistant epitope (Ber-TRAF1A) and investigated the expression of TRAF1, an intracellular component of TNFR signaling, in LyP and ALCL. We could show a strong TRAF1 expression in the tumor cells of most LyP cases (42/49, 84%). In contrast, tumor cells of primary and secondary cALCL revealed TRAF1 expression in only a few cases (3/41, 7%) as shown for sALCL without skin manifestation. The data indicate that TRAF1 expression reliably distinguishes LyP from primary or secondary cALCL. This might be of crucial diagnostic importance and has a strong impact on the treatment decision for patients with cALCL and LyP.

Two of the main problems associated with administration of receptor-targeted toxins in tumor therapy are severe systemic side effects and low transfer of the toxins into the cytosol after binding to the tumor cell surface. To improve chimeric toxins in this respect we have developed a molecular adapter that links the toxic moiety and ligand. The adapter is designed to improve cytosolic uptake, retain the toxin inside the cytosol and detoxify the drug after cell death. The plant toxin saporin linked either directly or via the adapter to epidermal growth factor (EGF) served to evaluate efficacy to inhibit tumor growth and reduce side effects in vivo. The lethal dose for BALB/c mice was three times less for the adapter-containing toxin (SA2E) than for the adapter-free construct (SE). Furthermore, SE only reduced the average weight of induced tumors by 33% whereas SA2E-treated mice exhibited 71% reduction with an almost complete suppression in 60% of the cases. Additionally, severe side effects like hyperalgesia, alopecia and death were drastically reduced in SA2E-treated animals. Tumors without target receptor were only slightly affected by SA2E and the reduction in side effects less pronounced indicating specific depletion from the blood by target receptor expressing cells.

In this study, the expression of activated caspase-3 by the tumor cells of classical Hodgkin lymphoma (cHL), the Hodgkin-Reed-Sternberg (HRS) cells, is confirmed. This raises the question why caspase-3 does not kill HRS cells. There are only a few molecules, which are able to directly inhibit caspase-3. One of them is cIAP2. We show that cIAP2 is expressed in the HRS cells in 20 of 23 cHL cases by in situ hybridization. Suppression experiments with cIAP2 antisense RNA show that down-regulation of cIAP2 significantly reduces apoptosis resistance in cHL cell lines. cIAP2 overexpression appears to be unique for HRS cells since the tumor cells of non-Hodgkin lymphomas are nearly cIAP2-negative. We demonstrate that cIAP2 is inducible by CD30 stimulation in cHL cell lines of T-cell origin and anaplastic large cell lymphoma cell lines, whereas cHL cell lines of B-cell origin constitutively express cIAP2. Inhibition of cIAP2 expression by cIAP2 antisense RNA decreases resistance to apoptosis. The results indicate that cIAP2 contributes to the apoptosis resistance of HRS cells, mainly by inhibiting effector caspases. According to these findings, a therapeutical application of inhibitors of apoptosis proteins antagonists in cHL appears promising.

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.

Complex cytogenetic abnormalities confer dismal prognoses in myeloid malignancies. Even bone marrow transplantation from siblings or matched unrelated donors offer minimal chances for cure, suggesting that these cases are not only refractory to chemotherapy but also resist the graft-vs.-leukemia effect. We herein describe the first permanent, factor-independent c-kithi+ cell line CS-1 derived from an unrelated donor stem cell transplanted patient with relapsed acute myeloid leukemia (AML)-M5a of high-risk karyotype [monosomy 7, t(2;11)(q31;p13), t(10;12)(q24;q24)]. Having the same karyotype, CS-1 exhibits an autonomous growth pattern and responds to stem cell factor (SCF). CS-1 did not induce T cell activation in mixed-lymphocyte-tumor-cultures (MLTCs) and, when used as third party stimulators, decreased T cell proliferation in mixed-lymphocyte reactions (MLRs). Cytokines added exogenously or secreted from bystander T cells caused CS-1 to differentiate into dendritic cells (DCs). CS-1-derived DCs, in contrast to DCs originating from non-malignant CD34+ progenitor cells, had virtually no T cell stimulatory effect, indicating that CS-1 is both immunosuppressive and poorly immunogenic. These properties may partially be due to the detected downregulation of costimulatory molecules and appear to involve a soluble factor. CS-1 cells injected subcutaneously (s.c.) to non-obese diabetes/severe combined immunodeficient (NOD/SCID) mice produced solid tumors, disseminating into bone marrow and spleen. The data show that transforming AML blasts with high-risk karyotype into DCs is insufficient to restore their immunogenicity and that the CS-1 cell line is useful to identify tumor-related immunosuppressive mechanisms in vitro and in vivo. © 2003 Wiley-Liss, Inc.

The human CD30 molecule is expressed transiently at very low levels on intrafollicular and perifollicular T and B cell blasts in lymphoid tissues, but is specifically upregulated on certain tumor cells, e.g. Hodgkin and Reed-Sternberg (H-RS) cells. With its specific expression pattern and easy accessibility on the surface of H-RS cells CD30 is a valuable diagnostic marker and holds considerable promise as a target for in vivo immunotherapy. Knowledge of epitopes on the CD30 molecule is expected to facilitate the design of novel non-immunogenic anti-CD30 reagents. Therefore, we have mapped the epitopes of several monoclonal antibodies (mAb) applying a peptide array of overlapping CD30-derived peptides. For the mAb Ber-H2, two linear epitopes with identical sequence were found, while the mAb Ki-2 and the single chain Fv fragment R4-4 each recognized a single linear antigenic determinant, respectively. On the other hand, the mAb Ki-1 bound to a discontinuous epitope composed of two regions, one located near the N-terminus and the other near the membrane-spanning region of CD30. Using molecular modeling, it was possible to visualize the location of the epitopes on exposed loop regions of the molecule within the N-terminal domain. Finally, the results obtained with the mAb Ki-1 imply that the ends of the N- and C-terminal parts of the extracellular portion of CD30 are in close vicinity of each other, suggesting a flower-like structure for the membrane-bound homotrimeric CD30 molecule.

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.

Originally, expression of the CD30 antigen was shown to be typical of the tumour cells of Hodgkin's 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.

One of the most peculiar immunohistological characteristics of the tumour cells of Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), and embryonal carcinoma of the testis is the expression of the CD30 antigen. Physiologically, CD30 expression is restricted to a few activated lymphocytes in normal lymphoid tissue and a small population of decidual cells. To clarify the reasons behind this highly restricted expression pattern and to learn about the combination of transcription factors involved in this regulation in Hodgkin's lymphoma and other CD30+ malignancies, the 5′-flanking regulatory region of the cd30 gene was analysed. The major transcription start site was determined to be 270 bases upstream of the translational start codon in the Hodgkin's lymphoma-derived cell lines L591 and L428. Reporter gene assays revealed that the CD30 promoter (−413 to 84) induces a 50- to 1000-fold higher luciferase expression in CD30+ human lymphoid cell lines (Co, Jurkat, and the Hodgkin's lymphoma-derived cell line L540) than in CD30− human lymphoid cell lines (DG75, SUP-T1, and U698M), CD30− human carcinoma cell lines (HeLa and MCF-7), or COS1 cells. Deletion analysis defined a TATA-less, minimal promoter sequence from −164 to 84. The transcription factor Sp1 and members of the Ets family induce CD30 expression, whereas the transcription factor Sp3 diminishes its induction. These data suggest that a high Sp1/Sp3 expression ratio and a peculiar expression pattern of the Ets transcription factors are involved in the overexpression of CD30 and might contribute to the transformation of CD30+ tumour cells. Copyright © 2000 John Wiley & Sons, Ltd.

Apaf-1, the human C. elegans Ced-4 homologue, plays a crucial role in mitochondrial mediated apoptosis. To determine whether a mutant Apaf-1 is involved in the pathogenesis of malignant lymphomas, we performed sequence analysis of its transcriptional gene product. Therefore, cDNAs coding for Apaf-1 were examined in six lymphoma derived cell lines, in three non-lymphoid tumor cell lines, as well as in peripheral blood lymphocytes and in tissue from heart, kidney, and liver by RT-PCR. These studies did not disclose the expected tumor related alterations but led instead to the identification of three novel forms of Apaf-1. These forms vary in length, but contain all functional domains formerly characterized in the Apaf-1 protein. The results indicate that the previously published sequence is not the Apaf-1 form most widely expressed.

Interleukin 15 (IL-15) shares many functional properties with interleukin 2 (IL-2), although both cytokines probably also exert distinct functions. In order to screen for functional differences between IL-2 and IL-15 with respect to the control of T cell functions, we have stimulated human T lymphoblasts (hTBl) with IL-2 and/or IL-15 and have assessed the resulting changes in the following parameters: T cell proliferation; expression of various relevant surface markers; cytokine and receptor (alpha-chain) transcription; and IL-2 and IL-15 activity. Both cytokines equally upregulate standard activation markers such as CD25 and CD95 and downregulate CD27. However, IL-2 upregulates CD30, TNF receptor type II and CD40L expression significantly stronger than IL-15. IL-15 potentiates Con A-induced IL-2 secretion. Even though hTBl transcribe the IL-15 gene, they do not secrete IL-15 activity. These observations suggest that both cytokines can differentially regulate T cells, e.g. T cell functions relevant to the control of cell cycle progression and apoptosis, and/or that they can stimulate different T cell subsets. Moreover, IL-15 may potentiate IL-2-driven T cell responses.

Classical Hodgkin's lymphoma (cHL)-affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (HRS) cells, which are disseminated within a massive infiltrate of reactive cells. In particular, the innate immune infiltrate is deemed to support tumour growth by direct cell-cell interaction. Since they are rarely found in close proximity to the malignant cells in situ, we investigated whether cHL-derived extracellular vesicles might substitute for a direct cell-cell contact. We studied the crosstalk of the transmembrane proteins CD30 and CD30 ligand (CD30L) because they are selectively expressed on HRS and innate immune cells, respectively. Here, we showed that HRS cells released both the ectodomain as a soluble molecule (sCD30) and the entire receptor on the surface of extracellular vesicles. The vesicle diameter was 40-800 nm, as determined by cryo- and immune electron microscopy. In addition to CD30, typical extracellular vesicle markers were detected by mass spectrometry and flow cytometry, including tetraspanins, flotillins, heat shock proteins and adhesion molecules. In contrast to sCD30, vesicles caused a CD30-dependent release of interleukin-8 in CD30L(+) eosinophil-like EoL-1 cells and primary granulocytes from healthy donors, underscoring the functionality of CD30 on vesicles. In extracellular matrix (ECM)-embedded culture of HRS cells, a network of actin and tubulin-based protrusions guided CD30(+) vesicles into the micro-environment. This network targeted CD30(+) vesicles towards distant immune cells and caused a robust polarization of CD30L. Confocal laser scanning microscopy of 30 µm sections showed a CD30 vesicle-containing network also in cHL-affected lymphoid tissue of both mixed-cellularity and nodular sclerosing subtypes. This network might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30-CD30L interaction in trans. The tubulin backbone of the network may provide a target for the therapy of cHL with antitubulin-based CD30 antibody constructs.

CD30, a member of the tumor necrosis factor receptor (TNFR) superfamily, is consistently expressed by tumor cells of anaplastic large-cell lymphoma (ALCL). CD30 stimulation induces massive caspase-dependent cell death of ALCL cells in case of canonical NFκB inhibition or proteasome inhibition. However, CD30, a TNFR lacking a death domain (DD), is unable to recruit a death inducing complex containing TRADD (TNFR1-associated DD-protein) or FADD (FAS-associated DD-domain protein) together with the receptor-interacting protein 1 (RIP1) and caspase-8. Thus, the mechanism explaining CD30-induced cell death of lymphocytes remains obscure. Here, we demonstrate that blockage of RIP1 by siRNA or pharmacological inhibition of RIP1 by Necrostatin-1 almost completely prevented CD30-induced cell death. In addition, we revealed CD30-induced accumulation of RIP1 at the cytoplasma membrane of NFκB-inhibited ALCL cells by confocal laser scanning microscopy. Finally, primary ALCL cases can be subdivided into two groups based on the presence or absence of RIP1 as revealed by immunohistology. Taken together, our study identified RIP1 as a crucial mediator of CD30-induced cell death that bears features of apoptosis as well as necroptosis. RIP1 expression in ALCL tumor cells might eligible for the therapeutic application of CD30 antibodies in combination with NFκB/proteasome inhibitors that should result in CD30-induced cell death.

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.

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 OX40 (hOX40) antigen. The analysis of the expression of hOX40 by immunohistochemical 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-Hodgkin's lymphoma (B-NHL) revealed a faint immunostaining with the Ber-ACT35 mAb. Furthermore, most of the hOX40+ T-cells are CD4+.

Targeted toxin-based therapeutics are hindered by poor intracellular uptake, limited stability and non-specific immune stimulation. To address these problems, ligand-targeted toxins in combination with low dose saponin mixtures have been adapted and tested in vivo in the past, however, undefined saponin raw mixtures are not suitable for use in clinical development. In the present work we therefore used a targeted toxin (Sap3-EGF, i.e. saporin fused to epidermal growth factor) in combination with a structurally defined isolated saponin m/z 1861 (SO-1861). In vitro evaluation confirmed a 6900-fold enhancement in the cytotoxic efficacy of Sap3-EGF against TSA-EGFR target cells. The required dose of the targeted toxin was appreciably reduced and there was a highly synergistic effect observed. An ex vivo hemolysis assay showed no or very less hemolysis up to 10 μg/mL of SO-1861. In the acute toxicity studies SO-1861 was found to be non-toxic up to a dose of 100 μg/treatment. The enzymes aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase did not show any statistically significant liver damage, which was further confirmed by histological examination. Additionally, creatinine was also similar to the control group thus ruling out damage to kidney. In vivo studies in a syngeneic BALB/c tumor model characterized by EGFR overexpression were done by applying 30 μg SO-1861 and 0.1 μg Sap3-EGF per treatment. A more than 90% reduction (p < 0.05) in the average tumor volume was observed by this combined therapy.

A20 (TNFAIP3) is a nuclear factor-κB (NF-κB)-inducible component of tumour necrosis factor and Toll-like receptor intracellular signal transduction. It negatively regulates NF-κB, and has been identified as a tumour suppressor. Several studies have described A20 inactivation by deletion of the A20 locus at 6q23, inactivating mutations, and/or methylation of the A20 promoter in various lymphoma entities. We generated a monoclonal antibody against the C-terminus of A20 (Ber-A20) and investigated full-length A20 expression of normal lymphoid tissue and lymphomas for the first time. We identified loss of A20 expression in tumour cells of 24% of classical Hodgkin lymphoma, 27% of diffuse large B-cell lymphoma, 20% of chronic lymphocytic leukaemia, 19% of follicular lymphoma, 13% of mantle cell lymphoma and 8% of primary mediastinal B-cell lymphoma cases by immunohistology. Loss of A20 expression rarely occurred in T-cell non-Hodgkin lymphoma. Our data are in agreement with cytogenetic and molecular analyses. Among 21 cases of ocular adnexal marginal zone lymphomas with known A20 mutation status, we detected complete absence of A20 expression, whereas cases with wild-type A20 were weakly A20-positive. We demonstrate that A20 loss can be detected by immunohistology with a sensitivity similar to that of complex molecular and genetic methods.

Tissue distribution and expression on mitogen and virally stimulated lymphocytes render the ACT35 molecule a human lymphocyte activation antigen which as yet could not be clustered. Expression cloning of the ACT35 antigen from a pCDM8 library of the HUT-102 cell line revealed strong homology of the cDNA and its encoded protein sequence with the formerly described rat OX40 antigen. The 1.4-kb nucleotide sequence and the deduced 277-amino acid sequence of the single transmembrane protein were 65% and 63% identical, in human and in rat, respectively. Conservation included one N-linked glycosylation site and one protein kinase C phosphorylation site. When expressed in COS-1 cells, the cDNA presented properties comparable to the native ACT35 antigen and the rat OX40 molecule (relative molecular mass 48,000). Thus, the ACT35 protein corresponds to the hitherto unknown human OX40 antigen and is, therefore, another member of the tumor necrosis factor/nerve growth factor receptor (TNFR/NGFR) family. After applying fluorescence in situ hybridization, the human ACT35/OX40 gene could be mapped to chromosome band 1p36 and is, thus, linked to the genes for TNFR II and CD30.

Endogenous β-galactose-binding lectins have many biological functions, but their biological significance in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) remains unclear. By immunohistochemistry, we analyzed the expression of galectin-1 and galectin-3 in HL and ALCL cases as well as in cell lines, and investigated the pharmacological effects of galectin-1 treatment with and without CD30 pre-stimulation of HL and ALCL cell lines. The galectin-3-negative human embryonic kidney cell line (HEK-293) was transfected with galectin-3 cDNA. Galectin-3 is differentially expressed in HL and ALCL. CD30 stimulation of the ALCL cell line Karpas 299 activates NF-κB without induction of apoptosis. Galectin-1 treatment of Karpas 299 induces cell death, which is significantly increased by CD30 pre-stimulation. The CD30-mediated increase of galectin-1-induced cell death is to some extent caspase independent and does not influence the expression of tumor necrosis factor-associated factor 1 (TRAF1), TRAF2, and cellular inhibitor of apoptosis 2 protein (cIAP2), as revealed in Karpas 299 cells. In other cell lines except Karpas 299, CD30 pre-stimulation did not significantly enhance galectin-1-induced cell death. Galectin-3 transfection of HEK-293 cells resulted in cell surface expression of galectin-3, associated with marked cell aggregation. CD30-targeted therapy in combination with galectin-1 treatment may induce effective killing of ALCL cells but not of HL cells.

In man, Hodgkin's 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.

Summary: 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.

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 131I-labeled L19 small immunoprotein (131I-L19SIP). In 2 relapsed Hodgkin lymphoma patients131I-L19SIP radioimmunotherapy induced a sustained partial response, qualifying ED-B FN as a promising target for antibody-based lymphoma therapies.

We characterized cDNA clones specific for the extracellular matrix glycoprotein undulin. Two sets of cDNA clones were isolated from a human placental lambda gt11 expression library and from a rhabdomyosarcoma cell line encoding two partially identical carboxyl-terminal polypeptides of 843 (Un1) and 443 (Un2) amino acids suggesting differential splicing of a single gene transcript. Northern blot analysis of human rhabdomyosarcoma cell poly (A) RNA with cDNA specific for Un1 identified transcripts of approximately 4.2, 6.5, and 8.5 kilobases, whereas a probe specific for Un2 detected a single mRNA of approximately 5 kilobases. Since a monoclonal antibody that is reactive with a sequence encoded by Un1 and not by Un2 detects the bands considered characteristic for undulin in Western blots, the mRNAs related to Un1 may code for the major part of the undulin molecule. The protein sequences deduced from Un1 and Un2 reveal an amino-terminal differentially spliced von Willebrand factor A domain, characteristic of proteins that interact with interstitial collagens, which is linked to fibronectin-like type III homology units by a unique sequence of 57 amino acids. Whereas Un2 encodes two complete and one incomplete type III homologies followed by a unique acidic carboxyl-terminal domain of 118 amino acids, Un1 codes for seven complete and one truncated type III homologies, followed by a short proline-rich carboxyl-terminal segment of 23 amino acids. Considering the 298 amino acids occurring in identical segments, the 989 different amino acid positions deduced from clones Un1 and Un2 represent an estimated 40% of the overall undulin sequence. In the context of 1) rotary shadowing electron microscopy data showing undulin as a structure composed of nodules that are interconnected by flexible rods of varying size, 2) the presence of three major bands of Mr 270,000, 190,000, and 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 3) common antigenic epitopes and similar peptide maps (Schuppan, D., Cantaluppi, M.C., Becker, J., Veit, A., Bunte, T., Troyer, D., Schuppan, F., Schmid, M., Ackermann, R., and Hahn, E.G. (1990) J. Biol. Chem. 265, 8823-8832), our finding of differentially spliced type III homology units, as found in tenascin and fibronectin, suggests that undulin is another member of the fibronectin-tenascin family of extracellular matrix glycoproteins. Furthermore, as in fibronectin and tenascin, undulin bears an additional subset of interactive domains tailored to specific structural and functional roles in development and differentiation.

High CD30 expression in classical Hodgkin's lymphoma and anaplastic large cell lymphoma (ALCL) suggests an important pathogenic role of this cytokine receptor. To test this hypothesis, we investigated CD30 signaling in Hodgkin's and ALCL cell lines by different approaches: 1) CD30 stimulation, 2) CD30 down-regulation, and 3) a combination of both. The effects were determined at the RNA (microarray and real-time quantitative RT-PCR), protein (electrophoretic mobility shift analysis, immunoblot, and flow cytometry), and cellular/functional (proliferation and apoptosis) levels. We demonstrate that Hodgkin's cells are virtually CD30 unresponsive. Neither CD30 stimulation nor CD30 silencing of Hodgkin's cells had any significant effect. In contrast, CD30 stimulation of ALCL cells activated nuclear transcription factor-kappaB (NF-kappaB), induced major transcriptional changes, and decreased proliferation. These effects could be abrogated by down-regulation of CD30. Stimulation of CD30 in ALCL cells, stably transfected with a dominant-negative NF-kappaB inhibitor, induced pronounced caspase activation and massive apoptosis. Our data indicate that 1) CD30 signaling is not effective in Hodgkin's cell lines but is effective in ALCL cell lines, 2) CD30 is probably not significantly involved in the pathogenesis of classical Hodgkin's lymphoma, and 3) CD30 stimulation triggers two competing effects in ALCL cells, namely activation of caspases and NF-kappaB-mediated survival. These data suggest that CD30-targeted therapy in ALCL should be combined with NF-kappaB inhibitors to induce effective cell killing.

Although the therapy of Hodgkin lymphoma and anaplastic large cell lymphoma has been considerably improved during the last decades, high therapeutic toxicity, relapses, secondary tumors, and primary treatment failure(s) occur. Both malignancies are well suited for CD30-targeted immunotherapy because of their strong CD30 expression. We constructed an immunotoxin composed of a single chain variable fragment of a CD30 antibody fused to the human pancreatic ribonuclease, showing CD30-specific binding and ribonucleolytic activity resistant to the inhibitor RNasin. This immunotoxin revealed CD30-specific anti-tumor activity in BALB/c mice that were challenged with CD30-positive or CD30-negative syngeneic tumor cells.

Control of apoptosis via death ligands plays a basic role for lymphocyte homeostasis and lymphoma development. In this study, cutaneous T-cell lymphoma (CTCL) cell lines revealed pronounced resistance to death ligands as compared to cell lines of T-cell acute lymphoblastic leukemia (T-ALL). The proapoptotic activity of tumor necrosis factor (TNF)-alpha was blocked, sensitivity to TNF-related apoptosis-inducing ligand was significantly reduced, and 1/4 CTCL cell lines was resistant to CD95 activation. In parallel, there was no activation of effector caspase-3 and initiator caspase-8 in nonresponsive CTCL cells, whereas caspase-10 was cleaved selectively in sensitive CTCL cells. No indication for a responsibility of typical downstream regulators of apoptosis was obtained, but loss of CD95 was found in 1/4, loss of TNF-R1 in 3/4, loss of caspase-10 in 2/4, loss of Bid in 1/4, and overexpression of cellular flice inhibitory protein was found in 4/4 CTCL cell lines. This clearly indicates an inhibition of apoptosis early in the extrinsic cascade, namely at the formation of the death-inducing signaling complex. Parallels with regard to expression of apoptosis regulators were seen in peripheral blood mononuclear cells and biopsies of CTCL patients. This study may indicate defects in apoptosis in CTCL and may help to guide CTCL therapy.

Aims : To investigate T-bet expression profiles in various lymphoid tissue diseases caused by intracellular pathogens and to compare them in disorders without an infective aetiology. Murine and in vitro experiments have shown that the expression/induction of T-bet, the master regulator of Th1 differentiation, can be achieved by obligate intracellular pathogens and high interferon (IFN)-γ levels. Methods : Lymph node biopsies were analysed immunohistochemically employing single and double labelling for T-bet and CD20, CD4, CD8 and CD30 detection. Results : In disorders associated with high IFN-γ levels and intracellular pathogens (infectious mononucleosis, HIV-associated lymphadenopathy, cat-scratch disease, and toxoplasmic lymphadenitis), T-bet-expressing CD4 cells were accompanied by significant numbers of T-bet-positive CD8 and B cells. A similar profile was also found in histiocytic necrotizing (Kikuchi) lymphadenitis, a disease of unknown cause. In contrast, T-bet expression in disorders without an infective aetiology was observed in only a small portion of lymphocytes. Conclusions : Increased T-bet expression does not only identify intracellular infections in lymphoid tissue associated with high IFN-γ levels, but also implies that, under these conditions, it becomes induced in B cells, which apparently support the Th1 response. T-bet expression in Kikuchi lymphadenitis underscores the hypothesis that it is caused by an intracellular microorganism.

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.

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.

Complex cytogenetic abnormalities confer dismal prognoses in myeloid malignancies. Even bone marrow transplantation from siblings or matched unrelated donors offer minimal chances for cure, suggesting that these cases are not only refractory to chemotherapy but also resist the graft-vs.-leukemia effect. We herein describe the first permanent, factor-independent c-kit(hi+) cell line CS-1 derived from an unrelated donor stem cell transplanted patient with relapsed acute myeloid leukemia (AML)-M5a of high-risk karyotype [monosomy 7, t(2;11)(q31;p13), t(10;12)(q24;q24)]. Having the same karyotype, CS-1 exhibits an autonomous growth pattern and responds to stem cell factor (SCF). CS-1 did not induce T cell activation in mixed-lymphocyte-tumor-cultures (MLTCs) and, when used as third party stimulators, decreased T cell proliferation in mixed-lymphocyte reactions (MLRs). Cytokines added exogenously or secreted from bystander T cells caused CS-1 to differentiate into dendritic cells (DCs). CS-1-derived DCs, in contrast to DCs originating from non-malignant CD34(+) progenitor cells, had virtually no T cell stimulatory effect, indicating that CS-1 is both immunosuppressive and poorly immunogenic. These properties may partially be due to the detected downregulation of costimulatory molecules and appear to involve a soluble factor. CS-1 cells injected subcutaneously (s.c.) to non-obese diabetes/severe combined immunodeficient (NOD/SCID) mice produced solid tumors, disseminating into bone marrow and spleen. The data show that transforming AML blasts with high-risk karyotype into DCs is insufficient to restore their immunogenicity and that the CS-1 cell line is useful to identify tumor-related immunosuppressive mechanisms in vitro and in vivo.

The human CD30 molecule is expressed transiently at very low levels on intrafollicular and perifollicular T and B cell blasts in lymphoid tissues, but is specifically upregulated on certain tumor cells, e.g. Hodgkin and Reed-Sternberg (H-RS) cells. With its specific expression pattern and easy accessibility on the surface of H-RS cells CD30 is a valuable diagnostic marker and holds considerable promise as a target for in vivo immunotherapy. Knowledge of epitopes on the CD30 molecule is expected to facilitate the design of novel non-immunogenic anti-CD30 reagents. Therefore, we have mapped the epitopes of several monoclonal antibodies (mAb) applying a peptide array of overlapping CD30-derived peptides. For the mAb Ber-H2, two linear epitopes with identical sequence were found, while the mAb Ki-2 and the single chain Fv fragment R4-4 each recognized a single linear antigenic determinant, respectively. On the other hand, the mAb Ki-1 bound to a discontinuous epitope composed of two regions, one located near the N-terminus and the other near the membrane-spanning region of CD30. Using molecular modeling, it was possible to visualize the location of the epitopes on exposed loop regions of the molecule within the N-terminal domain. Finally, the results obtained with the mAb Ki-1 imply that the ends of the N- and C-terminal parts of the extracellular portion of CD30 are in close vicinity of each other, suggesting a flower-like structure for the membrane-bound homotrimeric CD30 molecule.

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.

The CD30 antigen is a member of the tumor necrosis factor receptor (TNFR) family which is overexpressed on the surface of the tumor cells of Hodgkin’s lymphoma, anaplastic large cell lymphoma (ALCL), and embryonal carcinoma of the testis. In this study the entire cd30 gene which is more than 24 000 bp long and organized in eight exons was characterized by analyzing cosmid and phage λ clones from human placental libraries with long-range polymerase chain reaction (PCR) and sequencing. Differences to other genes of the TNFR family were detected in the region encoding the extracellular domain of the cd30 gene. In nearly all other TNFR genes, the coding region of each cysteine-rich repeat is interrupted by one intron, i.e., the 3–4 cysteine-rich repeats of these receptors are encoded by at least 4–5 exons, whereas the six cysteine-rich repeats of the cd30 gene are encoded by two exons, i.e., each of these exons encode three cysteine-rich repeats. In addition, we also found a genetic polymorphism of tetranucleotide ATCC-repeats in the 5′ part of the CD30 promoter. This region was amplified by PCR from seven CD30 overexpressing human lymphoid cell lines and five human tissues with an absent or very low CD30 expression. The amplification products showed length differences of more than 550 bp. The number of the ATCC-repeats was higher in CD30 + cell lines than in normal tissues. Comparison of the individual PCR products in reporter gene assays revealed that the CD30 promoter activity increased with the length of this polymorphic region up to eightfold. The data suggest that the number of ATCC-repeats in the 5′ region of the CD30 promoter modulates the regulation of CD30 expression.

Anaplastic large cell lymphoma (ALCL) represents a generally recognized group of large cell lymphomas. Defining features consist of a proliferation of predominantly large lymphoid cells with strong expression of the cytokine receptor CD30 and a characteristic growth pattern. With the use of molecular and clinical criteria, 3 entities of ALCL have been identified: primary systemic anaplastic lymphoma kinase (ALK)+ ALCL, primary systemic ALK− ALCL, and primary cutaneous ALCL. ALK expression is caused by chromosomal translocations, most commonly t(2;5). ALK+ ALCL predominantly affects young male patients and, if treated with chemotherapy, has a favorable prognosis. It shows a broad morphologic spectrum, with the “common type,” the small cell variant, and the lymphohistiocytic variant being most commonly observed. The knowledge of the existence of these variants is essential in establishing a correct diagnosis. ALK− ALCL occurs in older patients, affecting both genders equally and having an unfavorable prognosis. The morphology and the immunophenotype of primary cutaneous ALCL show an overlap with that of lymphomatoid papulosis. Both diseases have an excellent prognosis, and secondary systemic dissemination is only rarely observed. The described ALCL entities usually derive from cytotoxic T cells. In contrast, large B-cell lymphomas with anaplastic morphology are believed to represent not a separate entity but a morphologic variant of diffuse large B-cell lymphoma. Malignant lymphomas with morphologic features of both Hodgkin disease and ALCL have formerly been classified as Hodgkin-like ALCL . Recent immunohistologic studies, however, suggest that ALCLs Hodgkin-like represent either cases of tumor cell–rich classic Hodgkin disease or (less commonly) ALK+ ALCL or ALK− ALCL.

Interleukin-15 (IL-15) is a potent inhibitor of several apoptosis pathways. One prominent path toward apoptosis is the ligand-induced association of TNF receptor 1 (TNFR1) with death domain adaptor proteins. Studying if and how IL-15 blocks TNFR1-mediated apoptosis in a murine fibroblast cell line (L929), we show here that IL-15 blocks TNFR1-induced apoptosis via IL-15Ralpha chain signaling. The intracellular tail of IL-15Ralpha shows sequence homologies to the TRAF2 binding motifs of CD30 and CD40. Most important, binding of IL-15 to IL-15Ralpha successfully competes with the TNFR1 complex for TRAF2 binding, which may impede assembly of key adaptor proteins to the TNFR1 complex, and induces IkappaBalpha phosphorylation. Thus, IL-15Ralpha chain stimulation is a powerful deflector of cell death very early in the apoptosis signaling cascade, while TNF-alpha and IL-15 surface as major opponents in apoptosis control.

Several members of the tumour necrosis factor receptor (TNF-R) superfamily can induce cell death. For TNF-R1, Fas/APO-1, DR3, DR6, TRAIL-R1 and TRAIL-R2, a conserved 'death domain' in the intracellular region couples these receptors to activation of caspases. However, it is not yet known how TNF receptor family members lacking a death domain, such as TNF-R2, CD40, LT-betaR, CD27 or CD30, execute their death-inducing capability. Here we demonstrate in different cellular systems that cytotoxic effects induced by TNF-R2, CD40 and CD30 are mediated by endogenous production of TNF and autotropic or paratropic activation of TNF-R1. In addition, stimulation of TNF-R2 and CD40 synergistically enhances TNF-R1-induced cytotoxicity. These findings describe a novel pro-apoptotic mechanism induced by some members of the TNF-R family.

The translocation t(2;5)(p23;q35), discovered in CD30+ anaplastic large cell (ALC) lymphomas, creates a potentially oncogenic fusion gene, part of which is contributed by a novel tyrosine kinase, ALK. Absence of ALK expression from normal hematolymphoid cells provides a basis for the morphologic assessment of t(2;5). The distribution of the t(2;5) in ALC lymphomas and Hodgkin's disease (HD), as assayed by nonmorphologic methods, is controversial. We used in situ hybridization and/or immunohistology to show ALK gene products in 85 ALC lymphomas, 82 HD cases, 40 other lymphoproliferations, as well as in 6 HD- and 4 ALC lymphoma-derived cell lines. ALK gene products were restricted to t(2;5)-positive ALC lymphoma cell lines and tumor cells of 16 primary non-B cell, common-type ALC lymphomas. These were mainly from young patients with initial lymphonodal disease. ALK expression was not detectable in any other specimen, including all cases of HD and HD-like type ALC lymphoma as well as secondary ALC lymphomas. Full congruence was noted for labeling results obtained with both methods. In agreement with cytogenetic analyses, but at variance with recently published studies, ALK gene expression distinguishes a subset of ALC lymphomas from other CD30+ lymphomas, including HD. The results do not support concepts attributing a significant role to the t(2;5) in the development of HD.