Elisabeth Sievers

Activated γδ T cells express the natural cytotoxicity receptor natural killer p44 and show cytotoxic activity against myeloma cells

γδ T cells account for up to 10% of T lymphocytes in the peripheral blood of healthy donors. They can be activated by cytokines such as interleukin (IL)‐2, IL‐12 and IL‐15, express natural killer (NK) cell markers such as NKG2D and show cytotoxic activity against several tumour cells, including multiple myeloma. Here, we present activated polyclonal γδ T cells from healthy donors with an NK T cell‐like phenotype expressing the natural cytotoxicity receptor NKp44. Natural cytotoxicity receptors NKp30, NKp44 and NKp46 have been regarded as specific NK receptors; only two γδ T cell clones described so far expressed NKp44. Isolated polyclonal γδ T cells cultured for 7 days according to the cytokine‐induced killer cell (CIK) protocol with additional IL‐15 revealed a surface expression of NKp44 of 8 ± 7% (n = 22). This could be confirmed by detection of NKp44 mRNA by reverse transcription–polymerase chain reaction (RT–PCR). γδ T cells exhibited a marked cytotoxic activity against myeloma cells, which could be reduced by inhibition of NKp44. To our knowledge, this is the first description of the expression of NKp44 on polyclonal γδ T cells.

CD40ligand‐expressing dendritic cells induce regression of hepatocellular carcinoma by activating innate and acquired immunity in vivo

Dendritic cells (DCs) are professional antigen‐presenting cells able to prime T‐cells against tumor‐associated antigens (TAA), but their potential to induce hepatocellular carcinoma (HCC) regression is still limited. CD40/CD40L interaction is essential for DC activation and induction of antigen‐specific T‐cells. In this study, transduction of TAA‐pulsed DC with a CD40L‐encoding adenovirus (Ad‐CD40L) was used to improve the immune response induced by DC toward HCC. Bone marrow–derived DC from C3H/HeNcrl mice were cultured with granulocyte‐macrophage colony‐stimulating factor and interleukin‐4. On day 6, tumor‐lysate pulsed DCs were infected with adenoviruses. HCCs were induced by inoculation of mice with Hepa129‐cells subcutaneously. When tumor‐volume was 100 to 400 mm3, DCs were injected intratumorally, subcutaneously, or intravenously. Ad‐CD40L transduction exerted CD40/CD40L interactions between DCs, increasing DC immunostimulation with up‐regulation of CD80/CD86‐ and interleukin‐12 (IL‐12) expression. Intratumoral injection of CD40L‐DC was superior to intravenous or subcutaneous treatments, yielding tumor elimination in almost 70% of mice. Moreover, all tumor‐free animals were protected against hepatic tumor cell rechallenge. In a preventive setting, subcutaneous injection of CD40L‐expressing DCs protected 50% of mice for more than 3 months toward tumor cell challenge. The induced immune response seemed to be dependent on cross‐priming with Th1‐lymphocytes in the lymph nodes, because transduced DCs were redetected in lymphoid tissues. In addition, immunohistochemistry of tumors indicated a significant tumor infiltration with CD4+, CD8+ T cells and natural killer (NK) cells. Tumor‐infiltrating lymphocytes were tumor‐specific, as shown in interferon‐gamma (IFN‐γ) enzyme‐linked immunosorbent spot and T‐cell proliferation assays. Conclusion: Transduction of DCs with Ad‐CD40L increases significantly the stimulatory capacity of DCs. Intratumoral injection of DCs activates both acquired and innate immunity, inducing complete regression of established tumors and long‐term immunity against tumor recurrence. This approach improves the antitumoral potential of DCs. (HEPATOLOGY 2008.)

SRC Signaling Is Crucial in the Growth of Synovial Sarcoma Cells

Synovial sarcoma is a soft-tissue malignancy characterized by a reciprocal t(X;18) translocation encoding a chimeric transcriptional modifier. Several receptor tyrosine kinases have been found activated in synovial sarcoma; however, no convincing therapeutic concept has emerged from these findings. On the basis of the results of phosphokinase screening arrays, we here investigate the functional and therapeutic relevance of the SRC kinase in synovial sarcoma. Immunohistochemistry of phosphorylated SRC and its regulators CSK and PTP1B (PTPN1) was conducted in 30 synovial sarcomas. Functional aspects of SRC, including dependence of SRC activation on the SS18/SSX fusion proteins, were analyzed in vitro. Eventually, synovial sarcoma xenografts were treated with the SRC inhibitor dasatinib in vivo. Activated phospho (p)-(Tyr416)-SRC was detected in the majority of tumors; dysregulation of CSK or PTP1B was excluded as the reason for the activation of the kinase. Expression of the SS18/SSX fusion proteins in T-REx-293 cells was associated with increased p-(Tyr416)-SRC levels, linked with an induction of the insulin-like growth factor pathway. Treatment of synovial sarcoma cells with dasatinib led to apoptosis and inhibition of cellular proliferation, associated with reduced phosphorylation of FAK (PTK2), STAT3, IGF-IR, and AKT. Concurrent exposure of cells to dasatinib and chemotherapeutic agents resulted in additive effects. Cellular migration and invasion were dependent on signals transmitted by SRC involving regulation of the Rho GTPases Rac and RhoA. Treatment of nude mice with SYO-1 xenografts with dasatinib significantly inhibited tumor growth in vivo. In summary, SRC is of crucial biologic importance and represents a promising therapeutic target in synovial sarcoma.

Patient-derived dendritic cells transduced with an a-fetoprotein-encoding adenovirus and co-cultured with autologous cytokine-induced lymphocytes induce a specific and strong immune response against hepatocellular carcinoma cells

Abstract: Background/Aims: Breaking immunologic tolerance towards the hepatocellular carcinoma (HCC)‐associated α‐fetoprotein (AFP) antigen is possible. The use of this potential for the treatment of immunocompromised HCC patients is limited. In this study, we analyzed whether dendritic cells (DCs) from HCC patients transduced with a human AFP (hAFP)‐expressing adenovirus and co‐cultured with cytokine‐induced killer (CIK) cells can induce a strong specific immune response against HCC‐cells.

Phosphatidylinositol-3′-kinase/AKT signaling is essential in synovial sarcoma

Synovial sarcomas account for 5–10% of all malignant soft tissue tumors. They have been shown to express different membranous growth factor receptors, many of them signaling via intracellular kinase cascades. In our study, the functional role of PI3K/AKT signals in synovial sarcoma is analyzed with regard to tumor biology and therapeutic applicability. Immunohistochemical stainings of (Ser473)‐phosphorylated (p)‐AKT, its targets p‐(Ser9)‐GSK‐3β and p‐(Ser2448)‐mTOR and the cell cycle regulators Cyclin D1 and p27KIP1 were performed in 36 synovial sarcomas. The PIK3CA gene was screened for mutations. In vitro, four synovial sarcoma cell lines were treated with the PI3K inhibitor LY294002. Phosphorylation of AKT, GSK‐3β and mTOR was assessed, and cellular proliferation and apoptosis were analyzed to functionally characterize the effects of PI3K inhibition. Finally, coincubations of LY294002 with cytotoxic drugs were performed. Most tumors showed significant expression levels of p‐AKT, p‐GSK‐3β and p‐mTOR, indicating activation of the PI3K/AKT signaling cascade in synovial sarcomas; Cyclin D1 and p27KIP1 were differentially expressed. Mutations in the PIK3CA gene could be excluded. In vitro, PI3K inhibition diminished synovial sarcoma cell growth accompanied by reduced phosphorylation of AKT, GSK‐3β and mTOR. Mechanistically, PI3K pathway inhibition lead to enhanced apoptosis and decreased cellular proliferation linked to reduced Cyclin D1 and increased p27KIP1 levels. Simultaneous treatment of synovial sarcoma cell lines with LY294002 and cytotoxic drugs resulted in additive effects. In summary, PI3K signaling plays an essential role in growth control of synovial sarcomas and might be successfully targeted in multimodal therapeutic strategies.

SS18-SSX fusion protein-induced Wnt/β-catenin signaling is a therapeutic target in synovial sarcoma

Synovial sarcoma is a high-grade soft tissue malignancy characterized by a specific reciprocal translocation t(X;18), which leads to the fusion of the SS18 (SYT) gene to one of three SSX genes (SSX1, SSX2 or SSX4). The resulting chimeric SS18-SSX protein is suggested to act as an oncogenic transcriptional regulator. Despite multimodal therapeutic approaches, metastatic disease is often lethal and the development of novel targeted therapeutic strategies is required. Several expression-profiling studies identified distinct gene expression signatures, implying a consistent role of Wnt/β-catenin signaling in synovial sarcoma tumorigenesis. Here we investigate the functional and therapeutic relevance of Wnt/β-catenin pathway activation in vitro and in vivo. Immunohistochemical analyses of nuclear β-catenin and Wnt downstream targets revealed activation of canonical Wnt signaling in a significant subset of 30 primary synovial sarcoma specimens. Functional aspects of Wnt signaling including dependence of Tcf/β-catenin complex activity on the SS18-SSX fusion proteins were analyzed. Efficient SS18-SSX-dependent activation of the Tcf/β-catenin transcriptional complex was confirmed by TOPflash reporter luciferase assays and immunoblotting. In five human synovial sarcoma cell lines, inhibition of the Tcf/β-catenin protein–protein interaction significantly blocked the canonical Wnt/β-catenin signaling cascade, accompanied by the effective downregulation of Wnt targets (AXIN2, CDC25A, c-MYC, DKK1, CyclinD1 and Survivin) and the specific suppression of cell viability associated with the induction of apoptosis. In SYO-1 synovial sarcoma xenografts, administration of small molecule Tcf/β-catenin complex inhibitors significantly reduced tumor growth, associated with diminished AXIN2 protein levels. In summary, SS18-SSX-induced Wnt/β-catenin signaling appears to be of crucial biological importance in synovial sarcoma tumorigenesis and progression, representing a potential molecular target for the development of novel therapeutic strategies.

Glycoprotein B from strain 17 of herpes simplex virus type I contains an invariant chain homologous sequence that binds to MHC class II molecules

Major histocompatibility complex class I (MHCI) molecules are major targets of virus evasion strategies because they introduce antigens from the biosynthesis pathway into the antigen‐processing and presentation pathways for immune recognition by CD8+ T cells. Little is known about viral strategies that interfere with the MHC class II (MHCII) antigen presentation pathway. We identified a six amino acid sequence from type I herpes simplex virus (HSV‐1) glycoprotein B (gB) that is identical to a sequence of human leucocyte antigen D (HLA‐D) ‐associated invariant chain (Ii). In addition, this gB sequence is adjacent to a highly conserved HLA‐DR1 binding motif. Both viral sequences together resemble the class II binding site of human Ii, consisting of a MHCII groove binding segment and a promiscuous binding site. We cloned gB from HSV‐1 strain 17 and demonstrate association of the virus envelope protein to three HLA‐DR allotypes. With chimeric Ii/gB fusion proteins we identified gB sequences that mediate promiscuous or allotype‐specific binding to the HLA‐DR peptide‐binding domain. Mutation of two Lys residues in the viral segment of Ii/gB abolished promiscuous binding to HLA‐DR heterodimers. The result indicates promiscuous binding of the virus sequence to HLA‐DR molecules and suggests a potential for HSV‐1 to manipulate antigen processing and presentation.

Control of mitogenic and motogenic pathways by miR-198, diminishing hepatoma cell growth and migration

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths, worldwide. MicroRNAs, inhibiting gene expression by targeting various transcripts, are involved in genomic dysregulation during hepatocellular tumorigenesis. In previous studies, microRNA-198 (miR-198) was shown to be significantly downregulated in HCV-positive hepatocellular carcinoma (HCC). Herein, the function of miR-198 in hepatocellular carcinoma cell growth and gene expression was studied. In hepatoma cell-types with low levels of liver-specific transcription factor HNF1α indicating a low differentiation grade, miR-198 expression was most downregulated. However, miR-198 treatment did not restore the expression of the liver-specific transcription factors HNF1α or HNF4α. Importantly, overexpression of miR-198 in Pop10 hepatoma cells markedly reduced cell growth. In agreement, comprehensive gene expression profiling by microarray hybridisation and real-time quantification revealed that central signal transducers of proliferation pathways were downregulated by miR-198. In contrast, genes mediating cellular adherence were highly upregulated by miR-198. Thus, the low expression of E-cadherin and claudin-1, involved in cell adhesion and cell-cell contacts, was abolished in hepatoma cells after miR-198 overexpression. This definite induction of both proteins by miR-198 was shown to be accompanied by a significantly impaired migration activity of hepatoma Pop10 cells. In conclusion, miR-198 acts as a tumor suppressor by repression of mitogenic and motogenic pathways diminishing cell growth and migration.

Targeting Wnt pathway in lymphoma and myeloma cells.

The present study investigated the apoptotic effects of ethacrynic acid (EA) and the antifungal agent ciclopiroxolamine (cic), another drug that inhibits Wnt/beta-catenin signalling, on the myeloma cell line OPM-2 and three lymphoma cell lines OCI-LY8-LAM-53, SU-DHL-4 and Raji in vitro in comparison to normal peripheral blood mononuclear cells (PBMC) derived from healthy people. In contrast to PBMC, cic and EA led to a significant decrease of viability in lymphoma and myeloma cell lines. In conclusion, our results showed a significant selective induction of apoptosis by cic and EA in lymphoma and myeloma cells. The Wnt/beta-catenin pathway has been shown to play an important role in the regulation of cell proliferation, differentiation and apoptosis (Miller et al, 1999; You et al, 2002; Cadigan & Liu, 2006). It was recently demonstrated that the Wnt pathway is activated in lymphoma (Lu et al, 2004). Therefore, the Wnt/beta-catenin signalling molecules are attractive candidates for developing targeted therapies for lymphoma. Recently, we confirmed that the diuretic agent ethacrynic acid (EA) inhibited Wnt/beta-catenin signalling (J.X. Liu, T. Endo, I.G.H. Schmidt-Wolf, H. Zhou, J.E. Castro, T.J. Kipps, D.A. Carson and D. Lu, unpublished data). EA is already used clinically as a diuretic agent. Glutathione-Stransferase (GST), which is overexpressed in human tumours in form of GST-P, couples glutathione (GSH) with electrophilic compounds and detoxifies the cell (Aizawa et al, 2003). GSH acts as a reducing agent and antioxidant. The binding of EA to GSH can enhance the cytotoxicity of chemotherapeutic agents (Nagourney et al, 1990). Ciclopiroxolamine (cic) is a synthetic antifungal agent used topically for the treatment of yeast infections in humans and is degraded by glucoronidation (Hoffman et al, 1991). It acts as chelator of polyvalent metal cations (e.g. Feand Al) resulting in the inhibition of the metal-depending enzymes occurring in the metabolism of the cell. Furthermore, it blocks the cell cycle near the G1/S phase boundary (Hoffman et al, 1991). Recently, we used a 96-well plate-based TOPflash reporter system to screen the Gen-plus drug library (Microsource, Gaylordsville, CT, USA), which contained 960 compounds. This screen identified EA and cic as Wnt/beta-catenin inhibitors (J.X. Liu, T. Endo, I.G.H. Schmidt-Wolf, H. Zhou, J.E. Castro, T.J. Kipps, D.A. Carson and D. Lu, unpublished data). Given that the canonical Wnt signalling pathway is activated in lymphoma and myeloma cells (Lu et al, 2004), we investigated whether EA and cic could induce apoptosis and decrease viability of lymphoma and myeloma cell lines. EA and cic were titred in various lymphoma and myeloma cell lines and in PBMC derived from healthy individuals in order to check for toxicity. Titration of EA and cic revealed that 30 lmol/l EA and 10 lmol/l cic were the most effective concentrations to initialise cell death in lymphoma and myeloma cell lines without deteriorating the viability of normal PBMCs significantly (data not shown). The effect of dimethyl sulphoxide (DMSO) as a toxic solvent was only observed in the myeloma cell line OPM-2 (see below). As control, PBMC were investigated for fluorescence-activated cell sorting (FACS) analysis. A toxic effect toward cells induced a shift from viable, DiOC6-positive cells to apoptotic, DiOC6-negative cells. Viability of the cell lines and PBMC decreased slowly over time. After 72 h, the relative viability for the lymphoma cell lines SU-DHL-4, LAM-53 and Raji in the presence of EA (30 lmol/l) was 97Æ6 ± 1Æ1%, 91Æ0 ± 4Æ9% and 78Æ9 ± 2Æ1% respectively (Fig 1). These values were similar to values of the control of PBMC with 89Æ5 ± 3Æ1%. In contrast to PBMC and lymphoma cell lines, the myeloma cell line OPM-2 was highly sensitive towards DMSO and showed a decreased relative viability to 66Æ8 ± 2Æ3%. In the presence of cic (10 lmol/l), viability of the lymphoma cell lines was reduced to 55Æ9 ± 1Æ6% for SU-DHL-4, 32Æ4 ± 3Æ3% for LAM-53 and 21Æ7 ± 1Æ4% for Raji (all statistically significant with a P-value < 0Æ05) in contrast to PBMC, with 94Æ4 ± 1Æ8% viability. Similar to lymphoma cell lines, cic had a strong effect on cell vitality of the myeloma cell line OPM-2 reducing the relative viability by up to 29Æ4 ± 4Æ7% (P-value < 0Æ05). The decrease in viability was less pronounced using EA as compared to cic cell cultures (Fig 1). The combination of EA and cic showed similar values to those obtained with cic alone for SU-DHL-4 and LAM-53 cell lines. In the case of Raji cells, EA inhibited the toxic effect of cic from 21Æ7 ± 1Æ4% up to 59Æ3/)2Æ3%. In addition, in OPM myeloma cells the combination of cic and EA showed low values, with 27Æ9 ± 4Æ6% viability after 72 h of culture (P-value < 0Æ05; Fig 1). The effect of EA was studied in primary cultures derived from patients with chronic lymphocytic leukaemia. Similar data as for cell lines (Fig 1) were obtained for primary cells (data not shown). In conclusion, our results showed a significant induction of apoptosis by cic and EA in lymphoma and myeloma cells. Taken together with our previous results (Lu et al, 2004), our data suggest that EA and cic can inhibit Wnt/beta-catenin signalling in lymphoma and myeloma cell lines. In addition, EA was also shown to be effective in primary cultures derived from patients with chronic lymphocytic leukaemia. Our results are in accordance with a recent report (Sukhdeo et al, 2007) that the canonical Wnt signalling pathway is activated in multiple myeloma through constitutively active beta-catenin. Correspondence

A recombinant anti‐carcinoembryonic antigen immunoreceptor with combined CD3ζ‐CD28 signalling targets T cells from colorectal cancer patients against their tumour cells

Background and aims: The prognosis of metastatic colorectal cancer is still poor, raising the need for alternative therapeutic approaches, particularly by manipulating the antitumour immune response. Advanced tumour stages, however, are frequently accompanied by functional T cell defects which may be critical for a T cell based anticancer immunotherapy. The aim of this study was to address whether T cells from colorectal cancer patients with advanced tumour stages can be specifically antigen activated against their autologous tumour cells. Methods: T cells were isolated from colorectal cancer patients and retrovirally transduced to express a recombinant immunoreceptor that has an extracellular binding domain for carcinoembryonic antigen (CEA) and an intracellular CD3ζ signalling domain with and without CD28 costimulation for T cell activation. Results: Peripheral blood T cells from colorectal cancer patients were successfully engineered to express the anti-CEA immunoreceptor on the cell surface. On coincubation with autologous CEA+ tumour cells, T cells with anti-CEA immunoreceptor are specifically activated to secrete interferon γ (IFN-γ) and to lyse autologous tumour cells whereas T cells without immunoreceptor are not. T cells equipped with combined CD3ζ-CD28 signalling receptor are more efficiently activated to secrete IFN-γ compared with T cells with CD3ζ signalling receptor. Induction of interleukin 2 secretion on targeting towards autologous tumour cells requires triggering of T cells by the CD3ζ-CD28 costimulatory receptor. Conclusions: T cells from advanced colorectal cancer patients can be tumour specifically activated with high efficiency by engraftment with a combined CD3ζ-CD28 immunoreceptor to break tolerance against autologous tumour cells.