Wolfram Brugger

Dendritic Cell Immunogenicity Is Regulated by Peroxisome Proliferator-Activated Receptor γ

Dendritic cells (DC) are the most potent APCs known that play a key role for the initiation of immune responses. Ag presentation to T lymphocytes is likely a constitutive function of DC that continues during the steady state. This raises the question of which mechanism(s) determines whether the final outcome of Ag presentation will be induction of immunity or of tolerance. In this regard, the mechanisms controlling DC immunogenicity still remain largely uncharacterized. In this paper we report that the nuclear receptor peroxisome proliferator-activated receptor γ (PPAR-γ), which has anti-inflammatory properties, redirects DC toward a less stimulatory mode. We show that activation of PPAR-γ during DC differentiation profoundly affects the expression of costimulatory molecules and of the DC hallmarker CD1a. PPAR-γ activation in DC resulted in a reduced capacity to activate lymphocyte proliferation and to prime Ag-specific CTL responses. This effect might depend on the decreased expression of costimulatory molecules and on the impaired cytokine secretion, but not on increased IL-10 production, because this was reduced by PPAR-γ activators. Moreover, activation of PPAR-γ in DC inhibited the expression of EBI1 ligand chemokine and CCR7, both playing a pivotal role for DC migration to the lymph nodes. These effects were accompanied by down-regulation of LPS-induced nuclear localized RelB protein, which was shown to be important for DC differentiation and function. Our results suggest a novel regulatory pathway for DC function that could contribute to the regulated balance between immunity induction and self-tolerance maintenance.

German Cancer Society Neuro‐Oncology Working Group NOA‐03 multicenter trial of single‐agent high‐dose methotrexate for primary central nervous system lymphoma

The prospective multicenter NOA‐03 trial, conducted by the Neuro‐Oncology Working Group (NOA) of the German Cancer Society, was initiated to define the feasibility and efficacy of single‐agent high‐dose methotrexate therapy without concomitant radiotherapy in immunocompetent patients with primary central nervous system lymphoma. Thirty‐seven patients (median age, 60 years) received 179 biweekly courses of 8g/m2 methotrexate. Response was assessed after 3 and 6 courses. We had planned to enter 105 patients into the trial. Since fewer than the projected 18 of 37 patients achieved a complete response after an intermediate analysis, the trial was closed. In intention‐to‐treat analysis, 11 of 37 patients (29.7%) achieved complete response, whereas 14 of 37 patients (37.8%) were found to have progressive disease. The median relapse‐free survival among complete response patients was 13.7 months. Multivariate logistic regression analysis revealed that corticosteroid application during the first methotrexate course was associated with complete response. The regimen was well tolerated, but, unlike previously reported results, the activity of high‐dose methotrexate was only moderate.

Dendritic cells in cancer vaccines.

Dendritic cells (DC) are recognized as the most potent antigen-presenting cells with the ability to stimulate naive resting T cells and to initiate primary immune responses. Encouraging results in vaccination studies in animal models and the development of protocols to generate sufficient numbers of human DC for clinical application have led to attempts to verify the feasibility and efficacy of this approach in patients in the context of Phase I/II vaccination trials. This review aims to present a concise overview of the current knowledge in DC development and biology and describes the recent data of the first published DC-based vaccination studies. These preliminary trials indicate that immunotherapies utilizing DC-presenting tumor-associated antigens can safely be administered to patients with cancer and induce significant immunologic and clinical responses.

Functional response of leukaemic blasts to stromal cell-derived factor-1 correlates with preferential expression of the chemokine receptor CXCR4 in acute myelomonocytic and lymphoblastic leukaemia

The chemokine stromal cell‐derived factor‐1 (SDF‐1) that is released by bone marrow (BM) stromal cells and contributes to stem cell homing may also play a role in the trafficking of leukaemic cells. We analysed SDF‐1‐induced intracellular calcium fluxes in leukaemic blasts from the peripheral blood of patients with newly diagnosed acute myeloid leukaemia (AML) and lymphoblastic leukaemia (B‐lineage ALL), determined the effect of BM stromal cell‐conditioned medium on in vitro transendothelial migration (TM) and measured expression of the SDF‐1 receptor, CXCR4, by flow cytometry. AML FAB M1/2 blasts did not show calcium fluxes and TM was not stimulated. In myelomonocytic AML (M4/5), however, SDF‐1 induced significant calcium fluxes and TM was increased twofold by the conditioned medium. M3 and M4 blasts with eosinophilia (M4eo) showed intermediate activity and M6 blasts showed no functional activity. In ALL, strong calcium fluxes and increased TM (2.5‐fold) were observed. Accordingly, expression of CXCR4 was low in undifferentiated (M0) AML, myeloid (M1/2) AML and erythroid (M6) AML, but high [mean fluorescence (MF)u2003>u200350] in promyelocytic (M3) AML, myelomonocytic (M4/5) AML and B‐lineage ALL. We conclude that, in AML, SDF‐1 is preferentially active in myelomonocytic blasts as a result of differentiation‐related expression of CXCR4. Functional activity of SDF‐1 and high expression of CXCR4 in B‐lineage ALL is in accordance with the previously described activity of SDF‐1 in early B cells. SDF‐1 may contribute to leukaemic marrow infiltration, as suggested by increased CXCR4 expression and migratory response in BM‐derived blasts compared with circulating cells.

Expression of vascular endothelial growth factor (VEGF) and its receptors in human neuroblastoma

Angiogenic factors may play a role in the biology of neuroblastoma, a well vascularised tumour, which frequently spreads haematogenously. Therefore, we analysed expression of vascular endothelial growth factor (VEGF) in six human neuroblastoma cell lines and five primary neuroblastomas. High VEGF levels (1-3 ng/10(6) cells/day) were found in the supernatant of all cell lines examined (SK-N-LO, SK-N-SH, LS, SH-SY5Y, IMR-32, Kelly). VEGF peptide was also detected in tissue homogenates from four of five primary tumours. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that VEGF165 is the major isoform produced by neuroblastomas. In addition, all cell lines and primary tumours expressed the mitogenic VEGF receptor FLK-1, whilst the non-mitogenic receptor FLT-1 was less frequently positive, suggesting that the tyrosine kinase FLK-1 is involved in malignant transformation of neuroblastoma cells. However, neutralising antibodies to VEGF did not inhibit growth of neuroblastoma cell lines, which argues against a role of VEGF as an autocrine growth factor, at least for cell lines in vitro. We conclude that neuroblastoma cells produce VEGF, which may contribute to tumour vascularisation, growth and invasion.

Regulation of transendothelial migration of hematopoietic progenitor cells.

Abstract: Transendothelial migration of hematopoietic progenitor cells occurs in the bone marrow during mobilization and homing, and may therefore play a key role in the trafficking of hematopoietic stem cells. We hypothesize that adhesion molecules, chemokines, and paracrine cytokines are involved in this multifactorial process. As suggested in several studies, downregulation of adhesion molecules (e.g., integrins) may contribute to mobilization of progenitors due to a decreased avidity to bone morrow stromal and endothelial cells, which express the corresponding ligands. Using an in vitro model of transendothelial migration, we have shown that only a small number of more mature, committed progenitors migrates spontaneously under the control of adhesion molecules of the beta‐2‐integrin family and their corresponding endothelial/ stromal ligands. However, transendothelial migration of progenitors in vitro is substantially enhanced by the chemokine stromal cell‐derived factor‐1 (SDF‐1), which is constitutively produced by bone marrow stromal cells. More primitive progenitors also respond to this chemokine. In addition, the ligand for SDF‐1, the chemokine receptor CXCR‐4, is expressed in greater levels on bone marrow CD34+ cells as compared to mobilized progenitors, suggesting that downregulation of chemokine receptors occurs during progenitor mobilization. Indeed, bone marrow CD34+ cells migrate more avidly in response to SDF‐1 than mobilized progenitors. Paracrine cytokines may also play a role in hematopoietic stem cell trafficking, since growth factor‐stimulated hematopoietic cells produce cytokines that act on endothelial cells (e.g., vascular endothelial growth factor, VEGF), modifying their proliferation, motility, permeability, and fenestration. We conclude that transendothelial migration of hematopoietic progenitor cells is regulated by adhesion molecules, paracrine cytokines, and chemokines. Cytotoxic therapy as well as exogenously administered hematopoietic growth factors may affect adhesion molecule expression, the local cytokine and chemokine milieu, and chemokine receptor expression, which indirectly results in mobilization of hematopoietic stem cells.

Expression of Novel Surface Antigens on Early Hematopoietic Cellsa

Abstract: The purpose of this report is to demonstrate the expression of very recently identified surface antigens on CD34+ and AC133+ bone marrow (BM) cells. Coexpression analysis of AC133 and defined antigens on CD34+ BM cells revealed that the majority of the CD164+, CD135+, CD117+, CD38low, CD33+, and CD71+ cells resides in the AC133+ population. In contrast, most of the CD10+ and CD19+ B cell progenitors and a fraction of the CD71high population are AC133−, indicating that CD34+AC133+ cells are enriched in primitive and myeloid progenitor cells, whereas CD34+AC133− cells mainly consist of B cell and late erythroid progenitors. This corresponds to the highly reduced percentage of CD10+ B cells and the absence of CD71high erythroid progenitors on AC133+ selected BM cells. A portion of 0.2‐0.7% of the AC133+ selected cells do not coexpress CD34. These cells are very small and define a uniform CD71−, CD117−, CD10−, CD38low, CD135+, HLA‐DRhigh, CD45+ population with unknown delineation. Four color analysis on CD34+CD38− BM cells revealed that virtually all of these primitive cells express AC133. Using an improved liposome‐enhanced labeling technique for the staining of weakly expressed antigens, subsets of this population could be identified which express the angiopoietin receptors TIE (67.6%) and TEK (36.8%), the vascular endothelial growth factor receptors FLT1 (7%), FLT4 (3.2%), and KDR (10.4%), or the receptor tyrosine kinases HER‐2 (15.4%) and FLT3 (CD135; 77.6%). Our results suggest that the CD34+CD38− population is heterogeneous with respect to the expression of the analyzed receptor tyrosine kinases.

Cyclopentenone prostaglandins induce caspase activation and apoptosis in dendritic cells by a PPAR-γ-independent mechanism: Regulation by inflammatory and T cell–derived stimuli

OBJECTIVEnDendritic cells (DC) are professional antigen-presenting cells playing a pivotal role in the induction of immunological responses. There is evidence that DC survival during ongoing immune responses is finite. However, little is known about the mechanisms regulating apoptosis in these cells. Here, we have investigated the effects of the anti-inflammatory cyclopentenone prostaglandins on human monocyte-derived DC.nnnMATERIALS AND METHODSnPhenotype of DC was determined by flow cytometry and their allostimulatory potential in mixed leukocyte reaction. Induction of apoptosis in DC was monitored by staining with annexin-V-FITC and propidium iodide, propidium iodide staining of cell nuclei, and fluorimetric assay of caspase activity. Induction of maturation in DC was obtained by stimulation with TNF-alpha, LPS, IFN-gamma, CD40-ligand, or different combinations of these stimuli. PPAR-gamma expression in DC was determined by RT-PCR.nnnRESULTSnExposure of immature DC to cyclopentenone prostaglandins blunted their allostimulatory capacity and skewed their phenotype by downregulating CD1a and costimulatory molecules. These effects were due to activation of caspases and induction of apoptotic cell death in DC by cyclopentenone prostaglandins. Mature DC showed enhanced susceptibility to apoptosis via cyclopentenone prostaglandins as compared with immature DC. Although DC express PPAR-gamma, the corresponding receptor for some of these metabolites, PPAR-gamma activation by a synthetic high-affinity agonist failed to impair DC viability.nnnCONCLUSIONSnCyclopentenone prostaglandins induce apoptosis of human DC by a PPAR-gamma-independent mechanism. Since these compounds are released during an inflammatory event and show anti-inflammatory properties, they may contribute to the downregulation of DC function through apoptotic cell death.

Dose-intense therapy with etoposide, ifosfamide, cisplatin, and epirubicin (VIP-E) in 100 consecutive patients with limited- and extensive-disease small-cell lung cancer

BACKGROUNDnWe conducted a phase I/II trial to assess the feasibility and activity of VIP-E chemotherapy in small-cell lung cancer. End-points were treatment-related morbidity and mortality, response to treatment. duration of response, and survival.nnnPATIENTS AND METHODSnTwo cycles of combination chemotherapy followed by granulocyte colony-stimulating factor (G-CSF) were given at a dose of etoposide (500 mg/m2), ifosfamide (4000 mg/m2), cisplatin (50 mg/m2), and epirubicin (50 mg/m2) to 100 consecutive patients with SCLC. Thirty patients (19 with LD, and 11 with ED SCLC) proceeded to VIC-E high-dose chemotherapy with autologous peripheral blood stem cell transplantation (PBSCT) at a cumulative dose of etoposide 1500 mg/m2, ifosfamide 12,000 mg/m2, carboplatin 750 mg/m2 and epirubicin 150 mg/m2 (VIC-E). Surgical resection of primary tumor was attempted at the earliest feasible point. Thoracic irradiation was given after completion of chemotherapy. RESULTS of conventional-dose VIP-E: 97 patients were evaluable for response. Objective response rate was 81% in LD-SCLC (33% CR, 48% PR; excluding patients in surgical CR) and 77% in ED-SCLC (18% CR, 58% PR). Treatment mortality was 2%. Median survival was 19 months in LD-SCLC and 6 months in ED-SCLC. Two-year survival was 36% in LD and 0% in ED SCLC. RESULTS OF HIGH-DOSE VIC-E: All 30 patients improved on or maintained prior responses. Four patients (13%) died of treatment-related complications. Median survival was 26 months in LD-SCLC and 8 months in ED-SCLC. Two-year survival was 53% in LD and 9% in ED SCLC.nnnCONCLUSIONnVIP-E chemotherapy is an effective induction therapy for SCLC. Compared with traditional protocols such as ACO or carboplatin/etoposide, response rates are slightly improved, while survival is not different. In the LD SCLC subgroup, high-dose chemotherapy improved response rates and survival, especially for patients in surgical CR prior to high-dose therapy. In ED SCLC, however, higher response-rates did not translate into improved survival. Selected LD-SCLC patients with good partial or complete remissions after prior therapy may benefit from HDC and PBSCT.

Approaches to Dendritic Cell‐Based Immunotherapy after Peripheral Blood Stem Cell Transplantation

Abstract: High‐Dose chemotherapy with peripheral blood progenitor cell transplantation (PBPCT) is a potentially curative treatment option for patients with both hematological malignancies and solid tumors, including breast cancer. However, based on a number of clinical studies, there is strong evidence that minimal residual disease (MRD) persists after high‐dose chemotherapy in a number of patients, which eventually results in disease recurrence. Therefore, several approaches to the treatment of mrd are currently being evaluated, including treatment with dendritic cell (DC)‐based cancer vaccines. DCs, which play a crucial role with regard to the initiation of T‐lymphocyte responses, can be generated ex vivo either from CD34+ hematopoietic progenitor cells or from blood monocytes. They can be pulsed in vitro with tumor‐derived peptides or proteins, and then used as a professional antigen‐presenting cell (APC) vaccine for the induction of antigen‐specific T‐lymphocytes in vivo. This paper summarizes our preclinical studies on the induction of primary HER‐2/neu specific cytotoxic T‐lymphocyte (CTL) responses using peptide‐pulsed DC. As HER‐2/neu is overexpressed on 30‐40% of breast and ovarian cancer cells, this novel vaccination approach might be particularly applicable to advanced breast or ovarian cancer patients after high‐dose chemotherapy and autologous PBPCT.