Alexander von Rücker

Interactions Between Dendritic Cells and Cytokine-induced Killer Cells Lead to an Activation of Both Populations

Dendritic cells (DCs) are major antigen-presenting cells. They are capable of capturing and processing tumor antigens, expressing lymphocyte costimulatory molecules, and secreting cytokines to initiate immune responses. Here, the authors tested the effect of cytokine-induced killer (CIK) cells, a population that includes CD3 + CD56 + cells (natural killer T cells), with regard to their capacity to immunomodulate DCs. Cytokine-induced killer cells were cocultured with autologous DCs generated from peripheral blood mononuclear cells. Expression of markers typical for both populations was measured using flow cytometry, and secretion of interleukin (IL)-12 was determined using enzyme-linked immunosorbent assays. Cytotoxicity assays were performed to investigate the role of IL-12 and the importance of cell–cell interactions. Considering this, receptors for IL-12 and CD40 were blocked and cocultures were performed with cell culture inserts. Coculture of CIK cells led to a significant increase of DC-specific, costimulatory, and antigen-presenting molecules in DC cultures. In addition, coculture resulted in a dramatically increase of IL-12 secretion by DCs and to a significant increase in cytotoxic activity of CIK cells toward carcinoma cells. Blockage of IL-12 uptake decreased the cytolytic activity of CIK cells. Cytokine secretion was shown to be important for activation of CIK cells, and also cellular interactions between DCs and effector cells caused a higher cytolytic capacity. Interactions between DCs and CIK cells caused changes in the surface molecule expression of both populations, led to an increase of IL-12 secretion, and rendered an improved cytotoxic activity. The natural killer T cell subpopulation seems to be responsible for this effect. Therefore, coculture of DCs with CIK cells may have a major impact on immunotherapeutic protocols for patients with cancer.

Cellular characteristics of peripheral blood lymphocytes and tumour-infiltrating lymphocytes in patients with gynaecological tumours.

Abstract Immunotherapy of gynaecological cancer with tumour-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) has become a valid treatment modality with varying degrees of success in obtaining an antitumour response. TIL consist of lymphocytes, mainly T cells and minor populations of natural killer cells or B cells. Conventional cytogenetic studies of tumour cells from patients with breast and ovarian cancer have shown multiple chromosomal abnormalities including chromosomes 7 and 12. This study was designed to analyse the surface further, as well as investigate the intracellular, characteristics of TIL by multicolour flow cytometry and the cytogenetic features by fluorescence in situ hybridization. Tumour cell, peripheral blood and TIL samples from 25 patients (15 ovarian tumours, 8 breast cancers, 1 uterine sarcoma, 1 cervical carcinoma) were analysed for their phenotype, the expression of major cytokines [interleukin-2 (IL-2), IL-4 and interferon γ (IFNγ)], their proliferation rate, their cytotoxic ability and for the presence of numerical aberrations of chromosomes 7 and 12. All the tumour cells showed a high frequency of numerical aberration in chromosomes 7 and 12, especially trisomies or tetrasomies and combined aberrations. Trisomies of both chromosomes also occured at a low percentage in TIL and PBL.

Stem cell marker expression in small cell lung carcinoma and developing lung tissue

Histopathologic and clinical findings suggest that small cell lung cancer is derived from a multipotent proximal airway epithelial cell. In order to investigate the histogenetic origin of small cell lung cancer, we compared stem cell marker expression in human fetal lung tissue, human adult bronchial tissue, and a cohort of 64 small cell lung cancers. Supporting derivation of a multipotent precursor cell, 87.5% (56/64) of small cell lung cancers showed a dot-like expression of podocalyxin-like protein 1 (PODXL-1), a marker of embryonic and hematopoetic stem cells. Of small cell lung cancers, 98.4% (63/64) ubiquitously expressed Bmi-1, a key player in self-renewal of stem cells. Oct4 and AP2gamma were not expressed. Although podocalyxin-like protein 1 did not correlate with p53 or Wilms tumor suppressor 1, known regulators of podocalyxin-like protein 1, we could demonstrate demethylated CpG islands in the podocalyxin-like protein 1 promoter in small cell lung cancer, indicating epigenetic regulation. During fetal lung development and within adult bronchial mucosa, Bmi-1 was expressed ubiquitously. In contrast, podocalyxin-like protein 1 was detected in few stromal cells during the pseudoglandular phase (n = 7) and, importantly, in clustered epithelial cells within proximal bronchi and the trachea during the canalicular phase (n = 10). Interestingly, podocalyxin-like protein 1 was not expressed in normal or metaplastic adult bronchial epithelium (n = 36) but was expressed in sparse epithelial cells in half of the cases of normal tumor adjacent bronchial mucosa (20/40). Taken together, we show that small cell lung cancers and clustered epithelial cells in developing proximal bronchi share the expression of stem cell markers, suggesting a possible histogenetic link.

Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells

Abstract. Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-γ secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.

CPG ISLAND HYPERMETHYLATION OF CELL-FREE SERUM DNA INDICATES WORSE OUTCOME IN PATIENTS WITH BLADDER CANCER

912 CPG ISLAND HYPERMETHYLATION OF CELL-FREE SERUM DNA INDICATES WORSE OUTCOME IN PATIENTS WITH BLADDER CANCER Patrick J Bastian*, Jorg Ellinger, Nadja El Kassem, Lukas C Heukamp, Swapna Matthews, Figen Cubukluoz, Philip Kahl, Frank Perabo, Alexander von Rucker, Stefan C Muller. Munich, Germany, and Bonn, Germany. INTRODUCTION AND OBJECTIVE: CpG island (CGI) hypermethylation is a frequent event in bladder carcinogenesis and progression. Our study was designed to investigate the diagnostic and prognostic value of CGI hypermethylation in cell-free serum DNA of bladder cancer (BCA) patients. METHODS: The study cohort consisted of 45 patients with

Absent immunoglobulins in HIV-related Burkitt lymphoma/leukaemia.

Dear Editor: Burkitt lymphoma (BL) behaves in a very aggressive fashion when presenting as acute leukaemia [1]. It is classified into three groups: endemic, sporadic and immunodeficiencyassociated BL [2]. Histologically, BL can occur in three variants: classic, plasmacytoid and atypical Burkitt/Burkittlike lymphoma [3]. All forms of BL are associated with translocations of the c-myc gene on chromosome 8. In this report, we describe a 59-year-old human immunodeficiency virus (HIV)-positive male patient who initially presented with leukopaenia and anaemia (haematocrit 24, red blood cells 3.0 T/l, haemoglobin 8.9 g/dl, white blood cells 1.9 g/l, platelets 149 g/l) in the outpatient clinic department. Further clinical history included opportunistic cryptococcal meningitis and cytomegalovirus infection. Therefore, HIV infection was classified as CDC C3. The patient was treated with highly active anti-retroviral therapy. Eight months later, the patient was admitted with a 1month history of epistaxis, shortness of breath on exertion, fatigue and weight loss. There was no history of fever or night sweats. On physical examination, marked pallor, weakness and splenomegaly without lymphadenopathy were noted. Chest radiograph showed an acute pneumonia, which was complicated by acute prerenal failure. At this point, the patient demonstrated macrocytic anaemia (haemoglobin 6 g/l, mean corpuscular volume 104 fl) and thrombocytopenia (14 g/l) with a white blood cell count in the lower range (5.9 g/l) and the differential blood count revealing 38% blasts. CD4 count was 140/μl. The bone marrow showed an infiltration rate of greater than 90% by leukaemic cells. Epstein–Barr virus (EBV)-IgG (anti-EBNA 1) was positive by enzyme-linked immunosorbent assay, supporting previous EBV infection. After clinical stabilization, the patient underwent intensive combined chemotherapy including a prephase therapy with cyclophosphamide and prednisolone followed by a highdosed chemotherapy consisting of methotrexate, ifosfamide, teniposide, cytarabine, dexamethasone and additionally intrathecal methotrexate application. After the first cycle of chemotherapy, the patient developed a generalised tonic–clonic seizure for the first time, which did not respond to anti-epileptic drugs. Cranial computed tomography showed a subdural haemorrhage, and the patient died shortly after this event. Eight months before hospital admission, the bone marrow was normocellular with left-shifted myelopoesis and sparse scattered small B and T lymphocytes including a small lymphoid aggregate with CD20-positive small B cells. However, on hospital admission, the bone marrow showed the typical histological findings of BL with atypical medium-size lymphocytes arranged diffusely in a ‘starry sky pattern’ with numerous tingible body macrophages (Fig. 1a). High cell turnover was further supported by many mitotic figures and abundant spontaneous cell death. The atypical lymphoid cells expressed CD10 (Fig. 1b) and CD79a, whereas IgM was not expressed by immunohistoAnn Hematol (2008) 87:255–256 DOI 10.1007/s00277-007-0389-0