Andreas Draube

Constitutive Expression of c-FLIP in Hodgkin and Reed-Sternberg Cells

Crosslinking of the transmembrane receptor CD95/Fas leads to activation of a signaling cascade resulting in apoptosis. c-FLIP is a recently described protein that potently inhibits Fas-mediated apoptosis and has been shown to be a key factor in germinal center B cell survival. Because Hodgkin and Reed-Sternberg cells in classical Hodgkins disease (cHD) are also resistant to Fas-mediated apoptosis we studied the role of c-FLIP in classical HD. High levels of c-FLIP protein were identified in two Fas-resistant Hodgkin-derived cell lines. In contrast to other tumor cells, inhibition of protein synthesis by cycloheximide did not lead to down-regulation of c-FLIP protein in these HD cell lines. Furthermore, Fas-mediated apoptosis was only partially restored suggesting that normal regulation of c-FLIP was disrupted. The in vivo relevance of these findings was supported by demonstration of significant c-FLIP expression by immunohistochemistry in 18 of 19 evaluable cases of primary HD. Taken together, c-FLIP is constitutively expressed in HD and may therefore be a major mechanism responsible for Fas-resistance in HD.

CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential

Cellular adjuvants such as dendritic cells (DC) are in the focus of tumour immunotherapy. In DC‐vaccine trials, induction of tumour antigen‐specific immunity is observed frequently and well‐documented clinical responses have been reported. However, the overall response rate is less than 3%, therefore alternative strategies are being investigated. CD40‐activated B cells (CD40‐B) have been characterized previously as an interesting alternative because they present antigen efficiently and can be expanded by several logs from small amounts of peripheral blood. To determine the central technical challenges of cell‐based vaccines we performed a single‐patient analysis of 502 patients from DC‐based tumour vaccine trials and identified at least three factors contributing to their limited efficiency: (1) lack of cell numbers; (2) lack of documented purity thus high contamination of bystander cells; and (3) lack of quality control and thus heterogeneous or unknown expression of important surface molecules such as major histocompatibility complex (MHC) and chemokine receptors. Based on these findings we re‐evaluated the CD40‐B approach in cancer patients. Here, we show that proliferation of B cells from cancer patients is equivalent to that observed in healthy donors. Purity is always > 90% after 2 weeks and remains stable for several weeks. They have comparable antigen‐presenting capability determined phenotypically and by allogeneic mixed lymphocyte reaction. Expression of CCR7 and CD62L was detected in all samples and B cells migrated towards the relevant homing chemokines. Taken together, CD40‐B cells from cancer patients can be expanded in virtually unlimited numbers at high purity and full function concerning antigen‐presentation and migratory properties.

Immunomagnetic enrichment and detection of isolated tumor cells in bone marrow of patients with epithelial malignancies.

The detection of isolated tumor cells (ITC) in the bone marrow of patients with epithelial malignancies is an independant prognostic factor for several entities as breast cancer, colorectal cancer or non-small lung cancer. However, with conventional immunocytology using Ficoll density gradient and APAAP staining, only a small proportion of the bone marrow samples can be scanned for cytokeratin-positive (CK+) cells. To improve detection rates, we evaluated the enrichment of ITC by magnetic activated cell sorting (MACS) compared to regularly stained cytospins. Recovery experiments with a CK+ breast cancer cell line (SKBR3) were performed to calculate the MACS enrichment rate. Bone marrow was obtained by aspiration from 20 patients with carcinomas of epithelial origin and from 17 controls. ITC were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to cytokeratin 7 and 8. MACS of SKBR3 seeded in peripheral blood revealed average recovery rates of 62% and 48% and average enrichment factors of 104-fold and 8139-fold of the CK+ cells after one and after two separations, respectively. After immunomagnetic enrichment, CK+ cells were detected in 16 of 20 (80%) cancer patients, whereas only 7 (35%) patients showed CK+ cells without magnetic enrichment (P=0.002). Ten of twelve (83%) patients with metastatic disease (stage M1) and six of eight (75%) patients without any overt metastases (M0) had CK+ cells in their bone marrow. None of the negative controls showed any CK+ cells. Enrichment with magnetically labeled anti cytokeratin antibodies increases the detection rate of epithelial cells in bone marrow of cancer patients compared to immunocytology.

Clinicopathological Characteristics of RET Rearranged Lung Cancer in European Patients

Introduction Rearrangements of RET are rare oncogenic events in patients with non–small cell lung cancer (NSCLC). While the characterization of Asian patients suggests a predominance of nonsmokers of young age in this genetically defined lung cancer subgroup, little is known about the characteristics of non‐Asian patients. We present the results of an analysis of a European cohort of patients with RET rearranged NSCLC. Methods Nine hundred ninety‐seven patients with KRAS/EGFR/ALK wildtype lung adenocarcinomas were analyzed using fluorescence in situ hybridization for RET fusions. Tumor specimens were molecularly profiled and clinicopathological characteristics of the patients were collected. Results Rearrangements of RET were identified in 22 patients, with a prevalence of 2.2% in the KRAS/EGFR/ALK wildtype subgroup. Co‐occurring genetic aberrations were detected in 10 patients, and the majority had mutations in TP53. The median age at diagnosis was 62 years (range, 39–80 years; mean ± SD, 61 ± 11.7 years) with a higher proportion of men (59% versus 41%). There was only a slight predominance of nonsmokers (54.5%) compared to current or former smokers (45.5%). Conclusions Patients with RET rearranged adenocarcinomas represent a rare and heterogeneous NSCLC subgroup. In some contrast to published data, we see a high prevalence of current and former smokers in our white RET cohort. The significance of co‐occurring aberrations, so far, is unclear.

Activation of autologous leukemia-specific T cells in acute myeloid leukemia: monocyte-derived dendritic cells cocultured with leukemic blasts compared with leukemia-derived dendritic cells.

In acute myeloid leukemia (AML) blasts can be differentiated into dendritic cell (DC) like cells (AML‐DC). These cells have a mature DC‐like phenotype, are strong stimulators in mixed leukocyte reactions and can be used to generate leukemia‐specific cytotoxic T cells. However, recent reports about naturally existing leukemic DC with immunoregulatory dysfunctions in peripheral blood of AML patients caused concerns about the use of AML‐DC for therapeutic purposes.

The immunosuppressive factors IL-10, TGF-β, and VEGF do not affect the antigen-presenting function of CD40-activated B cells

BackgroundProgress in recent years strengthened the concept of cellular tumor vaccinations. However, a crucial barrier to successful cancer immunotherapy is tumor-mediated immunosuppression. Tumor-derived soluble factors such as IL-10, TGF-β, and VEGF suppress effector cells either directly or indirectly by disruption of dendritic cell (DC) differentiation, migration and antigen presentation. Human B cells acquire potent immunostimulatory properties when activated via CD40 and have been shown to be an alternative source of antigen-presenting cells (APCs) for cellular cancer vaccines. Nevertheless, in contrast to DCs little knowledge exists about their susceptibility to tumor derived immunosuppressive factors. Thus, we assessed whether IL-10, TGF-β, or VEGF do affect key aspects of the immunostimulatory function of human CD40-activated B cells.MethodsCell surface expression of adhesion and costimulatory molecules and the proliferation capacity of CD40-activated B cells were compared to untreated controls by flow cytometry. Migration towards important chemokines of secondary lymph organs was measured with or without exposure to the immunosuppressive cytokines. Finally, an influence on T cell stimulation was investigated by allogeneic mixed lymphocyte reactions. For statistical analysis Student’s t test or two-way analysis of variance followed by Bonferronis post-hoc test was used to compare groups. P values of <0.05 were considered statistically significant.ResultsNeither cell adhesion nor the expression of MHC class II and costimulatory molecules CD80 and CD86 was inhibited by addition of IL-10, TGF-β, or VEGF. Likewise, the proliferation of CD40-activated B cells was not impaired. Despite being exposed to IL-10, TGF-β, or VEGF the B cells migrated equally well as untreated controls to the chemokines SLC and SDF-1α. Most importantly, the capacity of CD40-activated B cells to stimulate CD4+ and CD8+ T cells remained unaffected.ConclusionOur findings suggest that key immunostimulatory functions of CD40-activated B cells are resistant to inhibition by the immunosuppressive factors IL-10, TGF-β, and VEGF. This supports considerations to use ex vivo generated CD40-activated B cells as a promising alternative or additional APC for cellular immunotherapy, especially in settings where these immunosuppressive cytokines are present in tumor environment.

High platelet contamination in progenitor cell concentrates results in significantly lower CD34+ yield after immunoselection.

BACKGROUND: Selection of CD34+ cells by specific immunoselection leads to a significant loss of those cells. The factors influencing the yield and purity are not well identified. The results of CD34+ selection from peripheral blood progenitor cells (PBPCs) with high and low platelet contamination that are harvested with two different cell separators are reported.

Efficient activation of autologous tumor-specific T cells: a simple coculture technique of autologous dendritic cells compared to established cell fusion strategies in primary human colorectal carcinoma

Different technologies have been employed to deliver the whole spectrum of tumor antigens (TAs) to dendritic cells (DCs) to be presented to T cells. These include whole tumor RNA-transfected DCs, preparations of DCs loaded with tumor-derived apoptotic bodies or tumor cell lysates, and DC tumor cell fusions. Early clinical trials have been conducted using such techniques. The presented study was aimed to revisit the necessity of tumor cell manipulation in DC-based immunotherapy strategies for colorectal carcinoma. We investigated a simple coculture method of autologous monocyte-derived DCs and human primary colorectal carcinoma (pCC) in comparison with 2 well-described cell fusion strategies for the efficacy of uptake, processing and presentation of TAs to autologous T cells. Before coculture or fusion, pCC had been cryopreserved without further manipulation. Fluorescence microscopy and flow cytometry analyses of fluorescent dye labeled cells were used for monitoring engulfment of pCC by DCs. The coculture procedure resulted in a double positive cell fraction of up to 22% and thus was comparable to that observed after cell fusion. More important, DCs after coculture with autologous pCC induced significant tumor-specific interferon-γ–producing autologous T cells in the same number of patients as DC/pCC fusions. Furthermore, tumor-specific major histocompatibility complex class I restricted cytotoxic T lymphocytes were generated by stimulation with DCs cocultured with pCC. In prior studies for human carcinomas coculture techniques were described to be inferior. In contrast, our data strongly suggest that at least for human pCC and autologous DCs this simple coculture method is similarly efficient compared to established fusion techniques.

Suppression of the tumorigenic growth of Burkitt's lymphoma cells in immunodeficient mice by cytokine gene transfer using EBV-derived episomal expression vectors

Epstein‐Barr virus (EBV)‐based expression vectors were tested for cytokine gene transfer‐mediated induction of an immune response against human lymphoma cells. These vectors express the EBV latent gene EBNA 1 and carry the EBV latent origin of replication (ori P) for episomal replication in transfected cells. In addition, 3 human immunoglobulin light chain enhancer elements augment expression in B‐cells. The suitability of these vectors for expression of cytokine genes in human lymphoma cells in vitro has been demonstrated. In order to extend these experiments in vivo, highly tumorigenic Burkitts lymphoma (BL) cells were transfected with different cytokine genes of human and murine origin cloned into the EBNA 1/ori P vectors. Tumorigenicity of the transfectants was measured after inoculation into nude mice. No effect on tumorigenicity was observed after hIL 6 transfection and an inconsistent effect after hTNFα transfection. In contrast, complete suppression of tumor outgrowth occurred in hIL 10 transfectants. This tumor suppressive effect, however, was restricted to the IL 10 transfectants themselves and not directed against non‐transfected cells. By comparison, mIL 4 transfected BL cells also were non‐tumorigenic. However, co‐inoculation of mIL 4 transfected and non transfected cells resulted in suppression of the tumorigenicity of the non‐transfected cells. Thus, highly tumorigenic BL cells in nude mice are sensitive to immune effector mechanisms triggered by cytokine expression. In this experimental model, EBNA 1/ori P expression vectors are a suitable tool for cytokine gene transfer mediated induction of an anti‐lymphoma immune response of the host. Int. J. Cancer 86:301–306, 2000.

The shared tumor associated antigen cyclin‐A2 is recognized by high‐avidity T‐cells

Cyclin‐A2, a key cell cycle regulator, has been shown to be overexpressed in various types of malignancies with little expression in normal tissue. Such tumor‐associated genes potentially are useful targets for cancer immunotherapy. However, high‐avidity cyclin‐specific T cells are considered to be thymically deleted. We identified at least one nonameric HLA‐A*0201 binding cyclin‐A2 epitope by a reverse immunology approach. Using a highly efficient T‐cell expansion system that is based on CD40‐activated B (CD40‐B) cells as sole antigen‐presenting cells we successfully generated cyclin‐A2 specific CTL from HLA‐A*0201+ donors. Interestingly, high‐avidity cyclin‐A2 specific CTL lines, which recognized peptide‐pulsed and antigen expressing target cells, were indeed generated by stimulation with CD40‐B cells when pulsed with low concentrations of peptide, whereas CD40‐B cells pulsed at saturating concentrations could only induce low‐avidity CTL, which recognized peptide‐pulsed target cells only. One high‐avidity CTL line was subcloned and CTL clones, whose peptide concentration required for half‐maximal lysis were less than 1 nM, could lyse cyclin‐A2 expressing tumor cells. Taken together, cyclin A2 is an attractive candidate for immune intervention in a significant number of cancer patients and high‐avidity T cells can be readily generated using CD40‐B cells as antigen‐presenting cells.