Immune cells regulate VEGF signalling via release of VEGF and antagonistic soluble VEGF receptor-1.

Vascular endothelial growth factor (VEGF) is an important regulator of physiological and pathological angiogenesis. Besides malignant and stromal cells, local immune cells shape VEGF signalling in the tumour microenvironment. Aminobisphosphonates such as zoledronic acid (Zol) are drugs known to inhibit osteoclast activity and bone resorption, but also have immunomodulatory and anti-tumour effects. These properties have been linked previously to the down-regulation of VEGF and interference with tumour neo-angiogenesis. It was therefore surprising to find that treatment with Zol in combination with low-dose interleukin (IL)-2 increased serum VEGF levels in cancer patients. In this study we aimed to characterize the effect of Zol and IL-2 on VEGF signalling of blood-derived immune cells in vitro. Upon stimulation with IL-2, T cells and natural killer (NK) cells increase production of VEGF consecutively to the release of proinflammatory interferon (IFN)-γ, and Zol accelerates this response specifically in γδ T cells. VEGF can, in turn, be antagonized by soluble VEGF receptor (sVEGFR)-1, which is released depending on stimulatory conditions and the presence of monocytes. Additionally, malignant cells represented by leukaemia and lymphoma cell lines produce VEGF and some release sVEGFR-1 simultaneously. Our findings indicate a mechanism by which the VEGF and the sVEGFR-1 production by immune cells regulates local VEGF signalling. Therefore, immunotherapeutic interventions may enable both pro- as well as anti-tumour effects via immune cell-mediated alterations of VEGF homeostasis.

First-line therapy of peripheral T-cell lymphoma: extension and long-term follow-up of a study investigating the role of autologous stem cell transplantation.

Current guidelines recommend consolidation with autologous stem cell transplantation (autoSCT) after induction chemotherapy for most patients with peripheral T-cell lymphoma (PTCL). This assumption is based on five prospective phase II studies, three of which included <50 patients with limited follow-up. Here we present the final analysis of the prospective German study. The treatment regimen consisted of four to six cycles of CHOP chemotherapy followed by mobilizing therapy and stem cell collection. Patients in complete remission (CR) or partial remission (PR) underwent myeloablative chemo(radio)therapy and autoSCT. From January 2001 to July 2010, 111 patients were enrolled in the study. The main subgroups were PTCL not specified (n=42) and angioimmunoblastic T-cell lymphoma (n=37). Seventy-five (68%) of the 111 patients received transplantation. The main reason for not receiving autoSCT was progressive disease. In an intent-to-treat analysis, the complete response rate after myeloablative therapy was 59%. The estimated 5-year overall survival, disease-free survival and progression-free survival rates were 44%, 54% and 39%, respectively. The results of this study confirm that upfront autoSCT can result in long-term remissions in patients with all major subtypes of PTCL and therefore should be part of first-line therapy whenever possible.

Clinical-scale single-step CD4(+) and CD8(+) cell depletion for donor innate lymphocyte infusion (DILI).

The ability to selectively deplete or enrich cells of specific phenotype by immunomagnetic selection to reduce the risk of GVHD holds significant promise for application in adoptive immunotherapy. Current clinical-scale approaches for T-cell depletion (e.g., CD34(+) selection, CD3(+) depletion), usually deplete gammadelta T cells, which may be advantageous in mediating graft-versus-tumor (GVT) effects and augmenting the innate immune response against infections. Here, we present a new method for depletion of T cells with potential GVHD reactivity by using a single-step immunomagnetic protocol, which efficiently depletes CD4(+) and CD8(+) alphabeta T cells under good manufacturing practice (GMP) conditions. Depletion from unstimulated leukapheresis products (n=6) containing up to 2.0 x 10(10) cells showed high efficiency (mean log depletion of CD4(+) cells: 4.12, CD8(+) cells: 3.77). In addition, immunomagnetic CD4/CD8 depletion resulted in passive enrichment of innate lymphocytes (mean recovery of natural killer (NK) cells: 38%, gammadelta T cells: 50%). We demonstrated that gammadelta/NK cells preserved their proliferative and cytotoxic capacity and conclude that simultaneous large-scale depletion of CD4(+)/CD8(+) T cells is feasible and can be performed under GMP conditions with high-depletion efficacy for alphabeta T cells and recovery of functionally intact innate effector lymphocytes for potential use in adoptive immunotherapy studies.

Effects of recombinant human thrombopoietin alone and in combination with erythropoietin and early-acting cytokines on human mobilized purified CD34+ progenitor cells cultured in serum-depleted medium.

The effects of recombinant thrombopoietin (TPO) alone and in combination with erythropoietin (EPO) and early-acting cytokines such as interleukin 3 (IL-3), stem cell factor (SCF) and GM-CSF on highly purified mobilized human CD34+ progenitor cells were studied in a serum-depleted culture system. Eight leukapheresis samples were cultured for seven days and analyzed; aliquots were replated and re-evaluated on day 12. Three-color flow cytometry was used together with morphologic analysis to determine proliferation and megakaryocytic or erythroid maturation. TPO alone was sufficient for cell survival and proliferation in serum-depleted medium. In the absence of other growth factors, almost all CD34+ cells differentiated along the megakaryocytic pathway within 12 days. Concomitantly, the progenitor cells gradually acquired the morphologic features of mature megakaryocytes. After exposure to TPO for one week, 50% of the cells still expressed CD34; by day 12 the remaining CD34+ cells (11%) were all coexpressing CD41. TPO alone did not support proliferation of glycophorin-A-positive cells. The addition of TPO to early-acting cytokines (EPO, GM-CSF, SCF and/or IL-3) not only increased the overall megakaryocyte expansion, but also generated a different maturation pattern of the CD41+ megakaryocyte progenitors. It further doubled the number of erythroid cells and c-kit+ cells in the second week of culture. Interestingly, the overall number of CD34+ cells was increased about fivefold when TPO was added to the early-acting cytokines, with a marked expansion of the CD34+/CD41+ and CD34+/CD117+ subpopulations. TPO can augment the pool of committed progenitors, thereby increasing the number of its own target cells and the number of EPO-responsive cells. These properties make TPO an interesting cytokine for the ex vivo expansion of human progenitor cells.

LW/SO cell line: a tool for studying the phenotypical characterization and commitment of hematopoietic stem cells.

We report our observations with the cell line LW/SO, which was recently derived from the bone marrow of a patient with acute myeloid leukemia. Based on the morphological and histochemical examination, the leukemic cells were classified primarily as FAB type M4. However, 2 years later, in relapse, the cells changed their morphology and were hence specified as FAB type M2 (slightly positive for acid phosphatase and Sudan black). The cells established have now been in culture for approximately 11 months and display nearly 100% CD4/5/7/15/25/71/120a,b at varying densities. Some of them spontaneously and reversibly become either CD34 + /38- or CD34 - /38+, yet the majority of the cells remain negative for both. All attempts to separate the cells with a distinct phenotype by limiting dilution or sorting through a flow cytometer failed repeatedly. The subsets, enriched up to 98% (regardless of their primary immunophenotype CD34 - / 38-, CD34 + /38-, or CD34 - /38+), soon displayed a phenotypical constellation similar to that before sorting. The ratio of CD34- to CD34+ seems to be influenced by the cell density: The greater the cell-to-cell contact, the lower the percentage of CD34-expressing cells. Some of the cells apparently differentiate into T-cell phenotype and acquire CD3 and T-cell receptor (TCR) alpha/beta molecules. While the quantity of CD34-expressing cells significantly increased in the presence of dexamethasone (10(-7) M), and some of them additionally acquired CD33 antigen, the percentage of CD3-positive cells was enhanced by adding 1% DMSO in medium. In contrast, cytokines such as IL-1, IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, or SCF (c-kit ligand) altered neither the proliferation capacity nor the phenotypical constellation of LW/SO cells (each tested alone). Although normal karyotype was obtained from the bone marrow cells, the LW/SO cells revealed a homogeneous chromosomal composition of 45, X, -X, der(9) inv(9) (p12q13) del(9) (p22?). These data suggested that LW/SO cells might be the leukemic counterpart of putative pre-CD34-positive progenitors. In order to substantiate this assumption, we analyzed the expression of other so-called T-cell markers on CD34+ cells from peripheral blood stem cell aphereses of five patients who later underwent high-dose chemotherapy and subsequent stem cell retransfusion. These data clearly revealed that a considerable amount of CD34+ hematopoietic progenitors co-express CD2/4/(5)/(7)/25 at an early stage of differentiation, and support the notion that CD34-negative LW/SO cells with the surface markers CD4/5/7/25 are probably phenotypical representatives of pluripotent stem cell. Hence, not all CD34-negative populations with so-called T-cell surface markers should be considered T-cells; some may constitute the ancestor of CD34 antigen-expressing progenitors.

Regulation of the density of the stem cell factor receptor (c-kit) by tumor necrosis factor alpha on a human myeloid cell line.

The GM/SO cell line bears a high level of stem cell factor receptors (SCF-R) i.e. c-kit protein, and therefore constitutes a potential model for studying the regulation of this crucial receptor on myeloid cells. In this study we evaluated the effect of tumor necrosis factor alpha (TNF-alpha) on the expression of SCF-R by flow cytometry. In contrast to 1 hour of preincubation, the experiments carried out after 24 hours preincubation revealed that TNF-alpha, if added alone, reduced the density of SCF-R on GM/SO cells in a dose-dependent manner. However, if combined with GM-CSF, which per se downregulates the SCF-R on these cells as well, TNF-alpha antagonized the effect of GM-CSF and slightly increased the density of SCF-R. Yet the cells incubated for 24 hours in medium without cytokines invariably expressed a higher level of SCF-R than the cells incubated in the presence of TNF-alpha and GM-CSF, either alone or in combination. In contrast to these cytokines, stem cell factor (SCF), which was also tested simultaneously in all experiments, downregulated its own natural receptor on these cells also after a preincubation of 1 hour. Furthermore, prolonged exposure of GM/SO cells to TNF-alpha for 5-7 days yielded a monocyte-macrophage-like morphology of some cells as these cells displayed an apparent glass and plastic adherence. In contrast, no such morphological changes could be observed in the presence of GM-CSF or SCF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) reduces the density of stem cell factor receptors (c-kit oncogene product) on a GM-CSF-dependent human myeloid cell line.

By employing a monoclonal antibody against the stem cell factor receptor (SCF-R), c-kit oncogene product, we analysed in flow cytometric technique the density of SCF-R on GM/SO cells which were incubated under various culture conditions. These experiments revealed that there is an inverse correlation between the SCF-R density on the cells and the doses of granulocyte-macrophage colony-stimulating factor (GM-CSF) in culture medium; the lower the dose, the higher the density of SCF-R on the cells. More detailed analyses showed that, in contrast to SCF which rapidly downregulates its own receptor, GM-CSF does not alter the measurable level of SCF-R in an exposition period of 60 minutes, which suggests that the internalization or shedding of the receptor is not the mechanism of action. Since the most striking difference regarding density of SCF-R between GM-CSF-treated and untreated cells was observed on day 2, the modulation of c-kit oncogene protein by GM-CSF likely occur prior to expression of protein onto the cell surface. In order to exclude the possibility that altered cell viability due to insufficient GM-CSF content in culture medium might be responsible for the increased SCF-R densities on GM-CSF-dependent cells, we subsequently generated a GM-CSF-independent subclone which still responded to GM-CSF as well as the dependent did. The experiments carried out with this subclone confirmed the results presented above. Thus our data suggest that GM-CSF is directly involved in the regulation of SCF receptor density on GM/SO cells.