Transformation of mature mouse B cells into malignant plasma cells in vitro via introduction of defined genetic elements.

An experimental system where defined alterations in gene function or gene expression levels in primary B cells would result in the development of transformed plasma cells in vitro would be useful in order to facilitate studies of the underlying molecular mechanisms of plasma cell malignancies. Here, such a system is described in which primary murine B cells rapidly become transformed into surface CD138+ , IgM-/low , CD19- IgM-secreting plasma cells as a result of expression of the transcription factors IRF4 and MYC together with simultaneous expression of BMI1, mutated p53 or silencing of p19Arf , and suppression of intrinsic apoptosis through expression of BCLXL. Analysis of gene expression patterns revealed that this combination of transforming genes resulted in expression of a number of genes previously associated with terminally differentiated B cells (plasma cells) and myeloma cells, whereas many genes associated with mature B cells and B-cell lymphomas were not expressed. Upon transplantation, the transformed cells preferentially localized to the bone marrow, presenting features of a plasma cell malignancy of the IgM isotype. The present findings may also be applicable in the development of novel methods for production of monoclonal antibodies.

Expression of the Epstein-Barr virus-encoded Epstein-Barr virus nuclear antigen 1 in Hodgkin's lymphoma cells mediates Up-regulation of CCL20 and the migration of regulatory T cells.

In approximately 50% of patients with Hodgkin's lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.

Immunomagnetic separation of normal rat testicular cells from Roser's T-cell leukaemia cells is ineffective.

Elimination of contaminating malignant cells is crucial to avoiding cancer relapse in association with transplantation of autologous spermatogonial stem cells. In the clinical setting, there is presently no effective procedure for separating testicular cells from cancer cells. Here, CD4, a selective surface marker for Roser's rat leukaemic T-cells, was utilized to eliminate cancer cells from testicular cell samples from leukaemic piebald variegated (PVG) rats by magnetic-activated cell sorting (MACS). All animals receiving MACS-selected testicular cells died within 14-15 days. Only one-third of the contaminating leukaemic cells could be removed from testicular samples. An increase in antibody concentration enhanced the proportion of leukaemic cells removed from 27 to 49%. Variations in the cell size and expression of surface antigens on testicular leukaemic cells were the major obstacles to purification based on this marker. It is concluded that MACS does not prevent transmission of leukaemia to syngenic PVG rats when cells from leukaemic testes are used for testicular cell transplantation.

Three-dimensional culturing of the Hodgkin lymphoma cell-line L1236 induces a HL tissue-like gene expression pattern.

To overcome some limitations of in vitro established cell-line tumor models for Hodgkin lymphoma (HL), we explored whether culturing in a three-dimensional (3D) matrix could improve the quality of the model. We used a novel designer peptide based self-organizing matrix. The gene expression profile of the 3D-cultured HL derived cell-line L1236 was compared with that of suspension-cultured (2D) L1236, as well as to the gene expression profile found in HL tumor samples. To validate our results we also included a gene expression data set of laser captured Hodgkin-Reed - Sternberg (H-RS) cells. The gene expression profiles were analyzed using Affymetrix technology. We found that the 3D culture affected gene expression of a HL derived cell-line inducing a more tumor-related expression profile. 3D culture affected the expression of 500 genes in L1236, upregulating genes involved in immune response and apoptosis and downregulating genes involved in cell division. It also affected genes involved in actin filament polymerization.

Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model.

Current vaccine development includes optimization of antigen delivery to antigen presenting cells, such as dendritic cells (DC). Particulate systems have attracted increasing attention in the development of vaccine delivery systems. In the present study, we investigated DC uptake of model fluorescent polystyrene particles with a broad size range and variable surface properties. Localization of particles was investigated using confocal laser scanning microscopy and uptake was quantified by flow cytometry. Immature DC were generated from mononuclear cells isolated from human blood. The polystyrene particles interacted with the DC throughout the tested diameter range of 0.04-15 microm in a time- and concentration-dependent manner. The optimal particle diameter for fast and efficient acquisition by a substantial percentage of the DC was 0.5 microm and below. The surface of 1 and 0.1 microm polystyrene particles was covalently modified with different polyaminoacids/proteins, yielding particles with varying surface charge. Uptake of 1 microm particles was greatly enhanced when particles displayed a positive surface charge. In general, the present findings establish that particle diameters of 0.5 microm and below were optimal for DC uptake; however uptake of larger particles could be greatly enhanced by rendering the particle surface positive. Whether increased particle uptake is correlated with increased immune responses, remains to be established.

Stimulation of T-cell cytokine production and NK-cell function by IL-2, IFN-alpha and histamine treatment during remission of non-Hodgkin's lymphoma.

Limited therapeutic options remain for patients with relapsing lymphoma following chemotherapy and autologous stem cell transplantation (ASCT), hence motivating investigations of complementary treatments. The aim of the present study was to evaluate feasibility and immunological effects of an immunotherapy schedule administered during chemotherapy-induced remission of aggressive non-Hodgkins lymphoma (NHL). Repeated cycles of rIL-2, rIFN-alpha and histamine were administered to a patient with a grade III follicle center cell lymphoma, following relapse and high-dose chemotherapy with stem cell support. T-cell cytokine production and repertoire alterations were monitored by flow cytometry together with assessment of natural killer (NK) cell-mediated cytotoxicity. The treatment schedule induced significant increases in frequencies of CD4+ T cells expressing intracellular IFN-gamma or IL-4, thus a T helper (Th) 1 and Th 2 type of response were observed. CD8+T cells showed enhancement mainly of TNF-alpha production. Such induction of T-cell effector functions was accompanied by an augmentation of NK-cell cytotoxicity and a pronounced reduction of possibly regulatory CD57 expressing lymphocytes. The results indicate synergistic T- and NK-cell activation by tolerable doses of the combined immunotherapy, administered during remission after chemotherapy and ASCT in NHL.

Expansion of immunoglobulin autoreactive T-helper cells in multiple myeloma.

Activation and expansion of T helper (Th) cells followed by regulation of activation are essential to the generation of immune responses while limiting concomitant autoreactivity. In order to characterize T cells reactive towards myeloma-derived monoclonal immunoglobulin (mIg), an autologous coculture assay for single-cell analysis of mIg-responding cells was developed. When cultured with dendritic cells loaded with mIg, CD4(+) Th cells from patients with progressing multiple myeloma (MM) showed a proliferative MHC class II-dependent response. CD8(+) T-cell reactivity and Th1 activation were consistently low or absent, and Th2 and regulatory cytokines were expressed. The presence of such non-Th1 CD4(+) T cells in peripheral blood was independent of treatment status, while the frequencies of responding cells varied between patients and reached the same order of magnitude as those measured for tetanus toxoid-specific Th memory cells. Furthermore, investigations of T-cell subpopulations indicated a possible regulatory role on the mIg responsiveness mediated by suppressive CD25(high)FOXP3(+)CD4(+) T cells. It is proposed from the present results that a predominant in vivo activation of non-Th1 mIg-reactive CD4(+) T cells constitute an Ig-dependent autoregulatory mechanism in human MM, with possible tumor growth supporting or permissive effects.

Decontamination of leukemic cells and enrichment of germ cells from testicular samples from rats with Roser's T-cell leukemia by flow cytometric sorting.

Testicular germ cell transplantation is a novel strategy for preservation of fertility in prepubertal cancer patients, but the risk of reseeding tumor cells into cured patients presently limits clinical application of this approach. To date, no systematic evaluation of the limitations of surface marker-based decontamination of testicular samples with acute lymphoblastic leukemia has been performed. Here, surface markers for leukemic (CD4 and major histocompatibility complex class I) and germ cells (epithelia cell adhesion molecule) in testicular samples infiltrated with Roser's T-cell leukemia were identified. These markers were then used to delete leukemic cells and/or select for germ cells by flow cytometry (FACS). The resulting cell populations were analyzed by FACS, immunocytochemistry, or evaluation of leukemia transmission in syngeneic piebald variegated rats. Simple positive selection of germ cells or deletion of leukemic cells using specific surface markers was unable to effectively decontaminate testicular samples. The poor specificity of spermatogonial surface markers and aggregation of germ and leukemic cells limited the positive selection of germ cells, while immunophenotypic variation among lymphoblastic leukemia cells prevented adequate deletion of leukemic cells. Enzymatic treatment to disperse the testicular cells and feature of the intratesticular environment contributed to this immunophenotypic variation. Only germ cell selection in combination with leukemic cell deletion prevented leukemia transmission in association with intratesticular injection of the sorted cells. However, with such combined sorting, only 0.23% of the original testicular cells were recovered. With presently available techniques, flow cytometric purification of germ cells from a leukemic donor is not sufficiently effective or safe for clinical use.

Targeting vaccines to dendritic cells.

Dendritic cells (DC) are specialized antigen presenting cells (APC) with a remarkable ability to take up antigens and stimulate major histocompatibility complex (MHC)-restricted specific immune responses. Recent discoveries have shown that their role in initiating primary immune responses seems to be far superior to that of B-cells and macrophages. DC are localized at strategic places in the body at sites used by pathogens to enter the organism, and are thereby in an optimal position to capture antigens. In general, vaccination strategies try to mimic the invasiveness of the pathogens. DC are considered to play a central role for the provocation of primary immune responses by vaccination. A rational way of improving the potency and safety of new and already existing vaccines could therefore be to direct vaccines specifically to DC. There is a need for developing multifunctional vaccine drug delivery systems (DDS) with adjuvant effect that target DC directly and induce optimal immune responses. This paper will review the current knowledge of DC physiology as well as the progress in the field of novel vaccination strategies that directly or indirectly aim at targeting DC.

Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition.

Vaccine efficacy might be improved by exploiting the potent antigen presenting properties of dendrite cells (DCs), since their ability to stimulate specific major histocompatibility complex-restricted immune responses has been well documented during the recent years. In that light, we investigated how the interaction of antigen-containing liposomes with DCs was affected by the bilayer composition. Monocyte-derived human DCs and murine bone marrow-derived DCs were analysed and compared upon in vitro incubation with liposomes by flow cytometry and confocal microscopy. Anionic liposomes with a bilayer composition of phosphatidylcholine, cholesterol and phosphatidylglycerol or phosphatidylserine interacted with a limited fraction of the total DC population in case of both DC types. Inclusion of mannosylated phosphatidylethanolamine (Man-PE) for targeting to the mannose receptor (MR) increased the interaction of negatively charged liposomes with both human and murine DCs. This increase could be blocked in human DCs by addition of the polysaccharide mannan indicating that uptake might be mediated by the mannose receptor. Cationic liposomes containing trimethyl ammonium propane interacted with a very high percentage of both DC types and could be detected in high amounts intracellularly. In conclusion, liposome bilayer composition has an important effect on interaction with DCs and might be critical for the vaccination outcome.

Natural cytotoxicity to autologous antigen-pulsed dendritic cells in multiple myeloma.

To analyse autologous lymphocyte cytolytic activities of potential importance for cell-based immunotherapy in multiple myeloma (MM), in vitro differentiated dendritic cells (DCs) loaded with patient-specific monoclonal immunoglobulin (mIg) were used as autologous target cellsin cytotoxicity assays. Effector populations consisted of purified natural killer (NK) cells (CD56+, CD3-) and T cells (CD3+). The MM patients' NK cells cultured in the presence of interleukin 2 (IL-2) showed pronounced cytotoxic activity towards autologous mature DCs. Autologous MM DC targets displayed similar susceptibility to NK cell lysis, compared with allogeneic control DC targets, despite high surface expression of self major histocompatibility complex (MHC) antigens. However, some degree of classic MHC class I-mediated negative regulation was implicated in the NK-DC interactions, as indicated by class I blocking experiments. NK-mediated lysis was also discerned towards primary autologous MM cells. The results indicated that the major effector mechanism was mediated through the perforin-granzyme exocytosis pathway. In conclusion, NK cells from MM patients displayed significant and consistent cytotoxicity towards autologous mature DCs, suggesting that innate immunity could be implicated in MM and may influence the outcome of the administration of tumour antigen-pulsed DCs in treatment trials.