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.

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.

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.

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.

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.

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.

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.