Telomerase activity in plasma cell dyscrasias.

Activation of telomerase is essential for in vitro cellular immortalization and tumorigenesis. In the present study, we investigated telomerase activation and its implications in plasma cell dyscrasias including monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM) and plasma cell leukaemia (PCL). All 5 patients with MGUS exhibited normal levels of telomerase activity in their plasma cells. Elevated telomerase activity was found in the samples from 21/27 patients with MM and 4/4 with PCL. In addition, 4 myeloma cell lines all expressed high levels of telomerase activity. The expression of telomerase reverse transcriptase (hTERT) and telomerase RNA template (hTER) was positively associated with the levels of telomerase activity in MM/PCL. Tankyrase expression was upregulated, concomitant with the induction of hTERT and activation of telomerase in MM/PCL. The present findings indicate that MGUS cells may not be immortalized and that activation of telomerase plays a role in the malignant transformation from MGUS to MM.

Interleukin 6, tumour necrosis factor alpha, interleukin 1beta and interleukin 1 receptor antagonist promoter or coding gene polymorphisms in multiple myeloma.

Proinflammatory cytokines such as interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-alpha) and IL-1beta are considered to be involved in the pathogenesis of multiple myeloma (MM). In the present study, we examined a G/C polymorphism at position -174 in the promoter region of IL-6, a biallelic polymorphism at position -308 in the promoter region of TNF-alpha, the TaqI restriction fragment length polymorphism in exon 5 of IL-1beta and a variable number of identical tandem repeat polymorphisms in intron 2 of IL-1 receptor antagonist (IL-1Ra) genes. The alleles of these loci are known to influence the level of production of the cytokines and the IL-1Ra. Seventy-three patients with MM, 27 with monoclonal gammopathy of undetermined significance (MGUS) and 129 healthy individuals were included. No difference was found between patients and healthy controls or between MM and MGUS patients in the distributions of genotypes and frequencies of alleles of the IL-6 (-174), TNF-alpha (-308), IL-1beta TaqI and IL-1Ra gene polymorphisms. No associations between the polymorphisms at the loci under study and clinical factors such as age, sex, clinical stage at onset and M-protein type were observed. Our results indicate that the cytokine (IL-6, TNF-alpha and IL-1beta) and IL-Ra gene polymorphisms do not confer susceptibility to the development of MM.

Blood dendritic cells from myeloma patients are not infected with Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8).

In recent studies, the sequence of Kaposi's sarcoma-associated herpes virus (KSHV) or human herpes virus-8 (HHV-8) was detected in dendritic cells (DC) of patients with multiple myeloma (MM). A concern was raised whether there is an causal association between the viral infection and development of these tumors. In the present study, we have examined DC generated from blood adherent cells from 8 Swedish MM patients at different clinical stages and 2 patients with monoclonal gammopathy of undetermined significance. In addition, 6 myeloma cell lines and bone marrow cells from 2 MM patients were also studied. By polymerase chain reaction (PCR), including nested PCR, no virus DNA was demonstrable in the patients' DC or in myeloma cell lines or fresh bone marrow cells. Moreover, no antibody against KSHV was found in the serum of these 10 patients. Thus, our results indicate that blood-derived DC of MM patients in Sweden usually are not infected with KSHV/HHV-8. This study also suggests that KSHV/HHV-8 is not regularly associated with MM and consequently does not play a primary role in the pathogenesis of these tumors.

Multiple myeloma: VI International Workshop, June 14-18, 1997, Boston, Massachusetts, USA.

The VI International Workshop on Multiple Myeloma was organised by Professor Kenneth C Anderson, Dana Farber Cancer Institute, and held on June 14-18, 1997, in Boston, MA, USA. More than 500 participants from all over the world joined this workshop. Several subjects were introduced and one of the most exciting items of news was the identification of Kaposi's sarcoma-associated herpes virus in the bone marrow stromal cells of patients with MM. The aim of this overview is to give a glimpse of the subjects discussed and the statements made.

Idiotype immunization combined with granulocyte-macrophage colony-stimulating factor in myeloma patients induced type I, major histocompatibility complex-restricted, CD8- and CD4-specific T-cell responses.

Idiotypic structures expressed on the myeloma Ig protein might be regarded as a tumor-specific antigen. Five patients with IgG myeloma were immunized with the purified serum M-component by repeated intradermal injections together with soluble granulocyte-macrophage colony-stimulating factor (GM-CSF). All patients developed an idiotype (Id)-specific T-cell immunity, defined as blood T cells predominantly secreting interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) (type I cells). Id-specific DNA synthesis was induced in one patient. Delayed-type hypersensitivity against the Id was not evoked. The specific IFN-gamma/IL-2 T-cell response was inhibited (46% to 100%) by a major histocompatibility complex (MHC) class I monoclonal antibody (MoAb) in all five patients. A 5% to 37% inhibition by an MHC class II MoAb was seen in four patients. CD4+ as well as CD8+ T cells enriched by magnetic microbeads contained Id-specific cells. The T cells recognized peptides corresponding to the complementarity-determining regions 1, 2, and 3 of the heavy chain of the Id. There was a transient rise of B cells producing IgM anti-idiotypic antibodies in all patients. The results indicate that immunization of myeloma patients using the autologous M-component and soluble GM-CSF may evoke an Id-specific predominantly MHC class I-restricted type I T-cell response.

Anti-idiotypic T-cell activation in multiple myeloma induced by M-component fragments presented by dendritic cells.

The monoclonal immunoglobulin (Ig) (M-component) secreted by the tumour plasma cells in multiple myeloma (MM) has specific antigenic determinants (idiotypes; id) that can serve as tumour-specific antigens. The intact Ig molecule is a weak antigen, and small fragments of id protein might be more immunogenic for the induction of id-specific immunity. Dendritic cells (DC) have attracted attention as the most efficient antigen-presenting cells and promising adjuvants for immunotherapy in tumours. In this study the in vitro T-cell response against F(ab')2 and Fab fragments, heavy and light chains of the M-component was examined in five patients with MM clinical stage I. All fragments were able to stimulate T cells but F(ab')2 or Fab fragments and heavy chains induced a stronger response than light chains. DC induced a significantly stronger id-specific immune response than monocytes. Moreover, with DC as antigen-presenting cells, a predominant interferon (IFN)-gamma (type-1 T-cell) response was seen in all patients. Both IFN-gamma and interleukin (IL)-4 (type-1 and type-2 T-cell) responses were noted when monocytes were used. Our study suggests that DC pulsed with idiotypic fragments such as F(ab')2 fragment and heavy chain can be used for the induction of type-1 anti-idiotypic T-cell response for immunotherapy in MM.

Myeloma bone marrow plasma cells: evidence for their capacity as antigen-presenting cells.

Myeloma plasma cells constitute 10% to 90% of the total bone marrow cell count in patients with multiple myeloma (MM). These cells express a variety of cell surface markers, such as HLA-ABC and HLA-DR, and surface antigens that are necessary for professional antigen-presenting cells, including adhesion and costimulatory molecules. In this study, we examined the expression of major histocompatability complex (MHC) and costimulatory molecules on CD38(bright,++) plasma cells in bone marrow aspirates from eight MM patients. Small percentages of plasma cells expressed weak but detectable levels of HLA-DR, HLA-DQ, CD40, CD80, and CD86, which could be upregulated by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha. CD38++ plasma cell and CD38(dim,+) cells were sorted from freshly isolated bone marrow mononuclear cells and tested for their capacity to act as antigen-presenting cells. Indeed, both CD38++ plasma cells and CD38+ cells were able to stimulate allogeneic T cells and present the soluble antigens purified protein derivative and tetanus toxoid to autologous T cells. Recognition of the antigens led to T-cell proliferation and secretion of IFN-gamma and was MHC class-I and -II restricted. Antigen processing and presentation by CD38++ and CD38+ cells were abolished by treatment of the cells with chloroquine. Hence, our study provides for the first time evidence that myeloma plasma cells may act as antigen-presenting cells. Further studies are warranted to examine in detail the molecules required for inducing T-cell stimulation.

Modulation of anti-idiotypic immune response by immunization with the autologous M-component protein in multiple myeloma patients.

Multiple myeloma is characterized by a proliferation of clonal B lymphocytes and plasma cells. The idiotypic structure of clonal immunoglobulin (Ig) expressed on the tumour B-cell surface can be regarded as a tumour-specific antigen and, as such, a potential target for anti-idiotypic T and B-cells in an immune regulation of the tumour-cell clone. Active immunization using the autologous mono-clonal Ig as a 'vaccine' was shown to induce tumour-specific immunity in murine B-cell tumours and in human B-cell lymphoma. With the aim to induce or amplify an anti-idiotypic response in multiple myeloma, five stage I-III patients were repeatedly immunized with the autologous monoclonal IgG. Induction of idiotype-specific cellular immunity was analysed in vitro by an enzyme-linked immunospot assay (interferon-gamma and interleukin-4 secreting cells). B cells secreting anti-idiotypic IgM antibodies were also analysed. An anti-idiotypic T-cell response was amplified 1.9-5-fold in three of the five patients during immunization. The number of B cells secreting anti-idiotypic antibodies also increased in these three patients. In two of the patients induction of idiotype-specific immunity was associated with a gradual decrease of blood CD19+ B cells. The induced T-cell response was eliminated during repeated immunization. Further studies are warranted to optimize the immunization schedule in order to achieve a long-lasting T-cell immunity against idiotypic determinants on the tumour clone. A role for immunity in controlling the tumour clone remains to be established.

Idiotype-reactive T-cell subsets and tumor load in monoclonal gammopathies.

The presence of idiotype-reactive T-cell subsets and their relation to the tumor load were analyzed in 9 patients with monoclonal gammopathy of undetermined significance (MGUS), in 12 patients with multiple myeloma (MM) clinical stage I, and in 9 patients with MM stage II/III. An enzyme-linked immunospot assay was used to identify interferon-gamma (IFN-gamma)-, interleukin-2 (IL-2)-, or IL-4-secreting T cells after stimulation by F(ab')2 fragments of monoclonal IgG. The response to autologous IgG was significantly higher than that induced by isotypic monoclonal IgG. Comparable results were obtained in a proliferation assay (3H-thymidine incorporation). A total of 8 of 9 patients with MGUS, 7 of 12 patients with MM stage I, and 3 of 9 with MM stage II/III had T cells secreting IFN-gamma and/or IL-2 (T helper [Th1] type-1 cells), whereas cells secreting both Th1 and Th2 or Th0 types of cytokines were more frequent in patients with MM, particularly in those with MM stage II/III. The number and frequency of Th1-type cells were significantly higher in MGUS patients as compared with those of MM stage II/III. The results indicate that idiotype-reactive T cells of the Th1 and Th2 or Th0 subsets were present in MGs and might provide indirect evidence that idiotype-reactive Th1-type cells may have a regulatory impact on the human tumor B cells.

Idiotype-specific T cells in multiple myeloma stage I: an evaluation by four different functional tests.

Idiotype-specific T cells were characterized in patients with multiple myeloma stage I by analysing idiotype-induced DNA synthesis (3H-thymidine incorporation), IL-2 and IFN-gamma production at the single cell level (ELISPOT) (in vitro tests) and delayed type hypersensitivity (DTH) skin reaction (in vivo test). In seven out of eight patients at least one of the four tests was positive. In five patients three or more tests were positive. One patient was negative in all four tests. Six patients had both IL-2 and IFN-gamma-secreting cells and three of them also a DTH response. Furthermore, those three patients with a proliferative response also had IL-2 and IFN-gamma-secreting cells induced by the idiotype. The data indicate that part of the idiotype-specific T cell fraction belongs to the CD4 Th1 cell population. Whether CD8-specific T cells also were present could not be ruled out. The present study provides further support for the existence of idiotype-specific T cells in multiple myeloma. Such cells might be an important target for an immune-mediated therapeutic approach.

Anti-idiotypic B lymphocytes in patients with monoclonal gammopathies.

The prevalence of peripheral blood B cells secreting antibodies reacting with the F(ab')2 fragment of monoclonal IgG was studied in five patients with multiple myeloma (MM), nine patients with monoclonal gammopathy of undetermined significance (MGUS) and six healthy controls. An enzyme-linked immunospot assay allowed direct visualization of antibody producing B cells. All patients had B cells producing antibodies to autologous or allogeneic monoclonal IgG. Autoreactive cells were found more frequently than alloreactive cells in seven out of nine patients with MGUS and three out of four patients with MM. The same frequency of alloreactive cells in the patient groups was detected in healthy individuals. These findings show the existence of B cells producing anti-idiotypic antibodies which could be a part of an idiotypic network in monoclonal gammopathies.

T-cell stimulation induced by idiotypes on monoclonal immunoglobulins in patients with monoclonal gammopathies.

The stimulation of peripheral blood mononuclear cells by purified autologous and/or allogeneic monoclonal IgG was studied in five patients with multiple myeloma (MM), nine patients with monoclonal gammopathy of undetermined significance (MGUS) and six healthy individuals. Single cells secreting IFN-gamma or IL-2 were identified using an enzyme-linked immunospot assay. Patients' cells were preferentially stimulated by autologous monoclonal IgG at low concentrations (1-100 pg/ml), while 100 ng/ml or higher stimulated T cells both from patients and, to a lesser degree, healthy individuals. This biphasic dose-response of T-cell stimulation by autologous monoclonal IgG was reproduced in all patients. The numbers of cells secreting IFN-gamma and IL-2 in response to allogeneic IgG were significantly lower than the numbers obtained using autologous IgG in patients with MM and MGUS. Cells from healthy individuals were stimulated by allogeneic monoclonal IgG, but to a lesser extent. The results of this study support the presence of idiotype-reactive T cells in patients with MM and MGUS and also may suggest a general but less pronounced T-cell reactivity to monoclonal IgG among these patients.

Anti-idiotypic immunity as a potential regulator in myeloma and related diseases.

In this paper some recent and partly preliminary results on anti-idiotypic immunity against clonal B cells in human monoclonal gammopathies are summarized. B cell lines producing antibodies to idiotypic determinants on autologous monoclonal immunoglobulin could be propagated after activation with Epstein-Barr virus of peripheral blood lymphocytes from patients with MGUS and MM clinical stage I but not from untreated persons with advanced MM. Blood T lymphocytes from patients with MGUS and Waldenström's macroglobulinemia were activated to DNA synthesis and production of interleukins by the autologous M protein. In another series of experiments T cell clones raised from patients with MM clinical stage I and MGUS bound F(ab')2 fragments of the autologous M protein and were stimulated to DNA synthesis by the idiotope-bearing protein. Control experiments demonstrated the specificity for idiotypic determinants. Ten of eleven clones were CD4-/CD8+. Finally, using a panel of 8 mAbs to alpha/beta V region epitopes, we noted a clonal expansion of CD4+ and CD8+ T cells in MGUS and MM patients.

Establishment of idiotype bearing B-lymphocyte clones from a patient with monoclonal gammopathy.

B-lymphocyte clones bearing an idiotypic Ig structure, which could also be found on the serum M-component, have been isolated from peripheral blood of a patient with monoclonal gammopathy (MG) using an immune-rosetting technique and limiting dilution. The majority of the cells had the surface phenotype id+/CD19+/CD20+/CD21+ or id+/CD19+/CD20+/CD21-. Some of the clones originated from spontaneous outgrowth of lymphocytes and some were derived from in vitro Epstein-Barr virus-infected lymphocytes. Three of the B-lymphocyte clones and the patient's bone marrow plasma cells appeared to have clonal identity as shown by the same Ig heavy chain gene rearrangements in Southern blot analysis using a JH probe. The study further supports the notion that the peripheral blood of patients with MG contains B lymphocytes that belong to the tumor clone.

Peripheral blood T lymphocytes in patients with monoclonal gammopathies: expanded subsets as depicted by capacity to bind to autologous monoclonal immunoglobulins or reactivity with anti-V gene-restricted antibodies.

The presence of T cells binding F(ab')2 fragments of the idiotypic immunoglobulin was examined by immunofluorescence in peripheral blood of patients with monoclonal gammopathy. In 3 out of 11 tested patients, 1-15% idiotype-binding T cells of either CD4 or CD8 phenotype were found. In 1 patient both a CD4+ and a CD8+ idiotype-binding T-cell fraction were present. In 1 patient the idiotype-binding T cells also reacted with a mAb directed against the variable parts of the TCR alpha or beta chains, further indicating a clonal origin at the alpha/beta level. 3 patients had an expanded predominant T-cell receptor V gene usage based on the reactivity with the limited panel of TCR mAb, but these "clonal" T cells did not bind the idiotype. The study supports the existence of idiotype-specific T cells in peripheral blood of patients with monoclonal gammopathy. Such cells might have a regulatory role on the monoclonal B-cell clone and may be an important target for immunotherapy.

Anti-idiotypic antibodies in patients with monoclonal gammopathies: relation to the tumour load.

The production of anti-idiotypic antibodies from Epstein-Barr virus transformed peripheral blood lymphocytes was analysed in 12 patients with multiple myeloma and monoclonal gammopathy of undetermined significance. A low anti-idiotype production was found in patients with advanced disease (multiple myeloma stage III), whereas the production was high in patients with multiple myeloma stage I and monoclonal gammopathy of undetermined significance (MGUS) (P less than 0.01). The study supports the existence of an immunological network response in patients with monoclonal gammopathies.

Generation of T cell clones binding F(ab')2 fragments of the idiotypic immunoglobulin in patients with monoclonal gammopathy.

Lymphocytes from two patients with multiple myeloma stage I and one patient with monoclonal gammopathy of undetermined significance were found to proliferate specifically in response to low concentrations of F(ab')2 fragments of the autologous M component. T cell clones isolated from repeatedly stimulated cultures bound specifically the autologous idiotype and proliferated after addition of soluble idiotype and exogenous interleukin-2. The majority of clones were CD8+ and showed negligible staining for CD4. Idiotype-binding clones could not be isolated from cultures of lymphocytes from a healthy control stimulated under the same conditions. The study provides support for the existence of idiotype-reactive T cells in monoclonal gammopathies. Such cells might have a regulatory role on the tumor cell clone and may be important for a future therapeutic approach.

Immunoglobulin light chain patterns in the cerebrospinal fluid. A study with special reference to the occurrence of free light chains in cerebrospinal fluid with and without oligoclonal immunoglobulin G.

The immunoglobulin light chain patterns were studied in paired cerebrospinal fluid (CSF) and serum samples from 30 controls, 70 multiple sclerosis (MS) patients, 18 subjects with other inflammatory neurological diseases and 17 patients with other non-inflammatory neurological disorders. In MS, all CSF samples examined by two-dimensional gel electrophoresis exhibited clonally restricted light chain components. Isoelectric focusing and immunoblotting detected free light chains in around 90% of CSF samples from patients with MS or other inflammatory diseases. These components were clonally restricted, appeared in both mono- and dimeric forms and occurred in CSF samples with as well as without oligoclonal immunoglobulin G bands. Generally, the positive CSF samples contained kappa as well as lambda free lights chains. Such components were not detected in the sera, nor in the CSF samples from controls or patients with non-inflammatory diseases.