Analysis of the B-cell compartment in plasma cell leukemia and multiple myeloma: immunoglobulin gene rearrangement of EBV-infected B-cell lines.

Multiple myeloma (MM) is defined as a tumoral expansion of plasma cells occurring in the bone marrow and sometimes in the peripheral blood (plasma-cell leukemia, PCL). Many reports have demonstrated a clonal expansion of B cells bearing the same idiotypic determinants as the myeloma protein (idiotypic B cells) in MM, suggesting that they could belong to the malignant clone. In order to investigate whether the B-cell population is a malignant component or not, either in the peripheral blood of patients with PCL or in the bone marrow of patients with MM, we derived B-cell lines by infecting, with the Epstein-Barr virus (EBV), cultures in limiting dilution of mononuclear cells from six patients. A limiting dilution culture was used to prevent the elimination of slowly proliferating clones by the more rapidly dividing ones, and thus to get the most exact representation of the B-cell repertoire of these patients. The cloning efficiency of the EBV-infected cells was similar in patients and healthy individuals (range: 1 in 100 to 1 in 1650 B cells). All of the clones obtained from a single patient exhibited different clonal immunoglobulin gene rearrangements (IGR), proving the validity of our cloning technique. No tumoral clones (61 clones analysed) showed the IGR pattern specific of autologous myeloma cells. These results indicate that malignant plasma cells cannot be immortalized with EBV. These results show that, if malignant B cells (pre-switch or post-switch) exist, they could be present only in a minor population, and the corollary of this is that there is a major population of non-malignant B cells in the sites of tumoral proliferation of patients with MM. This is remarkable in view of numerous reports showing a profound defect of the polyclonal B lymphopoiesis in these patients, and even an absence of B lymphocytes. Thus, these results challenge the existence of a major compartment of malignant idiotypic B cells and favor the hypothesis of non-malignant B cells sharing cross-reactive idiotypes with the autologous myeloma protein.

Human natural killer cells suppress the proliferation of B cells.

We have investigated the suppressive effect of human natural killer (NK) cells on autologous B-cell proliferation. Removal of NK cells by anti-NK-cell monoclonal antibodies (CD16, Leu 11b; Leu 7) increased by 2-3-fold the proliferative response of purified B cells activated by anti-mu and B-cell growth factor (BCGF). The inhibitory effect of NK cells was observed using recombinant IL-2 or semi-purified BCGF-I as sources of BCGF. Moreover NK cells, highly purified by centrifugation on a Percoll discontinuous density gradient, suppressed the proliferative response of purified autologous B cells activated by anti-mu and BCGF. These results show a suppressive effect of human NK cells on B-cell proliferation in vitro.

No preferential use of the VH(V) family in human multiple myeloma.

A recently described immunoglobulin VH family (the VH(V) family) close to the DH and JH genes is preferentially rearranged in immature B-cell tumours. The question of the emergence of multiple myeloma (MM) from a tumorous pre-B cell is not yet resolved. To draw a comparison with chronic lymphocytic leukaemia (CLL), we studied the VH(V) rearrangements in 28 MM patients. A rearranged Hind III-Bam HI fragment of 9.5 kb was detected in only one patient instead of the rearranged fragment of 8.5 kb described in CLL. Rearrangements of a member of the VH(V) family in a 9.5 kb fragment were also observed in two out of 20 lymphoblastoid cell lines obtained from peripheral blood of MM patients. We report here that the VH(V) family is not preferentially involved in this pathology and that the size of the only rearrangement obtained is larger than the 8.5 kb fragment observed in CLL. These results do not favour the hypothesis of a pre-B cell involvement in MM.

Limiting dilution cloning of B cells from patients with multiple myeloma: emergence of non-malignant B-cell lines.

Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of slowly proliferating malignant plasma cells in the bone marrow (BM). Several reports have shown the existence of an abnormal B-cell compartment including proliferative idiotypic B cells (i.e., B cells bearing the same idiotypic determinants as the myeloma protein) in the BM and peripheral blood (PB) of patients with MM. In order to study whether this abnormal compartment can be grown in vitro, we cultured the PB and BM of 23 patients with MM using limiting dilution methods. Our purpose was to restrict the effect of suppressor cells and the possible overgrowth of the cultures by the more rapidly growing B cells, which occurs in bulk cultures. Spontaneously growing cells were obtained only from patients seropositive for the Epstein-Barr virus (EBV) and all the cultures were composed of B cells carrying the EBV genome. Thus, positive cultures were generated only in the presence of B cells latently infected with EBV in vivo. The mean frequency of these B cells (1 in 25,000 B cells) was as low in MM patients as in healthy donors. This low frequency indicated that malignant cells do not bear the EBV genome in vivo and that the in vivo regulation of the EBV infection is unaffected in patients with MM. No Ig-gene rearrangements, specific of the autologous myeloma cells, were found in the cell lines obtained from BM or PB. Thus, the putative malignant B cells or myeloma cells were not able to generate cell lines in vitro, either spontaneously or after endogenous infection with EBV.

No detectable malignant B cells in the peripheral blood of patients with multiple myeloma.

Previous studies have reported the presence of idiotypic B lymphocytes in the peripheral blood of patients with multiple myeloma (MM), suggesting that they may belong to the malignant clone. This led us to investigate by Southern blot analyses the presence of tumour-specific immunoglobulin-gene (Ig-gene) rearrangements in the peripheral-blood mononuclear cells of 21 MM patients. This method was shown to detect clonal cells when they represent as little as 2% of the cell population. B-cell-enriched fractions were also studied in nine cases. An occasional contamination by circulating malignant plasma cells was carefully evaluated using immunofluorescence. Clonal rearrangements were observed in only two cases, in which a contamination by myeloma cells was evident. In these cases the use of different endonucleases clearly demonstrated that these Ig-clonal rearrangements involved post-switched cells. No clonal rearrangement was found when contamination by myeloma cells was absent. Our results demonstrate the absence of detectable B cells involved in the myeloma clone in the peripheral blood of patients with MM.

The defect in peripheral blood B-cell activation in patients with multiple myeloma is not due to a deficiency in the production of B-cell growth and differentiation factors.

The suppression of B lymphopoiesis is a major feature of multiple myeloma (MM). In this disease, there is a striking defect in the response of peripheral blood B cells to pokeweed mitogen (PWM). Normally, B-cell activation depends on B-cell growth factors (BCGFs) and B-cell differentiation factors (BCDFs), produced by peripheral blood mononuclear cells. We therefore evaluated whether the production of these cytokines was defective in patients with MM. We have studied the production of BCGFs (using the anti-mu assay) and, particularly, interleukin-2 and interferon-gamma, two well-documented BCGFs. No defect in the production of BCGFs, interleukin-2, and interferon-gamma was found in patients with active (N = 14) or stable (N = 10) MM, compared with healthy donors (N = 13). The production of BCDFs (i.e., overall activity) was also evaluated and, more particularly, that of interleukin-6 (IL-6). This cytokine is a potent BCDF which is essential in the PWM-induced activation of B cells, acting at the terminal stages of B-cell differentiation. Again, no defect in the production of BCDFs and IL-6 was found in patients with MM. Therefore, the ability to secrete cytokines controlling the process of B-cell activation is not affected in such patients. This indicates that the profound failure of humoral immune response is not due to deficiency of peripheral blood mononuclear cells producing these factors.