Limiting dilution cloning of B cells from patients with multiple myeloma: Emergence of non‐malignant B‐cell lines

Abstract

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.

The bone marrow of multiple myeloma patients contains B cell populations at different stages of differentiation that are clonally related to the malignant plasma cell.

“…If B cells at various stages of differentiation are clonally related, it would be expected that they share common Ig gene rearrangements. Detection of clonal Ig gene rearrangements in mononuclear cells from blood and cultured precursor cells has been inconsistent (5)(6)(7)(17)(18)(19). In part this may be due to limits in blot hybridization sensitivities.…”

Cilia containing 9 + 2 structures grown from immortalized cells

“…We used a dilution method [32] to clone cells and establish cell lines. The surviving geneticin-resistant cells were harvested, diluted with LHC-9 medium, and seeded in multi-well dishes at 0 to 1 cell count per well.…”

Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to C mu sequence in immunoglobulin (IgG)- and IgA-secreting multiple myelomas.

“…Third, PBMC may show the same IgH gene rearrangement as myeloma cells (7,8). However, the existence of a myeloma cell precursor is still a subject of controversy (9,10).…”