Phorbol myristate acetate, but not CD40L, induces the differentiation of CLL B cells into Ab-secreting cells.

In this study, we investigated the capacity of chronic lymphocytic leukemia (CLL) B cells to undergo terminal differentiation into Ig-secreting plasma cells in T cell-independent and T cell-dependent responses. We used a two-step model involving stimulation with phorbol myristate acetate (PMA) and CD40L, together with cytokines (PMA/c and CD40L/c), for 7 days. We describe immunophenotypic modifications, changes in the levels of mRNA and protein for transcription factors and morphological and functional events occurring during the differentiation of CLL B cells into antibody-secreting cells (ASCs). The induction of differentiation differed significantly between the CD40L/c and PMA/c culture systems. The PMA/c culture system allowed CLL B cells to differentiate into IgM-secreting cells with an immunophenotype and molecular profile resembling those of preplasmablasts. By contrast, CD40L/c-stimulated cells had a phenotype and morphology similar to those of activated B cells and resembling those of the CLL B cells residing in the lymph node and bone marrow. These data suggest that the CLL B cells are not frozen permanently at a stage of differentiation and are able to differentiate into ASCs as appropriate stimulation are provided. The data presented here raise questions about the molecular processes and stimulation required for CLL B-cell differentiation and about the inability of CD40 ligand to induce differentiation of the CLL B cells.

TLR9 Ligand (CpG Oligodeoxynucleotide) Induces CLL B-Cells to Differentiate into CD20(+) Antibody-Secreting Cells.

B-cell chronic lymphocytic leukemia (CLL) is the most frequent adult leukemia in the Western world. It is a heterogeneous disease characterized by clonal proliferation and the accumulation of CD5(+) mature B lymphocytes. However, the normal counterpart from which the latter cells arise has not yet been identified. CD27 expression and gene expression profiling data suggest that CLL cells are related to memory B-cells. In vitro, memory B-cells differentiate into plasma cells when stimulated with CpG oligodeoxynucleotide (CpG). The objective of the present study was therefore to investigate the ability of CpG, in the context of CD40 ligation, to induce the differentiation of CLL B-cells into antibody-secreting cells (ASCs). CD20(+)CD38(-) CLL B-cells were stimulated with a combination of CpG, CD40 ligand and cytokines (CpG/CD40L/c) in a two-step, 7-day culture system. We found that the CpG/CD40L/c culture system prompted CLL B-cells to differentiate into CD19(+)CD20(+)CD27(+)CD38(-)ASCs. These cells secreted large amounts of IgM and had the same shape as plasma cells. However, only IgMs secreted by ASCs that had differentiated from unmutated CLL B-cells were poly/autoreactive. Class-switch recombination (CSR) to IgG and IgA was detected in cells expressing the activation-induced cytidine deaminase gene (AICDA). Although these ASCs expressed high levels of the transcription factors PRDM1 (BLIMP1), IRF4, and XBP1s, they did not downregulate expression of PAX5. Our results suggest that CLL B-cells can differentiate into ASCs, undergo CSR and produce poly/autoreactive antibodies. Furthermore, our findings may be relevant for (i) identifying the normal counterpart of CLL B-cells and (ii) developing novel treatment strategies in CLL.