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

Abstract

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.

Factors involved in CLL pathogenesis and cell survival are disrupted by differentiation of CLL B-cells into antibody-secreting cells

“…Thus, by analogy with the terminal differentiation program of normal B-cells into plasma cells, CLL B-cells increased their STAT3, IRF4, XBP1s and BLIMP1 expression and decreased their c-MYC, PAX5, BCL6, IRF8 and BACH2 expression. These findings correlates with our previous results and other literature data [ 14 , 33 – 36 ], suggesting that CLL B-cells (i) are able to restore the transcriptional program associated with plasma cell differentiation if appropriate stimulation is provided [ 34 – 36 ] and (ii) display relevant ASC features, including morphological changes, UPR induction [ 34 ] and initiation of secretory function.…”

“…ABT-199 and ABT-737) [ 3 , 4 ] and survivin [ 55 ]. As we have shown here and in recent work [ 34 ], levels of these targets (e.g. BCLxL and survivin) are exacerbated by terminal differentiation of leukemic cells; indeed, a number of the corresponding inhibitors appear to have potential as treatments for CLL [ 3 , 4 , 55 , 59 – 61 ].…”

“…In our previous work, we have characterized in a similar two-step, seven-day culture model the differentiation of CLL B-cells stimulated separately by PMA and CD40L [ 34 ]. As CD40L-CD40 interactions and cytokines are important components of the CLL microenvironment, in the present study, we studied the CLL B-cells' ability to differentiate into antibody-secreting plasma cells after stimulation with PMA at the same time as with CD40L.…”

“…Injection of intratumoral CpG and systemic treatment with ibrutinib resulted in the full, permanent regression of both local and distant tumors. Phorbol myristate acetate is a polyclonal activator of normal B-cells and CLL B-cells [ 34 , 82 ]. Our unpublished data and previously published data [ 82 ] show that PMA has a specific differentiation effect on CLL B-cells and has no effect on other B-cell malignancies.…”

“…Recent research has shown that CLL B-cells display a strong tendency to differentiate into ASCs and may thus be amenable to differentiation therapy [ 14 , 29 , 33 – 35 ]. In a two-step, 7-day culture system, our laboratory recently demonstrated that phorbol myristate acetate (PMA) and CpG oligodeoxynucleotide induces differentiation of CLL B-cells to an intermediate stage in the plasma cell differentiation process [ 34 , 35 ]. Using a similar culture systems, in this study we sought to investigate the impact of B-cell differentiation on the expression of factors that contribute to the physiopathology of CLL and/or are known to be deregulated in CLL B-cells (including LEF1, TCL1, ROR1, FMOD, TACI, PI3K, BTK and p27).…”