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.
Abstract The ETS-domain transcription factors divide into subfamilies based on protein similarities, DNA-binding sequences, and interaction with cofactors. They are regulated by extracellular clues ...and contribute to cellular processes, including proliferation and transformation. ETS genes are targeted through genomic rearrangements in oncogenesis. The PU.1/SPI1 gene is inactivated by point mutations in human myeloid malignancies. We identified a recurrent somatic mutation (Q226E) in PU.1/SPI1 in Waldenström macroglobulinemia, a B-cell lymphoproliferative disorder. It affects the DNA-binding affinity of the protein and allows the mutant protein to more frequently bind and activate promoter regions with respect to wild-type protein. Mutant SPI1 binding at promoters activates gene sets typically promoted by other ETS factors, resulting in enhanced proliferation and decreased terminal B-cell differentiation in model cell lines and primary samples. In summary, we describe oncogenic subversion of transcription factor function through subtle alteration of DNA binding leading to cellular proliferation and differentiation arrest. Significance: The demonstration that a somatic point mutation tips the balance of genome-binding pattern provides a mechanistic paradigm for how missense mutations in transcription factor genes may be oncogenic in human tumors. This article is highlighted in the In This Issue feature, p. 681
B-chronic lymphocytic leukemia (B-CLL), the most common human leukemia, is characterized by predominantly non-dividing malignant mature CD5+ B lymphocytes with an apoptosis defect. Various ...microenvironmental stimuli confer a growth advantage on these leukemic cells and extend their survival in vivo. Nevertheless, when cultured in vitro, CLL B-cells rapidly die from apoptosis. Certain cytokines may extend the survival capacity of CLL B-cells in vitro and individual anti-apoptotic effects of several cytokines have been reported. The potential cumulative effect of such cytokines has not been studied. We therefore investigated the effects on CLL B-cells survival in vitro of humoral factors, polyclonal lymphocyte activators and a combination of cytokines known for their anti-apoptotic effects. Purified CLL B-cells were cultured in the presence or absence of various soluble molecules and the leukemic cell response was assessed in terms of viability. Apoptotic cell death was detected by flow cytometry using annexinV and 7-amino-actinomycin. The survival of CLL B-cells in vitro was highly variable. When tested separately, cytokines (IL-2, -6, -10, -12, -15, -21, BAFF and APRIL) improved CLL B cell survival moderately; in combination, they significantly enhanced survival of these cells, even up to 7 days of culture. We also report that humoral factors from autologous serum are important for survival of these malignant cells. Our findings support the concept that the CLL microenvironment is critical and suggest that soluble factors may contribute directly to the prolonged survival of CLL B-cells. Therefore, the combination of cytokines we describe as providing strong resistance to apoptosis in vitro might be used to improve the treatment of CLL.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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