Interferon γ is a STAT1-dependent direct inducer of BCL6 expression in imatinib-treated chronic myeloid leukemia cells.

B-cell CLL/lymphoma 6 (BCL6) exerts oncogenic effects in several human hematopoietic malignancies including chronic myeloid leukemia (CML), where BCL6 expression was shown to be essential for CML stem cell survival and self-renewal during imatinib mesylate (IM) treatment. As several lines of evidence suggest that interferon γ (IFNγ) production in CML patients might have a central role in the response to tyrosine kinase inhibitor (TKI) therapy, we analyzed if IFNγ modulates BCL6 expression in CML cells. Although separate IFNγ or IM treatment only slightly upregulated BCL6 expression, combined treatment induced remarkable BCL6 upregulation in CML lines and primary human CD34+ CML stem cells. We proved that during combined treatment, inhibition of constitutive signal transducer and activator of transcription (STAT) 5 activation by IM allowed the specific enhancement of the STAT1 dependent, direct upregulation of BCL6 by IFNγ in CML cells. By using colony-forming assay, we found that IFNγ enhanced the ex vivo colony or cluster-forming capacity of human CML stem cells in the absence or presence of IM, respectively. Furthermore, inhibition of the transcriptional repressor function of BCL6 in the presence of IM and IFNγ almost completely blocked the cluster formation of human CML stem cells. On the other hand, by using small interfering RNA knockdown of BCL6, we demonstrated that in an IM-treated CML line the antiapoptotic effect of IFNγ was independent of BCL6 upregulation. We found that IFNγ also upregulated several antiapoptotic members of the BCL2 and BIRC gene families in CML cells, including the long isoform of MCL1, which proved to be essential for the antiapoptotic effect of IFNγ in an IM-treated CML line. Our results suggest that combination of TKIs with BCL6 and MCL1 inhibitors may potentially lead to the complete eradication of CML stem cells.

Functional characterization of murine CD25 expressing B cells.

B cells are an important part of both innate and adaptive immune system. Their ability to produce antibodies, cytokines and to present antigen makes them a crucial part in defence against pathogens. In this study, we have in naïve Naval Medical Research Institute mice functionally characterized a subpopulation of splenic B cells expressing CD25, which comprise about 1% of the total B cell compartment. Murine spleen cells were sorted into two highly purified B cell populations either CD19(+) CD25(+) or CD19(+) CD25(-). We found that CD25(+) B cells secreted higher levels of IL-6, IL-10 and INFgamma in response to different TLR-agonists, and were better at presenting alloantigen to CD4(+) T cells. CD25 expressing B cells spontaneously secreted immunoglobulins of IgA, IgG and IgM subclass and had better migratory ability when compared with CD25(-) B cells. In conclusion, our results demonstrate that CD25(+) B cells are highly activated and functionally mature. Therefore, we suggest that this population plays a major role in the immune system and may belong to the memory B-cell population.

Impact of short-term therapies with biologics on prothrombotic biomarkers in rheumatoid arthritis.

BACKGROUND: Imbalance of haemostasis in patients with rheumatoid arthritis (RA) contributes to future risk of cardiovascular diseases (CVD). Prothrombotic molecules, e.g. fibrinogen, D-dimer, and tPA are elevated in plasma of RA patients, being associated to CVD. There is no imformation about the influence of biological drugs, e.g. anti-CD20 and tumor necrosis factor (TNF) antibodies on these prothrombotic molecules. OBJECTIVE: To assess whether anti-TNF and anti-CD20 therapies modify the profiles of cardiovascular risk factors in patients with RA. METHODS: The expression of prothrombotic molecules in plasma was investigated in 10 RA patients before and after treatment with TNF-alpha antibodies and in another 12 RA patients before and after anti-CD20 treatment. RESULTS: Both anti-TNF and anti-CD20 infusions gave rise to clear clinical improvement. However, only anti-CD20 infusion significantly (p=0.05) reduced concentration of fibrinogen (p=0.05), D-dimer (p<0.001), as well as tPA levels (p<0.01). In contrast, in TNF antibody treated patients only tPA levels were significantly decreased following the treatment (p<0.05). CONCLUSIONS: Infusion of CD20 antibodies to the patients with active RA led to a clearly reduced plasma levels of predictors of CVD indicating that this treatment, apart from its anti-inflammatory properties, may reduce the risk for future CVD in RA.

The human immunomodulatory CD25+ B cell population belongs to the memory B cell pool.

We have shown that human CD20(+)25(+) B cells display immunomodulatory properties. The aim of this study was to investigate if CD25(+) B cells are found within the CD27 memory B cell population, and to analyse pattern of their cytokine production. B cells isolated from healthy subjects, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients were analysed regarding the frequency of CD25(+) B cells within certain B cell subsets. Purified CD25(+) B cells from healthy subject were used in vitro to evaluate their production of immunomodulatory cytokines. In healthy subjects the majority (60%) of memory B cells (CD20(+)27(+)) also co-expressed CD25 while only 10-20% of the naïve B cells (CD20(+)27(-)) and plasmablasts (CD20-27(+)) expressed CD25. In RA and SLE patients, we found that 51% and 48%, respectively, co-expressed CD25 in the memory population, whereas only 11% and 9% co-expressed CD25 in the naïve B cell population. Phenotypic analysis of the CD20(+)25(+)27(+) and CD20(+)25(+)27(-) cells using CD10, CD24, CD38, CD45, CD71, CD80, CD86, CD95, CD138, BAFF-R, TACI, IgA, IgD, IgG and IgM showed that CD20(+)25(+)27(+) B cells preferentially represent highly activated, Ig class switched memory B cells. Cytokine profile analysis showed that CD25(+) B cells secreted significantly higher levels of IL-10 versus CD25(-) B cells. In contrast, TGF-beta1 secretion was similar between the CD25(+) and CD25(-) sub-populations. In conclusion, CD20(+)25(+) B cells constitute a unique subpopulation preferentially occurring among CD20(+)27(+) memory B cells. We suggest that CD25 can be used as a marker for a memory B cell subset.

CD25-expressing B-lymphocytes in rheumatic diseases.

B cells play an important role in the development of autoimmune diseases due to their production of autoantibodies, antigen-presenting capacity and production of pro-inflammatory cytokines. The purpose of the present study was to analyse B cells from rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients, with respect to their expression of the IL-2 receptor (IL-2R) subunit CD25. Using flow cytometry, we found that CD25(+) B cells from RA patients expressed significantly higher frequencies of CD122 and CD132 than CD25(+) B cells from control subjects, indicating a fully functional IL-2R. These CD25(+) B cells also expressed higher frequencies of the co-stimulatory molecule CD80, whereas IgM and IgA expression was decreased compared with CD25(+) B cells from healthy controls. In addition B cells from SLE patients co-expressed CD25 together with CD80, CD122, and CD132, but to a lower degree IgD and IgM, when compared with healthy controls. Taken together, our results indicate that CD25(+) B cells from RA and SLE patients are in a highly activated state, display a more mature phenotype and suggest that this B cell subset may be involved in the pathogenesis of RA and SLE.

B-cell CD25 expression in murine primary and secondary lymphoid tissue.

B cells are in analogy with T cells capable of expressing functional IL-2 receptors. IL-2R alpha-chain (CD25) positive T cells have been studied in detail but not much is known about CD25 positive B cells. The aim of this study was to examine the phenotypic properties of the CD25 expressing B cells collected from different lymphoid organs in mice. Samples were stained for various cell surface markers and analysed using flow cytometry. We found that approximately 49% of B cells in bone marrow, 16% in peritoneal cavity, 2% in spleen and 1% in lymph nodes express CD25. In contrast, CD25 expressing B cells were not found in the blood or in Peyer's patches. Phenotypic characterization showed that CD25+ B cells in spleen, lymph nodes and peritoneal cavity have higher expression of AA4.1, CD5, CD69, CD80, CD86, CD122, CD132, IgA, IgG and IgM on their surface in comparison with CD25- B cells. In contrast, expression of IgD and IA-IE was lower on CD25+ B cells in spleen and lymph nodes. In bone marrow, the expression of CD5, CD80, CD86, CD122, CD132, IgA, IgD and IgM was lower, while the expression of AA4.1, IgG and IA-IE was increased on CD25+ B cells compared with CD25- B cells. In conclusion, our results indicate that B cells expressing CD25 are phenotypically distinctly different from those that are CD25 negative. Our findings suggest that CD25+ B cells are more prone to efficient antigen presentation and display a more mature phenotype.