In humans, up to 40% of peripheral B cells express CD27 and have hypermutated variable regions in their Ig genes. The CD27+ B cells are considered to be derived from germinal center following specific antigenic stimulation. Actually, somatic hypermutation in Ig genes and CD27 expression are hallmarks of memory B cells. However, the blood IgM+IgD+CD27+ B cells were recently associated to splenic marginal zone B cells and proposed to be a subset distinct from germinal center-derived memory B cells showing premutated Igs. The results presented herein further weaken this bona fide association because B cells expressing surface IgG, but not CD27, were found in human blood. Representing 1–4% of all peripheral B cells and ∼25% of the IgG+ blood B cells, this population expressed mutated IgG genes showing antigenic selection characteristics but with lower mutation frequencies than that of CD27+IgG+ B cells. However, their morphology and phenotype were similar to that of CD27+IgG+ cells. Interestingly, the proportion of IgG2 over IgG3 transcripts was opposite in CD27−IgG+ and CD27+IgG+ cells, suggesting distinct functions or origins. Overall, these findings extend the memory B cell reservoir beyond the CD27+ compartment and could provide further insights into B cell disorders of unknown etiology.

• High-level miR-155 enhances BCR signaling, and is associated with poor prognosis in CLL.• Signals within the CLL microenvironment, such as CD154 or BAFF, can induce miR-155 and enhance BCR signaling.High-level leukemia cell expression of micro-RNA 155 (miR-155) is associated with more aggressive disease in patients with chronic lymphocytic leukemia (CLL), including those cases with a low-level expression of z-chain-associated protein of 70 kD. CLL with highlevel miR-155 expressed lower levels of Src homology-2 domain-containing inositol 5-phosphatase 1 and were more responsive to B-cell receptor (BCR) ligation than CLL with low-level miR-155. Transfection with miR-155 enhanced responsiveness to BCR ligation, whereas transfection with a miR-155 inhibitor had the opposite effect. CLL in lymphoid tissue expressed higher levels of miR155HG than CLL in the blood of the same patient. Also, isolated CD5 bright CXCR4 dim cells, representing CLL that had been newly released from the microenvironment, expressed higher levels of miR-155 and were more responsive to BCR ligation than isolated CD5 dim CXCR4 bright cells of the same patient.Treatment of CLL or normal B cells with CD40-ligand or B-cell-activating factor upregulated miR-155 and enhanced sensitivity to BCR ligation, effects that could be blocked by inhibitors to miR-155. This study demonstrates that the sensitivity to BCR ligation can be enhanced by high-level expression of miR-155, which in turn can be induced by crosstalk within the tissue microenvironment, potentially contributing to its association with adverse clinical outcome in patients with CLL. (Blood. 2014;124(4):546-554)

During secondary immune response, memory B lymphocytes proliferate and differentiate into Ig-secreting cells. In mice, the binding of CD40 by CD154 clearly enhances the activation and differentiation of memory B lymphocytes. In humans, the role of CD40-CD154 in the stimulation of memory B lymphocytes is not as obvious since in vitro studies reported positive and negative effects on their proliferation and differentiation in Ig-secreting cells. In this study, we examine the response of peripheral memory and naive cells in relation to the duration of CD40-CD154 interaction. We measured the proliferation and differentiation of both subsets stimulated with CD154 and IL-4 for short- (4–5 days) and long-term (>7 days) periods. Following short-term stimulation, memory B lymphocytes did not expand but represented the only subset differentiating into IgG- and IgM-secreting cells. A longer stimulation of this population led to cell death, while promoting naive B lymphocyte proliferation, expansion, and differentiation into IgM- or IgG-secreting cells. This prolonged CD40 stimulation also triggered naive B lymphocytes to switch to IgG and to express CD27 even in absence of somatic hypermutation, suggesting that these latter events could be independent. This study suggests that naive and memory B lymphocytes have distinct requirements to engage an immune response, reflecting their different roles in humoral immunity.

The chemokine CXCL12, via its receptor CXCR4, promotes increased survival of chronic lymphocytic leukemia (CLL) B cells that express high levels of -chainassociated protein (ZAP-70), a receptor tyrosine kinase associated with aggressive disease. In this study, we investigated the underlying molecular mechanisms governing this effect. Although significant differences in the expression or turnover of CXCR4 were not observed between ZAP-70 ؉ and ZAP-70 ؊ cell samples, CXCL12 induced greater intracellular Ca 2؉ IntroductionChronic lymphocytic leukemia (CLL) is a disease characterized by the accumulation of mature monoclonal B cells in the blood, secondary lymphoid tissue, and marrow. 1,2 Regardless of their apparent longevity in vivo, CLL B cells undergo apoptosis in vitro unless rescued by monocyte-derived nurse-like cells (NLCs) or marrow stromal cells. [3][4][5][6] In line with this hypothesis, the marrow is invariably infiltrated with CLL cells in patients, and the extent of infiltration correlates with clinical stage and prognosis. 5,7 These accessory cells also protect CLL cells from drug-induced apoptosis in vitro. 8 Thus, it has been postulated that CLL cells receive survival signals from these accessory cells, which constitute part of the CLL B-cell microenvironment in secondary lymphoid tissue and marrow. 6 Such niches could protect leukemia cells from spontaneous or drug-induced apoptosis in vivo, motivating the current study to better understand the survival pathways triggered by the microenvironment.Accessory cells such as NLCs protect CLL cells from apoptosis in vitro in part through the secretion of the stromal cell-derived factor-1␣ (renamed as CXCL12). 9,10 CXCL12 is a highly conserved chemokine that signals through the chemokine receptor CXCR4, which is expressed at high levels by CLL cells. 3,10,11 Although most noted for its role in directing cell migration, CXCL12 also provides survival stimuli to CLL cells and partially protects them from spontaneous or drug-induced apoptosis or both in vitro. 3,9 Further, the enhanced viability of these cells in the presence of CXCL12 can be blocked by antibodies to CXCL12 3 or peptide inhibitors of CXCR4. 8 In prior studies, it was found that treatment of CLL cells with CXCL12 induced activation of extracellular signal-regulated kinase (ERK). 8,12 In this study, we further examined the survival and signaling responses of CLL cells to CXCL12 to characterize the mechanism for the survival benefit. In addition, we compared the CXCL12-induced responses of CLL cells from 2 subgroups of patients, with high or low expression levels of -chain-associated protein of 70 kDa (ZAP-70), a tyrosine kinase whose high-level expression is correlated with increased risk of early disease progression and relatively short survival 12,13 . Methods Preparation of CXCL12CXCL12 was prepared as previously described. 14 Briefly, CXCL12 was expressed as a His-tag fusion protein and purified from inclusion bodies in BL21 Escherichia coli. Bacterial pellets were resuspended in 10mM Tris...