Pierre-Antoine Deglesne

Down-regulation of CXCR4 and CD62L in Chronic Lymphocytic Leukemia Cells Is Triggered by B-Cell Receptor Ligation and Associated with Progressive Disease

Progressive cases of B-cell chronic lymphocytic leukemia (CLL) are frequently associated with lymphadenopathy, highlighting a critical role for signals emanating from the tumor environment in the accumulation of malignant B cells. We investigated on CLL cells from 30 untreated patients the consequence of B-cell receptor (BCR) triggering on the membrane expression of CXCR4 and CD62L, two surface molecules involved in trafficking and exit of B-lymphocytes from lymph nodes. BCR stimulation promoted a strictly simultaneous down-regulation of CXCR4 and CD62L membrane expression to a variable extent. The variable BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the CLL cells to respond to BCR engagement in a given patient. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR engagement displayed a reduction in both migration toward CXCL12 and adhesion to lymphatic endothelial cells. Remarkably, the ability of CLL cells to respond to BCR ligation was correlated with unfavorable prognostic markers and short progression-free survival. In conclusion, BCR signaling promotes decrease of CXCR4 and CD62L membrane expression in progressive cases only. These results are consistent with the hypothesis that BCR-mediated signaling pathways favor accumulation of a proliferative pool within the lymph nodes of progressive CLL cases.

The degree of BCR and NFAT activation predicts clinical outcomes in chronic lymphocytic leukemia

B-cell antigen receptor (BCR)-mediated signaling plays a critical role in chronic lymphocytic leukemia (CLL) pathogenesis and gives an in vitro survival advantage to B cells isolated from patients with unfavorable prognostic factors. In this study, we undertook to elucidate the signaling intermediates responsible for this biologic alteration. In responding cells only, in vitro BCR engagement triggers global phosphorylation of Syk, activation of phospholipase Cγ2, and intracellular calcium mobilization, reflecting competency of BCR signaling. The calcium-calcineurin-dependent transcription factor NFAT2 is up-regulated and to some extent constitutively activated in all CLL B cells. In contrast, its DNA-binding capacity is enhanced on IgM stimulation in responding cells only. NFAT inhibition using the VIVIT peptide prevents induction of CD23 target gene and IgM-induced survival, converting responding cells to unresponsive status. At the opposite, ionomycin-induced NFAT activity allows survival of nonresponding cells. These results demonstrate that the functional heterogeneity relies on variability of protein levels establishing BCR-dependent thresholds and NFAT-dependent activation. Finally, status of the BCR-NFAT pathway for each patient reveals its relevance for CLL clinical outcome and points out to BCR-NFAT intermediates as promising functional therapeutic targets.

CD16 cross-linking induces increased expression of CD56 and production of IL-12 in peripheral NK cells

Human NK cells are classified into two populations according to the intensity of CD56 surface expression, as well as possession of CD16, FcRIII. CD56(dim)CD16(bright) make up 90% circulating NK cells, whereas CD56(bright)CD16(-/dim) comprises the remaining 10%. Here we report that peripheral NK cells upon CD16 cross-linking up-regulates the expression of activating markers and receptors such as CD25, CD69, NKp44, NKp30, CD40L and the intensity of CD56 expression. Additionally, co-culturing immature DCs with CD16 activated NK cells was found to significantly increase the expression of maturation markers on DCs. These results suggest that CD16 cross-linking on resting peripheral blood NK cells triggers the activation of these cells and induces the appearance of CD56(bright) NK cells. The latter were found capable of producing pro-inflammatory cytokines, IFN-gamma and TNF-alpha and notably IL-12.

Novel function of STAT1β in B cells: induction of cell death by a mechanism different from that of STAT1α

Alternate splicing of STAT1 produces two isoforms: α, known as the active form, and β, previously shown to act as a dominant‐negative factor. Most studies have dealt with STAT1α, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1‐deficient human B cell line was transfected to express STAT1α or STAT1β. STAT1α, expressed alone, enhanced cell death, potentiated the fludarabine‐induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1β, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1β‐expressing B cells, p53 was stricktly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1β in programmed cell death, which is independent of p53.

STAT1‐dependent IgG cell‐surface expression in a human B cell line derived from a STAT1‐deficient patient

STAT1 is a key effector of cytokines involved in the resistance to pathogens; its identified transcriptional targets mediate the innate immune response involved in the defense against viruses and bacteria. Little is known about the role of STAT1 in adaptive immunity, including its impact on BCR or surface Ig expression. Analysis of this point is difficult in humans, as STAT1 deficiency is extremely rare. SD patients die early in childhood from a severe immunodeficiency. Herein, a SD B cell line obtained from a SD patient was compared with a B cell line from a STAT1‐proficient subject in search of differences in surface Ig expression. In this SD B cell line, a complete absence of surface IgG was noted. The mRNA encoding the surface form of IgG was detected only in STAT1‐proficient B cells; the mRNAs encoding the secreted and the surface forms were detected in SD and STAT1‐proficient B cells. Re‐expression of STAT1 in SD B cells restored surface IgG expression and a functional BCR. Conversely, shRNA silencing of STAT1 in B cells reduced considerably the expression of the surface IgG. Although limited to one B cell line, these results suggest that STAT1 may play an essential role in surface IgG expression in human B cells. Possible mechanisms involve regulation of mRNA splicing, transcription, or both. These observations extend the role of STAT1 further in adaptive immunity, including the regulation of BCR expression.

Unexpectedly high frequency of European parentage in Venezuelan patients with chronic lymphocytic leukemia

Abstract There is insufficient information on the characteristics of chronic lymphocytic leukemia (CLL) in Latin American patients. Immunoglobulin variable-region heavy-chain (IGVH) gene usage and mutation status and prognostic factors were investigated in patients resident in Venezuela. The most frequently used IGVH family genes were: VH3 > VH1 > VH4 > VH5, with a high incidence of IGVH1.69 and IGVH3.21 genes, and 55.2% of IGVH genes were mutated. Analysis of HCDR3 (third complementarity-determining region of the heavy chain) revealed that 24% of Venezuelan HCDR3s belonged to a CLL stereotyped HCDR3. Results for prognostic factors were similar to those reported previously for Caucasian populations. Interestingly, we found an over-representation of people of European extraction among Venezuelan patients with CLL, suggesting the possibility of a higher frequency of susceptibility genes for CLL in Europeans in comparison with Latin American mestizos.