Extracellular vesicles and their associated miRNAs as potential prognostic biomarkers in chronic lymphocytic leukemia.

PURPOSE OF REVIEW: Many prognostic and predictive biomarkers have been proposed for chronic lymphocytic leukaemia (CLL). Here, we aim to discuss the evidence showing a prognostic potential for extracellular vesicles (EV) and their associated microRNAs (miRNAs). RECENT FINDINGS: EV are produced by several cells in the body as a physiological event; however, there is evidence suggesting that an elevated EV concentration is present in the circulation of CLL patients. Moreover, some studies have associated EV concentration with advanced Rai stage and unmutated CLL while others have demonstrated its potential as an independent prognostic factor for TTFT and OS. Finally, some studies have shown that CLL EV shared some dysregulated microRNAs with CLL cells and plasma. On the other hand, it was found that CLL EV has a distinctive microRNA expression profile. Until now, EV-associated miR-155 is the most studied miRNA. Despite methodological diversity and limitations in study design, unanimity in CLL EV concentration behaviour and miRNA content has been found.

Proteasome Dependent Actin Remodeling Facilitates Antigen Extraction at the Immune Synapse of B Cells.

Engagement of the B cell receptor (BCR) with surface-tethered antigens leads to the formation of an immune synapse (IS), where cell signaling and antigen uptake are tightly coordinated. Centrosome re-orientation to the immune synapse has emerged as a critical regulatory step to guide the local recruitment and secretion of lysosomes, which can facilitate the extraction of immobilized antigens. This process is coupled to actin remodeling at the centrosome and at the immune synapse, which is crucial to promote cell polarity. How B cells balance both pools of actin cytoskeleton to achieve a polarized phenotype during the formation of an immune synapse is not fully understood. Here, we reveal that B cells rely on proteasome activity to achieve this task. The proteasome is a multi-catalytic protease that degrades cytosolic and nuclear proteins and its dysfunction is associated with diseases, such as cancer and autoimmunity. Our results show that resting B cells contain an active proteasome pool at the centrosome, which is required for efficient actin clearance at this level. As a result of proteasome inhibition, activated B cells do not deplete actin at the centrosome and are unable to separate the centrosome from the nucleus and thus display impaired polarity. Consequently, lysosome recruitment to the immune synapse, antigen extraction and presentation are severely compromised in B cells with diminished proteasome activity. Additionally, we found that proteasome inhibition leads to impaired actin remodeling at the immune synapse, where B cells display defective spreading responses and distribution of key signaling molecules at the synaptic membrane. Overall, our results reveal a new role for the proteasome in regulating the immune synapse of B cells, where the intracellular compartmentalization of proteasome activity controls cytoskeleton remodeling between the centrosome and synapse, with functional repercussions in antigen extraction and presentation.

NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome.

Although B cell development requires expression of the B cell antigen receptor (BCR), it remains unclear whether engagement of self-antigen provides a positive impact for most B cells. Here, we show that BCR engagement by self-ligand during development in vivo results in up-regulation of the Nod-like receptor member Nod1, which recognizes the products of intestinal commensal bacteria. In anti-thymocyte/Thy-1 autoreactive BCR knock-in mice lacking self-Thy-1 ligand, immunoglobulin light chain editing occurred, generating B cells with up-regulated Nod1, including follicular and marginal zone B cells with natural autoreactivity. This BCR editing with increased Nod1 resulted in preferential survival. In normal adult mice, most mature B cells are enriched for Nod1 up-regulated cells, and signaling through Nod1 promotes competitive survival of mature B cells. These findings demonstrate a role for microbial products in promoting survival of mature B cells through up-regulated Nod1, providing a positive effect of BCR engagement on development of most B cells.

Membrane-incorporated immunoglobulin receptors increase the antigen-presenting ability of B cells.

Monoclonal antibodies specific for ovalbumin were conjugated to palmitate and inserted into the membrane of normal spleen B cells. Their presence in the membrane, as well as their ability to bind ovalbumin, was established by immunofluorescence. The so called anti-ovalbumin-'decorated' B cells were tested for their ability to act as antigen-presenting cells for ovalbumin-specific I-Ad-restricted T-cell hybridomas. It was found that the antibody-decorated B cells presented antigen more efficiently than non-decorated B cells.

Regulation of B cell antigen receptor signal transduction and phosphorylation by CD45.

CD45 is a member of a family of membrane proteins that possess phosphotyrosine phosphatase activity, and is the source of much of the tyrosine phosphatase activity in lymphocytes. In view of its enzymatic activity and high copy number, it seems likely that CD45 functions in transmembrane signal transduction by lymphocyte receptors that are coupled to activation of tyrosine kinases. The B cell antigen receptor was found to transduce a Ca(2+)-mobilizing signal only if cells expressed CD45. Also, both membrane immunoglobulin M (mIgM) and CD45 were lost from the surface of cells treated with antibody to CD45, suggesting a physical interaction between these proteins. Finally, CD45 dephosphorylated a complex of mIg-associated proteins that appears to function in signal transduction by the antigen receptor. These data indicate that CD45 occurs as a component of a complex of proteins associated with the antigen receptor, and that CD45 may regulate signal transduction by modulating the phosphorylation state of the antigen receptor subunits.

Dual molecular mechanisms mediate ligand-induced membrane Ig desensitization.

We have previously shown that ligation of murine B cell membrane IgM or IgD can lead to inactivation of the signal transducing ability of unligated Ag receptors. We describe further studies of the molecular basis of this desensitization. Consistent with the possibility that ligand induced desensitization is mediated by protein kinase C (PKC) are findings that demonstrate that both Ig binding ligands and PKC activators (DIC8 or PMA) induce desensitization in virtually all resting B cells. However, ligand-induced desensitization is longer lived than PMA- or DIC8-induced desensitization and insensitive to the PKC inhibitor staurosporine. Further, biochemical studies indicate that insufficient PKC activation is induced by ligation of membrane Ig to mediate the observed desensitization. Thus data indicate that PKC must play only a minor role in ligand-induced membrane Ig desensitization. Further studies explored the molecular source and target of effectors that mediate ligand-induced desensitization. Data indicate that phosphoinositide hydrolysis is neither necessary nor sufficient for ligand induction of desensitization. Finally, ligand-induced desensitization appears to be mediated by uncoupling of membrane Ig from G proteins that regulate phospholipase C because ligand desensitized cells are hyperresponsive to agents including ALF4- and mastoparan which activate G proteins leading to mobilization of Ca2+. Thus, the function of G proteins and further downstream elements that mediate Ca2+ mobilization is intact. Taken together, these data are most consistent with ligand-induced membrane Ig desensitization being mediated by a non-PKC, non phosphatidylinositol 4,5-bisphosphate hydrolysis involving mechanism that has as its target a structure that is very proximal to the receptor, such as the receptor itself or a transducer complex analogous to CD3.

Ligand-induced desensitization of B-cell membrane immunoglobulin-mediated Ca2+ mobilization and protein kinase C translocation.

Binding of ligand to B-cell membrane immunoglobulin (mIg) can lead to activation of a number of distinct biologic responses, including altered expression of genes encoding c-fos, c-myc, and Ia, as well as proliferation and immunologic tolerance. Tolerance could reflect a functional uncoupling of receptors from systems that generate intracellular second messengers (i.e., receptor desensitization). To better understand the molecular basis of immune regulation, we examined the ability of mIg to function as a signal transducer after the cell's initial contact with mIg-binding ligand. The results show that ligand binding to as little as 2-10% of mIgM or mIgD renders the cell unresponsive to ligand binding to the reciprocal isotype as judged by Ca2+ mobilization and protein kinase C translocation responses. This heterologous receptor desensitization lasts longer than 24 hr and does not reflect loss of receptor from the cell surface. Studies with the calcium ionophore ionomycin, 1,2-dioctanoyl-sn-glycerol, and the protein kinase inhibitor staurosporine indicate that both protein kinase C-dependent and protein kinase C-independent (staurosporine-insensitive) mechanisms mediate heterologous desensitization after mIg crosslinking.