The integrin leukocyte function-associated antigen-1 (LFA-1) is important in the promotion of B cell adhesion, thereby facilitating immunological synapse (IS) formation and B cell activation. Despite this significance, the associated signaling mechanisms regulating LFA-1 activation remain elusive. Here, we show that both isoforms of the small GTPase Rac expressed by primary B cells, Rac1 and Rac2, were activated rapidly downstream of Src-family kinases, guanine-nucleotide exchange factors Vav1 and Vav2, and phosphoinositide-3 kinase (PI3K) after BCR engagement. We identify Rac2, but not Rac1, as critical for B cell adhesion to intercellular adhesion molecule-1 (ICAM-1) and IS formation. Furthermore, B cells expressing constitutively active Rac2 are highly adhesive. We observe that Rac2-deficient B cells exhibit lower amounts of Rap1-GTP and severe actin polymerization defects, identifying a potential mechanism underlying their behavior. We postulate that this critical role for Rac2 in mediating B cell adhesion and IS formation might apply in all lymphocytes.

Journal of Cell Science 2280 activation (Lanzavecchia, 1985;Rock et al., 1984). Subsequently activated B cells can develop into plasma cells that are competent for large-scale production of soluble antibodies, or memory cells that provide long-lasting immunological memory.In this Commentary we highlight recent investigations of the mechanism underlying inside-out integrin activation in response to antigen stimulation through the BCR. Intriguingly, we observe a number of important differences in the mechanism that underlies inside-out activation according to the particular integrin activated and the cellular context of integrin expression. We discuss recent evidence that identifies key signalling molecules that govern the activity and distribution of LFA-1 and VLA-4 following engagement of the BCR. Finally, we propose a mechanism whereby these regulatory molecules mediate the cytoskeleton rearrangements that are required for the integrin activation and subsequent IS formation that occur during the antigen-recognition process. Antigen-induced BCR signallingThe BCR can be described as a receptor tyrosine kinase, and its stimulation by a specific antigen results in the proliferation and differentiation of B cells. The BCR comprises a membrane immunoglobulin (Ig) in complex with the heterodimeric Igα-Igβ sheath (Fig. 1B), which allows its stable expression in the membrane (Reth, 1989;Venkitaraman et al., 1991). The constituents of the BCR act in concert to couple the recognition of extracellular antigens with the initiation of intracellular signalling through the immunoreceptor tyrosine-based activation motifs (ITAMs) of the Igα-Igβ sheath (Hombach et al., 1990;Reth, 1989). These activated ITAMs subsequently act as recruitment sites for the assembly of a complex array of intracellular adaptors and signalling molecules in a structure known as the signalosome (DeFranco, 2001;Fruman et al., 2000). The precise composition of a signalosome is dictated by the nature and context of the antigenic stimulant, and determines the outcome of signalling through the BCR (Depoil et al., 2008;Weber et al., 2008). In the specific case of inside-out activation of integrins it would be expected that this outcome involves the direct targeting of the cytoplasmic domains of the integrin itself, in addition to the extensive reorganisation of the cytoskeleton that is necessary for the recognition of antigens that are presented on a spatially constrained surface.The mechanism of inside-out activation of integrins has been investigated extensively in T cells (reviewed in Kinashi, 2005). The Journal of Cell Science 121 (14) Fig. 1. Molecular mechanisms required for the activation of integrin-mediated B-cell adhesion in response to membrane-bound antigens. (A) Prior to antigen stimulation, the 'resting' B cell contains BCRs and inactive integrins that are distributed throughout the membrane. Following BCR engagement with antigen on the surface of an antigen-presenting cell (APC) that expresses integrin ligands, crosslinking of BCRs initiates int...

Human B-cell studies in vitro have routinely used B lymphocytes purified from spleen, blood or tonsils irrespective of potential differences in their immunological traits. In this study, we compared the functional responses of total (CD19(+)) and memory B cells (Bmem; CD19(+)/CD27(+)) isolated from blood and tonsils to different stimuli. Peripheral B cells showed enhanced survival and proliferation compared with their tonsillar equivalents when stimulated for 10 days. Stimulated B cells from both tissues secreted significantly greater amounts of cytokines than unstimulated controls demonstrating their functional responsiveness. Analysis of CD27 expression over time indicated that the conditions that promoted survival and proliferation of peripheral Bmem, caused massive tonsillar Bmem death. Purified tonsillar Bmem failed to expand but rapidly differentiated in antibody secreting cells and subsequently underwent apoptosis. In contrast, circulating Bmem showed delayed activation and differentiation, but exhibited a longer lifespan and active proliferation. In addition, short-term stimulation of tonsillar Bmem resulted in the production of more immunoglobulin G (IgG) than their peripheral counterparts. At later time points, however, IgG production from the different B cells was reversed. Our findings imply that the tissue located and peripheral Bmem have distinct behaviors, indicating organ dependent functional responses that should not be generalizable to all Bmem. This work provides a greater understanding of how Bmem location is coupled to specialized roles of B lymphocytes.

Despite compelling evidence that a large proportion of antigens encountered in vivo by B cells are membrane bound, the general view is that B cells are mainly activated by soluble antigens. This notion may have been biased somewhat over the years because the high affinity of the B-cell receptor (BCR) for soluble intact ligands allows efficient B-cell stimulation in vitro. In vivo, however, even soluble antigens are likely to be deposited on the surface of antigen-presenting cells, either by complement or Fc receptors in the form of immune complexes, thus becoming more potent stimulators of B-cell activation. In this framework, the BCR works in a complex environment of integrins and coreceptors, as well as the B-cell cytoskeleton. Over the last few years, we have focused on B-cell membrane-bound antigen recognition. Here, we discuss some of our findings in the context of what is currently known in this exciting new field.

Current treatment of retinoblastoma involves using the maximum dose of chemotherapy that induces tumor control and is tolerated by patients. The impact of dose and schedule on the cytotoxicity of chemotherapy has not been studied. Our aim was to gain insight into the cytotoxic and antiangiogenic effect of the treatment scheme of chemotherapy used in retinoblastoma by means of different in vitro models and to evaluate potential effects on multi-drug resistance proteins. Two commercial and two patient-derived retinoblastoma cell types and two human vascular endothelial cell types were exposed to increasing concentrations of melphalan or topotecan in a conventional (single exposure) or metronomic (7-day continuous exposure) treatment scheme. The concentration of chemotherapy causing a 50% decrease in cell proliferation (IC50) was determined by MTT and induction of apoptosis was evaluated by flow cytometry. Expression of ABCB1, ABCG2 and ABCC1 after conventional or metronomic treatments was assessed by RT-qPCR. We also evaluated the in vivo response to conventional (0.6 mg/kg once a week for 2 weeks) and metronomic (5 days a week for 2 weeks) topotecan in a retinoblastoma xenograft model. Melphalan and topotecan were cytotoxic to both retinoblastoma and endothelial cells after conventional and metronomic treatments. A significant decrease in the IC50 (median, 13-fold; range: 3–23) was observed following metronomic chemotherapy treatment in retinoblastoma and endothelial cell types compared to conventional treatment (p<0.05). Metronomic topotecan or melphalan significantly inhibited in vitro tube formation in HUVEC and EPC compared to vehicle-treated cells (p<0.05). Both treatment schemes induced apoptosis and/or necrosis in all cell models. No significant difference was observed in the expression of ABCB1, ABCC1 or ABCG2 when comparing cells treated with melphalan or topotecan between treatment schedules at the IC50 or with control cells (p>0.05). In mice, continuous topotecan lead to significantly lower tumor volumes compared to conventional treatment after 14 days of treatment (p<0.05). Continuous exposure to melphalan or topotecan increased the chemosensitivity of retinoblastoma and endothelial cells to both chemotherapy agents with lower IC50 values compared to short-term treatment. These findings were validated in an in vivo model. None of the dosing modalities induced multidrug resistance mechanisms while apoptosis was the mechanism of cell death after both treatment schedules. Metronomic chemotherapy may be a valid option for retinoblastoma treatment allowing reductions of the daily dose.

The comprehension of unconventional immune functions of tonsillar B cells, their role in tolerance induction and protective immune responses, is crucial to unveil the dynamic interactions of the upper aero digestive tract with polymicrobial commensal flora and pathogens, in health and disease. Here, we describe the kinetics of IL10 intracellular expression and compare it with that of cytokines known to be produced by tonsillar B cells. Additionally, we detected a relevant proportion of IL17-expressing tonsillar B cells, which has not previously been reported. We immunophenotyped tonsillar IL10-expressing B cells (B10) and observed IL10 production in activated B cells at every developmental stage. Finally, we identified a relationship between decreased B10 percentages, increased proportion of the germinal centre (GC) population and hypertrophied tonsils (HT). Our findings provide greater insight into the role of B10 in GC reactions and characterized their involvement in the pathogenesis of tonsillar dysfunction.

Digoxin has antitumor activity for retinoblastoma while exerting antiangiogenic activity in vitro at similar concentrations. Metronomic treatment showed no advantage in terms of dose for cytotoxic effect. Four biweekly injections of digoxin led to local toxicity to the retina but no systemic toxicity in rabbits.

Immune responses at the boundary between the host and the world beyond are complex and mucosal tissue homeostasis relies on them. Obstructive sleep apnea (OSA) is a syndrome suffered by children with hypertrophied tonsils. We have previously demonstrated that these tonsils present a defective regulatory B cell (Breg) compartment. Here, we extend those findings by uncovering the crucial role of resident pro-inflammatory B and T cells in sustaining tonsillar hypertrophy and hyperplasia by producing TNFα and IL17, respectively, in ex vivo cultures. Additionally, we detected prominent levels of expression of CD1d by tonsillar stratified as well as reticular epithelium, which have not previously been reported. Furthermore, we evidenced the hypertrophy of germinal centers (GC) and the general hyperplasia of B lymphocytes within the tissue and the lumen of the crypts. Of note, such B cells resulted mainly (IgG/IgM)+ cells, with some IgA+ cells located marginally in the follicles. Finally, by combining bacterial culture from the tonsillar core and subsequent identification of the respective isolates, we determined the most prevalent species within the cohort of OSA patients. Although the isolated species are considered normal oropharyngeal commensals in children, we confirmed their capacity to breach the epithelial barrier. Our work sheds light on the pathological mechanism underlying OSA, highlighting the relevance taken by the host immune system when defining infection versus colonization, and opening alternatives of treatment.