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We report 2 novel, cryptic chromosomal abnormalities in precursor B-cell acute lymphoblastic leukemia (BCP-ALL): a translocation, either t(X;14)(p22;q32) or t(Y;14)(p11; q32), in 33 patients and an interstitial deletion, either del(X)(p22.33p22.

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis, and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pH i in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na ؉ /H ؉ antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pH i decreased sharply, and recovery required both Na ؉ /H ؉ antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn 2؉ resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn 2؉ . Both the rate and extent of acidification in HVCN1-deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis. innate immunity ͉ ion channels ͉ phagocyte ͉ respiratory burst ͉ SNARF W e have reinvestigated the regulation of cytoplasmic pH, pH i , in human neutrophils during phagocytosis. For 3 decades, pH i during the respiratory burst has been characterized as a small (0.05-0.1 unit) transient drop in pH i that is followed by larger (0.16-0.60 unit) prolonged alkalinization caused by Na ϩ /H ϩ antiport activity, whether the stimulus is fMLF (fMetLeuPhe, a chemotactic peptide), PMA (a PKC-activating phorbol ester), phospholipase C, or OPZ (opsonized zymosan) (1-7). In most studies, soluble stimuli were applied to populations of neutrophils. When phagocytosis was studied, the responses of many cells were averaged (5, 8). A powerful new imaging approach, confocal microscopy using the fluorescent pH indicator SNARF-1 enhanced by shifted excitation and emission ratioing (SEER) (9, 10) enabled us to examine the responses of individual cells with high spatial and temporal resolution. We report here that the behavior of individual human neutrophils during phagocytosis differs markedly from the prevailing view. The fundamental pH i response is triphasic: rapid and profound acidification followed by rapid but variably complete recovery, and after longer times, slow acidification that may reflect the onset of apoptosis. ResultsCells That Phagocytose Become Acidic. Human neutrophils were allowed to adhere to a g...

Purpose In high-grade serous ovarian cancer (HGSOC), higher densities of both B cells and the CD8+ T-cell infiltrate were associated with a better prognosis. However, the precise role of B cells in the antitumor response remains unknown. As peritoneal metastases are often responsible for relapse, our aim was to characterize the role of B cells in the antitumor immune response in HGSOC metastases. Experimental Design Unmatched pre and post-chemotherapy HGSOC metastases were studied. B-cell localization was assessed by immunostaining. Their cytokines and chemokines were measured by a multiplex assay, and their phenotype was assessed by flow cytometry. Further in vitro and in vivo assays highlighted the role of B cells and plasma cell IgGs in the development of cytotoxic responses and dendritic cell activation. Results B cells mainly infiltrated lymphoid structures in the stroma of HGSOC metastases. There was a strong B-cell memory response directed at a restricted repertoire of antigens and production of tumor-specific IgGs by plasma cells. These responses were enhanced by chemotherapy. Interestingly, transcript levels of CD20 correlated with markers of immune cytolytic responses and immune complexes with tumor-derived IgGs stimulated the expression of the costimulatory molecule CD86 on antigen-presenting cells. A positive role for B cells in the antitumor response was also supported by B-cell depletion in a syngeneic mouse model of peritoneal metastasis. Conclusions Our data showed that B cells infiltrating HGSOC omental metastases support the development of an antitumor response.

Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pH i regulation. The "enhanced gating mode" appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-␦ phosphorylation sites in the human voltage-gated proton channel, H V 1 (Hvcn1), Thr 29 and Ser 97 , both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-␦. Enhanced gating was evaluated by expressing wild-type (WT) or mutant H V 1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT H V 1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr 29 is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes.Voltage-gated proton channels enable sustained superoxide anion (O 2 . ) production by NADPH oxidase during the respira- efflux through open voltage-gated proton channels (1-6). The activities of NADPH oxidase and voltage-gated proton channels are coordinated in several ways. The depolarization and pH i decrease resulting from NADPH oxidase activity both directly promote proton channel opening. In addition, interventions that activate NADPH oxidase profoundly enhance the gating properties of proton channels (3, 7). This "enhanced gating mode" consists of four changes in proton channel properties, each of which increases the likelihood of channel opening under any given set of conditions. The channels open faster (smaller activation time constant, act ) 3 and close more slowly (larger deactivation time constant, tail ), display increased maximum proton conductance (g H,max ), and manifest a 40-mV hyperpolarizing shift of the entire proton conductance-voltage relationship (g H -V). The enhanced gating mode improves the efficiency of NADPH oxidase by minimizing the depolarization required to open enough proton channels to fully compensate the electrical consequences of NADPH oxidase activity (i.e. the electron current) (5). Depolarization directly inhibits NADPH oxidase (4, 8).The enhanced gating mode is induced by PMA, an activator of PKC, and is prevented and at least partially reversed by the PKC inhibitor GFX (9, 10). Although these results suggest regulation by PKC phosphorylation, they do not clarify whether the target of PKC is an accessor...

Decay-accelerating factor (CD55) is a complement regulatory protein, which is expressed by most cells to protect them from complement-mediated attack. CD55 also binds CD97, an EGF-TM7 receptor constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells upon activation. Early results suggested that CD55 could further enhance T cell proliferation induced by phorbol ester treatment. The present study demonstrates that coengagement of CD55, using either cross-linking mAbs or its natural ligand CD97, and CD3 results in enhanced proliferation of human peripheral blood CD4+ T cells, expression of the activation markers CD69 and CD25, and secretion of IL-10 and GM-CSF. Recently, an increase in T cell responsiveness in CD55−/− mice was shown to be mediated by a lack of complement regulation. In this study, we show that direct stimulation of CD55 on CD4+ T cells with CD97 can modulate T cell activation but does not interfere with CD55-mediated complement regulation. Our results support a multifaceted role for CD55 in human T cell activation, constituting a further link between innate and adaptive immunity.

Electrophysiological studies have implicated voltage-gated proton channels in several specific cellular contexts. In neutrophils, they mediate charge compensation associated with the oxidative burst of phagocytosis. Molecular characterization of the hydrogen voltage-gated channel 1 (HVCN1) has enabled identification of unanticipated and diverse functions: HVCN1 not only modulates signaling from the B-cell receptor following B-cell activation and histamine release from basophils, but also mediates pH-mediated activation of spermatozoa, as well as acid secretion by tracheal epithelium. The importance of HVCN1 in pH regulation during phagocytosis was established by surprising evidence indicating its first-responder role. In this review, we examine recent findings from a functional perspective, and discuss the potential of HVCN1 as a therapeutic target for autoimmune and other diseases.