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Microglia are the tissue-resident macrophage population of the brain, specialized in supporting the CNS environment and protecting it from endogenous and exogenous insults. Nonetheless, their function declines with age, in ways that remain to be fully elucidated. Given the critical role played by microglia in neurodegenerative diseases, a better understanding of the aging microglia phenotype is an essential prerequisite in designing better preventive and therapeutic strategies. In this review, we discuss the most recent literature on microglia in aging, comparing findings in rodent models and human subjects.

Age, disease stage and treatment of patients from the cohort used for plasma IgG1,2,3 and 4 dosage

Supplementary Material and Methods describing in vivo, mass spectrometry and immunocytofluorescence experiments; Supplementary Figures 1,2 and 3 and Supplementary figures and tables legends.

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. Clin Cancer Res; 23(1); 250–62. ©2016 AACR.

Background and aims The presence of tertiary lymphoid structures (TLSs) may confer survival benefit to patients with pancreatic ductal adenocarcinoma (PDAC), in an otherwise immunologically inert malignancy. Yet, the precise role in PDAC has not been elucidated. Here, we aim to investigate the structure and role of TLSs in human and murine pancreatic cancer. Methods Multicolor immunofluorescence and immunohistochemistry were used to fully characterize TLSs in human and murine (transgenic [KPC (KrasG12D, p53R172H, Pdx-1-Cre)] and orthotopic) pancreatic cancer. An orthotopic murine model was developed to study the development of TLSs and the effect of the combined chemotherapy and immunotherapy on tumor growth. Results Mature, functional TLSs are not ubiquitous in human PDAC and KPC murine cancers and are absent in the orthotopic murine model. TLS formation can be induced in the orthotopic model of PDAC after intratumoral injection of lymphoid chemokines (CXCL13/CCL21). Coadministration of systemic chemotherapy (gemcitabine) and intratumoral lymphoid chemokines into orthotopic tumors altered immune cell infiltration ,facilitating TLS induction and potentiating antitumor activity of chemotherapy. This resulted in significant tumor reduction, an effect not achieved by either treatment alone. Antitumor activity seen after TLS induction is associated with B cell-mediated dendritic cell activation. Conclusions This study provides supportive evidence that TLS induction may potentiate the antitumor activity of chemotherapy in a murine model of PDAC. A detailed understanding of TLS kinetics and their induction, owing to multiple host and tumor factors, may help design personalized therapies harnessing the potential of immune-oncology., Graphical abstract

Monocyte migration to the sites of inflammation and maturation into macrophages are key steps for their immune effector function. Here, we show that mechanistic target of rapamycin complex 2 (mTORC2)-dependent Akt activation is instrumental for metabolic reprogramming at the early stages of macrophage-mediated immunity. Despite an increased production of proinflammatory mediators, monocytes lacking expression of the mTORC2 component Rictor fail to efficiently migrate to inflammatory sites and fully mature into macrophages, resulting in reduced inflammatory responses in vivo. The mTORC2-dependent phosphorylation of Akt is instrumental for the enhancement of glycolysis and mitochondrial respiration, required to sustain monocyte maturation and motility. These observations are discussed in the context of therapeutic strategies aimed at selective inhibition of mTORC2 activity.

Defective silencing of retrotransposable elements has been linked to inflammageing, cancer and autoimmune diseases. However, the underlying mechanisms are only partially understood. Here we implicate the histone H3.3 chaperone Daxx, a retrotransposable element repressor inactivated in myeloid leukaemia and other neoplasms, in protection from inflammatory disease. Loss of Daxx alters the chromatin landscape, H3.3 distribution and histone marks of haematopoietic progenitors, leading to engagement of a Pu.1-dependent transcriptional programme for myelopoiesis at the expense of B-cell differentiation. This causes neutrophilia and inflammation, predisposing mice to develop an autoinflammatory skin disease. While these molecular and phenotypic perturbations are in part reverted in animals lacking both Pu.1 and Daxx, haematopoietic progenitors in these mice show unique chromatin and transcriptome alterations, suggesting an interaction between these two pathways. Overall, our findings implicate retrotransposable element silencing in haematopoiesis and suggest a cross-talk between the H3.3 loading machinery and the pioneer transcription factor Pu.1., Gerber, Russ et al. show that the H3.3 chaperone Daxx, which represses endogenous retroviral and retrotransposable elements, acts as an epigenetic barrier to control haematopoietic progenitor plasticity and protect against PU.1-mediated inflammation.

Microglia are the innate immune cells of the brain, which maintain homeostasis by constantly scanning and surveying the environment with their highly ramified processes. In order to exert this function, they need to phagocytose synapses as well as debris and dead cells, a process that is further amplified in pathological conditions. Importantly, it has been shown that microglia phagocytic capacity is altered in the course of neurodegenerative disease, for which aging is one of the highest risk factors. Thus, understanding how phagocytosis is impaired during aging is a priority for future research. Advances in this area are expected to significantly contribute to our understanding of normal cognition during aging, as well as changes that take place in age-associated neurodegenerative diseases. In this review, we will summarize the current knowledge on how phagocytosis is executed and affected by aging or in age-associated neurological disorders, such as Alzheimer's disease (AD). Furthermore, we will summarize both protective and deleterious consequences of altered phagocytosis in AD and where relevant in other neurodegenerative diseases.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response. This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 µL basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFNγ and TNFα) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFNγ+, TNFα+ and CD107a+ CD4+ and CD8+ T cells was quantified. This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response. This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 µL basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFNγ and TNFα) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFNγ+, TNFα+ and CD107a+ CD4+ and CD8+ T cells was quantified. This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB–dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration.

Background: Heterozygous germline loss-of-function mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene are usually associated with acromegaly or gigantism due to a young-onset somatotropinoma. AIP functions as a tumor suppressor gene in the pituitary, while its role in other tumours is unknown. AIP is highly expressed in patients with Diffuse Large B Cell Lymphoma (DLBCL) compared to any other tumor type. DLBCL arises from germinal centre B cells which characteristically express the transcriptional repressor B cell lymphoma-6 (BCL6), key for germinal centre formation. Increased expression of BCL6 can lead to the development of DLBCL. Despite the obvious importance of BCL6 expression in GC B cells, the mechanisms by which it is regulated are still poorly understood. Aim: The aim of this study was to investigate the relationship of the high AIP expression, BCL6 and the pathobiology of DLBCL. Methods: We generated mice carrying a conditional homozygous deletion of Aip in T and B cells (Aipfl/fl;Rag1Cre/+). Co-localization of AIP and members of the BCL6 ubiquitination/proteasome pathway by immunofluorescence analysis. Ubiquitin-mediated proteasomal degradation study was done by immuno-precipitation (IP). Results: Our study found that AIP is highly expressed in DLBCL. Genetic deletion of Aip revealed that AIP supported BCL6 expression in mice. The ubiquitin E3 ligase FBXO11 has been previously shown to add ubiquitin to BCL6. We found that AIP could bind to the deubiquitinase UCHL1 and that UCHL1 could remove ubiquitin from BCL6 thereby supporting BCL6 expression by preventing FBXO11 mediated degradation of BCL6 recapitulating what we observed in Aip deficient B cells. Conclusions: The data presented here reveal AIP to be a novel positive regulator of BCL6 protein expression which is commonly up-regulated in DLBCL and prevents FBXO11 degradation of BCL6 by enabling the UCHL1 to remove ubiquitin from BCL6. Therefore, AIP is a potential novel therapeutic target to treat DLBCL. It appears that AIP function as a tumor suppressor in the pituitary and as an oncogene to the pathobiology of DLBCL. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

B cells are salient features of pancreatic ductal adenocarcinoma (PDAC) tumors, yet their role in this disease remains controversial. Murine studies have indicated a protumoral role for B cells, whereas clinical data show tumor-infiltrating B cells are a positive prognostic factor, both in PDAC and other cancers. This disparity needs to be clarified in order to develop effective immunotherapies. In this study, we provide new evidence that reconcile human and mouse data and highlight the importance of using relevant preclinical tumor models when assessing B cell function. We compared B cell infiltration and activation in both a genetic model of murine PDAC (KPC mouse) and an injectable orthotopic model. A pronounced B cell infiltrate was only observed in KPC tumors and correlated with T cell infiltration, mirroring human disease. In contrast, orthotopic tumors exhibited a relative paucity of B cells. Accordingly, KPC-derived B cells displayed markers of B cell activation (germinal center entry, B cell memory, and plasma cell differentiation) accompanied by significant intratumoral immunoglobulin deposition, a feature markedly weaker in orthotopic tumors. Tumor immunoglobulins, however, did not appear to form immune complexes. Furthermore, in contrast to the current paradigm that tumor B cells are immunosuppressive, when assessed as a bulk population, intratumoral B cells upregulated several proinflammatory and immunostimulatory genes, a distinctly different phenotype to that of splenic-derived B cells; further highlighting the importance of studying tumor-infiltrating B cells over B cells from secondary lymphoid organs. In agreement with the current literature, genetic deletion of B cells (μMT mice) resulted in reduced orthotopic tumor growth, however, this was not recapitulated by treatment with B-cell-depleting anti-CD20 antibody and, more importantly, was not observed in anti-CD20-treated KPC mice. This suggests the result from B cell deficient mice might be caused by their altered immune system, rather than lack of B cells. Therefore, our data indicate B cells do not favor tumor progression. In conclusion, our analysis of relevant preclinical models shows B cells to be active members of the tumor microenvironment, producing immunostimulatory factors that might support the adaptive antitumor immune response, as suggested by human PDAC studies.

Summary: B cell lymphoma-6 (BCL6) is highly expressed in germinal center B cells, but how its expression is maintained is still not completely clear. Aryl hydrocarbon receptor interacting protein (AIP) is a co-chaperone of heat shock protein 90. Deletion of Aip in B cells decreased BCL6 expression, reducing germinal center B cells and diminishing adaptive immune responses. AIP was required for optimal AKT signaling in response to B cell receptor stimulation, and AIP protected BCL6 from ubiquitin-mediated proteasomal degradation by the E3-ubiquitin ligase FBXO11 by binding to the deubiquitinase UCHL1, thus helping to maintain the expression of BCL6. AIP was highly expressed in primary diffuse large B cell lymphomas compared to healthy tissue and other tumors. Our findings describe AIP as a positive regulator of BCL6 expression with implications for the pathobiology of diffuse large B cell lymphoma. : BCL6 overexpression contributes to the pathobiology of diffuse large B cell lymphoma (DLBCL). Sun et al. find that the co-chaperone aryl hydrocarbon receptor interacting protein (AIP), whose high expression is associated with reduced survival of DLBCL patients, helps maintain BCL6 expression by facilitating the removal of ubiquitin from BCL6. Keywords: AIP, BCL6, FBXO11, UCHL1, ubiquitination, lymphoma

The identification of the HVCN1 gene, encoding the only mammalian voltage-gated proton channel, prompted a number of studies on how proton channels affect cellular functions. As their expression is mainly restricted to immune cells, it is not surprising that proton channels regulate different aspects of immune responses. In this review, I will examine the current knowledge of voltage-gated proton channels in both innate and adaptive responses and assess the remaining outstanding questions.

B regulatory cells are a newly described subpopulation of B cells that appear to play important roles in autoimmunity and more recently, in cancer. In this review we summarize our current knowledge of B regulatory cells, as well as the body of evidence pointing towards a role for B cells in general, and B regulatory cells in particular, in promoting tumor growth.

Proton channels are expressed in all the cells of the immune system to various degrees. While their function in phagocytic cells, immune cells that engulf bacteria and cell debris for clearance, has been the object of extensive research, the function of proton channels in non-phagocytic cells has remained more elusive until recently. Further studies have been supported by the discovery of the gene coding for the mammalian proton channel, HVCN1, which has prompted a new wave of research in this area. Recent findings show how proton channels regulate cell function in non-phagocytic cells of the immune system such as basophils and lymphocytes. WIREs Membr Transp Signal 2013, 2:65–73. doi: 10.1002/wmts.78 For further resources related to this article, please visit the WIREs website.

Cancers are not just masses of malignant cells but complex ‘rogue’ organs, to which many other cells are recruited and can be corrupted by the transformed cells. Interactions between malignant and non-transformed cells create the tumor microenvironment (TME). The non-malignant cells of the TME

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.

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. Clin Cancer Res; 23(1); 250–62. ©2016 AACR.

Electrophysiological studies have implicated voltage-gated proton channels in several specific cellular contexts. In neutrophils, they mediate charge compensation that is 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-dependent 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 that indicated its first-responder role. In this review, we discuss recent findings from a functional perspective, and the potential of HVCN1 as a therapeutic target for autoimmune and other diseases.

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.

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)(p22.33p22.33) or del(Y)(p11.32p11.32), in 64 patients, involving the pseudoautosomal region (PAR1) of the sex chromosomes. The incidence of these abnormalities was 5% in childhood ALL (0.8% with the translocation, 4.2% with the deletion). Patients with the translocation were older (median age, 16 years), whereas the patients with the deletion were younger (median age, 4 years). The 2 abnormalities result in deregulated expression of the cytokine receptor, cytokine receptor-like factor 2, CRLF2 (also known as thymic stromal-derived lymphopoietin receptor, TSLPR). Overexpression of CRLF2 was associated with activation of the JAK-STAT pathway in cell lines and transduced primary B-cell progenitors, sustaining their proliferation and indicating a causal role of CRLF2 overexpression in lymphoid transformation. In Down syndrome (DS) ALL and 2 non-DS BCP-ALL cell lines, CRLF2 deregulation was associated with mutations of the JAK2 pseudokinase domain, suggesting oncogenic cooperation as well as highlighting a link between non-DS ALL and JAK2 mutations.

It is now accepted that metabolic disorders such as type 2 diabetes (T2D) and obesity are characterized by alterations in immune cells. Indeed, immune cells have been shown to play a critical role in fueling the inflammation that accompanies increased fat deposition and insulin resistance. Both the innate (macrophages) and the adaptive arm (CD4 and CD8 T cells, in particular) significantly contribute to the chronic inflammatory state that characterizes these conditions. B cells, however, have until recently remained significantly understudied. This is not due to the fact that they do not play as significant a role, as more recent publications appear to show. Here, we briefly review the most recent literature and highlight the remaining outstanding questions.

T-cell defects, immune suppression, and poor antitumor immune responses are hallmarks of chronic lymphocytic leukemia (CLL), and PD-1/PD-L1 inhibitory signaling has emerged as a major immunosuppressive mechanism. However, the effect of different microenvironments and the confounding influence of aging are poorly understood. The current study uses the Eμ-TCL1 mouse model, which replicates human T-cell defects, as a preclinical platform to longitudinally examine patterns of T-cell dysfunction alongside developing CLL and in different microenvironments, with a focus on PD-1/PD-L1 interactions. The development of CLL was significantly associated with changes in T-cell phenotype across all organs and function. Although partly mirrored in aging wild-type mice, CLL-specific T-cell changes were identified. Murine CLL cells highly expressed PD-L1 and PD-L2 in all organs, with high PD-L1 expression in the spleen. CD3(+)CD8(+) T cells from leukemic and aging healthy mice highly expressed PD-1, identifying aging as a confounder, but adoptive transfer experiments demonstrated CLL-specific PD-1 induction. Direct comparisons of PD-1 expression and function between aging CLL mice and controls identified PD-1(+) T cells in CLL as a heterogeneous population with variable effector function. This is highly relevant for therapeutic targeting of CD8(+) T cells, showing the potential of reprogramming and selective subset expansion to restore antitumor immunity.

Alterations of mitochondrial metabolism and genomic instability have been implicated in tumorigenesis in multiple tissues. High-grade glioma (HGG), one of the most lethal human neoplasms, displays genetic modifications of Krebs cycle components as well as electron transport chain (ETC) alterations. Furthermore, the p53 tumor suppressor, which has emerged as a key regulator of mitochondrial respiration at the expense of glycolysis, is genetically inactivated in a large proportion of HGG cases. Therefore, it is becoming evident that genetic modifications can affect cell metabolism in HGG; however, it is currently unclear whether mitochondrial metabolism alterations could vice versa promote genomic instability as a mechanism for neoplastic transformation. Here, we show that, in neural progenitor/stem cells (NPCs), which can act as HGG cell of origin, inhibition of mitochondrial metabolism leads to p53 genetic inactivation. Impairment of respiration via inhibition of complex I or decreased mitochondrial DNA copy number leads to p53 genetic loss and a glycolytic switch. p53 genetic inactivation in ETC-impaired neural stem cells is caused by increased reactive oxygen species and associated oxidative DNA damage. ETC-impaired cells display a marked growth advantage in the presence or absence of oncogenic RAS, and form undifferentiated tumors when transplanted into the mouse brain. Finally, p53 mutations correlated with alterations in ETC subunit composition and activity in primary glioma-initiating neural stem cells. Together, these findings provide previously unidentified insights into the relationship between mitochondria, genomic stability, and tumor suppressive control, with implications for our understanding of brain cancer pathogenesis.

The juice of Euterpe oleracea Mart. fruit (Arecaceae), known as Acai in the Brazilian Amazon region, is dark purple with a high anthocyanin and phenolic content. The antioxidant and anti-radical properties of E. oleracea juice are well known; the chemical characterisation of its phenolic composition as well as its potential use as food ingredient and natural pigment have been previously studied. Cyanidin 3-O-glycoside, and various hydroxy-benzoic and hydroxy-cinnamic acids were detected in E. oleracea juice. The radical scavenging properties, measured as the mean of DPPH radical tests, were similar to those obtained by a common commercial bilberry juice. Therefore, novel natural colorants from E. oleracea juice could be considered as “functional” ingredients for their anti-oxidant and anti-radical activity. Yogurt is a typical fermented dairy product consumed all around the world; the yogurt flavouring is obtained both by means of natural ingredients like fruit juices and also by synthetic aromas. The aim of this work was to evaluate the use of E. oleracea fruit juice as a natural colorant for yogurt. The results obtained showed that yogurt enriched with Euterpe juice (10%, w/w) showed characteristics similar to those of typical commercial yogurt with bilberry juice. Aggregation of milk proteins in the E. oleracea containing yogurt was measured by SDS-PAGE. The protein profile of the E. oleracea containing yogurt was essentially identical to the untreated control yogurt. In conclusion, we suggest that E. oleracea juice could be used as a natural functional pigment for flavouring and colouring yogurt.

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1L, Thr(29). Furthermore, the association of HVCN1S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus define common subgroups of B-cell lymphoma but are rare in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Recent fluorescent in situ hybridization and molecular cloning studies have identified several novel IGH translocations involving genes that play important roles in normal hemopoiesis, including the cytokine receptor genes CRLF2 and EPOR, all members of the CCAAT enhancer-binding protein gene family, as well as genes not normally expressed in hemopoietic cells including inhibitor of DNA binding 4. IGH translocation results in deregulated target gene expression because of juxtaposition with IGH transcriptional enhancers. However, many genes targeted by IGH translocations are also more commonly deregulated in BCP-ALL as a consequence of other genetic or epigenetic mechanisms. For example, interstitial genomic deletions also result in deregulated CRLF2 expression, whereas EPOR expression is deregulated as a consequence of the ETV6-RUNX1 fusion. The possible clinical importance of many of the various IGH translocations in BCP-ALL remains to be determined from prospective studies, but CRLF2 expression is associated with a poor prognosis. Despite their rarity, IGH chromosomal translocations in BCP-ALL therefore define not only new mechanisms of B-cell transformation but also clinically important subgroups of disease and suggest new targeted therapeutic approaches.

Intra-tumor B cells and immunoglobulin signatures have been associated with a better clinical prognosis in ovarian cancer (Iglesia et al, 2014; Nielsen et al, 2012). However little is known about the mechanisms by which B cells influence the anti-cancer immune response. The aim of our research is to evaluate B cell dependent anti-cancer immune responses in peritoneal metastases in high grade serous ovarian cancer (HGSOC) patients. We have found that in these peritoneal metastases, B cells are mainly located in lymphoid structures and a high proportion of these (60%) display a CD27+ memory phenotype. HGSOC B cells are able to secrete Th1 related cytokines as well as high levels of IL8, which is a chemo-attractant for T cells, granulocytes and macrophages. Interestingly CD4+ and CD8+ T cells express high levels of the IL8 receptor, CXCR1, which suggests B cells help recruiting T cells to lymphoid structures. Plasma cells were also detected in the peritoneal metastases along with IgG deposits. Secreted IgGs were mainly located in stromal areas suggesting that the associated matrix may prevent their infiltration into tumour islets and/or that they target specific stromal antigens. Interestingly, we observed that neoadjuvant chemotherapy and residual disease after chemotherapy influence the type of IgG subclasses expressed in the omentum of HGSOC patients. Chemotherapy enhances the expression of IgG1, IgG2 and IgG3 immunoglobulins in omentum with residual disease. This supports the notion that B cells are involved in the anti-tumor immune response in HGSOC metastases. In conclusion, our data suggest that B cells actively participate to the anti-tumor immune response in HGSOC metastases. This response is preferentially Th1 oriented through the production of cytokines and dominant expression of IgG1 immunoglobulins. Enhancing the B cell dependent anti-tumor immune response could be a potential strategy for the treatment of HGSOC patients. Iglesia MD, Vincent BG, Parker JS, Hoadley KA, Carey LA, Perou CM, Serody JS (2014) Prognostic B-cell signatures using mRNA-seq in patients with subtype-specific breast and ovarian cancer. Clin Cancer Res 20: 3818-3829 Nielsen JS, Sahota RA, Milne K, Kost SE, Nesslinger NJ, Watson PH, Nelson BH (2012) CD20+ tumor-infiltrating lymphocytes have an atypical CD27- memory phenotype and together with CD8+ T cells promote favorable prognosis in ovarian cancer. Clin Cancer Res 18: 3281-3292 Citation Format: Anne Montfort, Steffen Boehm, Thomas Dowe, Joanne Topping, Michelle Lockley, Melania Capasso, Frances Balkwill. B cells actively participate to the anti-cancer immune response in high grade serous ovarian cancer metastases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 455. doi:10.1158/1538-7445.AM2015-455

One of the best established functions of proton channels is facilitating NADPH oxidase activity in phagocytes. NADPH oxidase moves electrons across the membrane, leaving protons in the cytoplasm. Proton channels extrude most of this acid, simultaneously compensating for the electrogenic action of the oxidase and preventing cytoplasmic acidification. Agonists that activate NADPH oxidase also produce an “enhanced gating mode” in proton channels, characterized by faster activation, increased conductance, and a negative shift of the gH-V relationship, all of which increase the likelihood of proton channel opening. The enhanced gating mode is the result of PKC activity (Morgan et al, 2007, J. Physiol. 579:327-344). We assessed proton channel responses in a murine B cell line, LK35.2. Nontransfected LK35.2 cells have no detectable proton current; cells transfected with the human proton channel gene, HVCN1, have proton currents that respond to PMA and the PKC inhibitor, GF109203X (GFX), when studied in perforated-patch configuration. The response of proton currents in these cells to PMA or GFX was qualitatively like that of native leukocytes, but smaller in amplitude. In contrast, there was no detectable PMA response of human or murine proton channels expressed in HEK-293 or COS-7 cells (Musset et al, 2008, J. Physiol. 586:2477-2486). Here we mutated two putative PKC phosphorylation sites on the human proton channel. When the mutant channels were expressed in LK35.2 cells, the response to PMA or GFX was ablated. These studies indicate that PKC phosphorylates the proton channel directly.Supported by the NIH-HLBI (HL61437), Philip Morris, and the Schmidtmann Foundation.

Background CLL-induced severe T-cell dysfunction and ineffective anti-tumor immune-responses are hallmarks of the disease, but the specific interactions remain poorly understood. The PD-1/PD-L1 axis is an important mediator of T-cell dysfunction in solid tumors, and we have previously demonstrated that T cells from CLL patients exhibit impaired immunological synapse (IS) formation, predominantly mediated by PD-L1 (CD274) expression on CLL cells. We have also shown that the corresponding T-cell ligand PD-1 (CD279) is also upregulated, probably as a result of chronic antigenic stimulation, and that T cells have similarities to exhausted T cells observed in the context of chronic viral infection. Recent studies demonstrated that ibrutinib has impressive clinical activity in CLL, and mechanisms of action include irreversible binding of essential components of both B-cell- and T-cell-receptor signaling and interactions with the tumor microenvironment. Modulation of PD-1/PD-L1 interactions might therefore be an additional potential mode of action of this drug. Using the well-established Eμ-TCL1 (TCL1) mouse model of CLL, our aims were to demonstrate that (1) altered expression of PD-L1 on CLL cells and PD-1 on T cells and CLL are causally related, (2) the second ligand of PD-1, PD-L2, is also involved in mediating T-cell dysfunction, (3) T-cell effector function and IS formation are directly linked to PD-1 expression and (4) PD-1 associated in vivo T-cell responses can be modulated by treatment with ibrutinib. Methods As we have previously demonstrated that the spleen is the major organ of disease and representative of T-cell changes in peripheral blood and lymph nodes, experiments were performed on spleens from young TCL1 and wild-type (WT) C57Bl/6 mice with established CLL after adoptive transfer (AT) of syngeneic CLL cells (n=10), and on matched litter-mates after AT of healthy mouse B cells (n=10). An additional 12 mice were randomized to treatment with 25 mg/kg/d ibrutinib in 10% HP-β-CD, vehicle control, or sterile water, all administered by gavage, three weeks after AT of syngeneic CLL cells, and sacrificed 20 days later at a pre-defined endpoint. Multicolor flow cytometry was used to characterize T-cell subsets, expression of PD-1, PD-L1 and PD-L2 and T-cell effector function. Entire population CD8 T cells, PD-1+ve and PD-1-ve CD8 T cells were flow-sorted and used in IS formation assays with healthy murine B cells as antigen-presenting cells. Results Our previous studies using aged TCL1 mice and age-matched WT controls indicated that CLL-related PD-1 upregulation on antigen-experienced CD44+ CD8 T cells is masked by aging. However, PD-1 expression could also be induced in young TCL1 and WT mice by AT of CLL cells but not healthy B cells, suggesting a causal relationship with disease. Both PD-L1 and PD-L2 surface expression on CLL B cells were significantly increased compared to healthy B cells. Using TCL1 mice at early stages of CLL development when a healthy CD19+ B-cell population is still present, we were able to confirm that PD-L2 expression is a unique feature of CLL cells, with PD-L2 being virtually absent on healthy B cells. We next compared effector function and the ability to form IS of PD-1+ve and PD-1-ve antigen-experienced CD44+ T-cell subsets in mice with CLL. While proliferation was equally impaired in these subsets, they were both able to degranulate but generally failed to localize granzyme B to the IS. Although subsets produced some IL2/TNFα/IFNγ cytokine responses, PD-1+ve cells had significantly impaired TNFα and slightly impaired IL2 and IFNγ production, and a highly significant impaired ability to form IS compared to PD-1-ve cells. Treatment with ibrutinib reduced PD-1 expression on antigen-experienced CD44+ CD8 T cells and promoted stronger IFNγ production of entire population CD8 T cells, but failed to restore proliferation and granzyme B relocation to the IS. Conclusion Our in vivo data suggests that CLL and PD-1/PD-L1-mediated T-cell dysfunction are causally related, but that phenotypic and functional T-cell changes are not absolute and might be at least partly reversible by ibrutinib treatment. We also show that the second ligand of PD-1, PD-L2, is also a critical mediator of PD-1 associated T-cell dysfunction in CLL. Disclosures: Riches: Celgene: Research Funding. Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

HVCN1 is the only mammalian voltage-gated proton channel, highly expressed in B cell cancers such as lymphoma and chronic lymphocytic leukaemia (CLL). In normal B cells, we showed that HVCN1 sustains ROS production by the NADPH oxidase, required for optimal B Cell Receptor (BCR) signalling. In malignant B cells, HVCN1 appears to sustain tumour growth, since HVCN1 downregulation impairs cell survival. We are currently addressing whether HVCN1 supports cancer cells via maintaining ROS production and BCR signalling. During our initial investigation on HVCN1 in B cells, we identified a shorter translational isoform of 253 aa, translated from a second initiation site 20 aa downstream from the first ATG. HVCN1 Short is specifically expressed by malignant B cells: we have evidence that it has different electrophysiological properties, such as conducting larger currents with faster activation kinetics after PMA stimulation. In addition, while HVCN1 Long is internalised with the BCR upon stimulation, HVCN1 short is not, indicating it could mediate proton currents constitutively, thus further helping tumour cells maintain their aberrant pH and sustaining BCR signalling.Support: Bennett Fellowship from Leukaemia and Lymphoma Research (ref n: 12002) to MC. NSF MCB-0943362, NIH R01-GM087507 to TD.

HVCN1 is a highly-conserved voltage-gated proton channel. Voltage-gated proton currents have been recorded in lymphocytes but their functions in B cells remain unknown. We isolated HVCN1 in a proteomic survey of plasma membrane proteins in mantle cell lymphoma (MCL) in leukemic phase. In normal lymphocytes, HVCN1 expression was restricted to the B-cell lineage; HVCN1 was highly expressed in mantle zone cells but down-regulated in germinal center (GC) cells undergoing receptor affinity maturation and class-switch recombination (CSR). Highest level expression was also observed in Chronic Lymphocytic Leukemia (CLL) cells from the peripheral blood. In MCL tumors, HVCN1 was expressed in circulating cells but absent from involved lymph nodes, whereas in diffuse large B cell lymphoma (DLBCL), its expression correlated with cases with a low proliferation index. Thus, in both primary and neoplastic B cells, HVCN1 expression appears to be associated with a non-proliferative phenotype. In human primary resting B cells and B cell lines, HVCN1 directly interacted with the B cell receptor (BCR) complex, as shown by Igβ and HVCN1 reciprocal immunoprecipitation experiments. We also found by confocal microscopy and subcellular fractionation, that upon BCR engagement the channel was internalized with the antigen receptor and the two proteins co-migrated to the endo-lysosomal, MHC class II (MHC-II) containing compartments (MIICs). When overexpressed in a hen egg lysozyme (HEL)-specific B cell clone, LK35.2, HVCN1 showed a basal phosphorylation which increased with HEL stimulation. The increased phosphorylation corresponded to an increase in proton conductance, termed “enhanced gating mode” and it was PKC dependent. We then asked whether HVCN1 over-expression could influence MHC II antigen presentation and if the effect could be mediated by changes in MIICs pH. Indeed, presentation of HEL peptides to a T cell clone was impaired in LK35.2 and A20 D1.3 cells, where HVCN1 had been re-introduced; effect was stronger for plate-bound antigen than for soluble antigen. The reduced antigen presentation was accompanied by an increase in endo-lysosomal pH, from pH4.9 ± 0.2 to 6.3 ± 0.1 (which may reflect HVCN1 channel-mediated proton flux out of the organelles), as measured with an anti-IgM antibody conjugated to a pH sensitive dye in HVCN1 over-expressing cells. Evidently, the presence of HVCN1 leads to increased endo-lysosomal pH, consistent with H+ current from the lysosomal compartment into the cytosol. Hence, active antigen presenting cells, like GC cells, might down-regulate HVCN1 expression to maximize the effect of antigen presentation. In order to investigate the role of HVCN1 in vivo, we used a HVCN1-deficient mouse line generated by genetrap insertion. These mice showed no obvious changes in numbers or composition of B-cell subpopulations. Immunization of HVCN1-deficient mice with a T-dependent antigen resulted in a defect in CSR to all IgG subclasses, particularly marked for the IgG2b, whereas in contrast, no differences were observed in IgM secretion, suggesting a pivotal role for HVCN1 during antigen-driven B-cell activation and subsequent CSR. HVCN1 may influence B-cell activation through alteration of reactive oxygen species (ROS) as HVCN1-deficient B cells showed reduced ROS production following BCR activation, a sign of suboptimal NADPH oxidase activity. It has been postulated that proton channels are required to counterbalance the electrogenic activity of NADPH oxidase during ROS production. Our data suggest that this mechanism also occurs in vivo and shed new light on the role of ROS in B cell activation and downstream effects.

We have recently described recurrent IGH@ translocations in BCP-ALL, which result in deregulated expression of the translocated oncogene. We carried out screening for novel IGH@ translocations in BCP-ALL by fluorescence in situ hybridization (FISH) and identified two cryptic translocations involving PAR1 of either sex chromosomes in 29 patients: t(X;14)(p22;q32) (n=18) and t(Y;14)(p11;q32) (n=7) (X or Y unknown, n= 4) and 2 BCP-ALL cell lines with t(Y;14)(p11;q32). The median age of the patients was 18 years (range 4–76 years) and median WBC was 70 ×109/l (range 1–342). Follow-up data was available for 16 patients. Among 5 children diagnosed 1996–2001, 1 failed to remit, 1 suffered an early death and 3 relapsed (16m, 54m, and 64m post diagnosis). To date, all 5 children diagnosed 2004–2005 are in first continuing complete remission (CCR) after 33–46 months, suggesting an improved outcome on the current childhood ALL treatment trial. All 6 adults were diagnosed 2004–2006: 2 died within a year (1 in remission and 1 post transplant); 2 relapsed and died within 1 year and 2 are in 1st CCR after 20 and 36 months. These observations indicate a variable outcome among adults. Translocation breakpoint cloning from IGHJ by long distance inverse PCR and subsequent FISH mapping identified CRLF2, also known as thymic stromal-derived lymphopoietin receptor, at Xp22 and Yp11, to be juxtaposed to IGH@ in these translocations. Breakpoints were cloned in 11 patients and clustered between 2–27kb centromeric of the 3′ UTR of CRLF2. Expression levels of CRLF2, measured by qRT-PCR in 4 patients and the cell lines, was 500–6,500 fold higher than the control cohort of BCP-ALL patients without the translocation. This overexpression confirmed CRLF2 to be the target gene. CRLF2 and IL2RGC bind to IL7RA to form functional receptors for TSLP and IL7, respectively. However, neither IL2RGC nor IL7RA were aberrantly expressed. The presence of associated genomic copy number alterations was shown by array-based comparative genomic hybridization and FISH. As described for BCP-ALL in general, the significant changes included deletions of the B cell differentiation genes: PAX5 and IKAROS, as well as the cell cycle control gene, CDKN2A. The cell lines revealed some biochemical insights into the functional consequences of this translocation involving PAR1. Cell surface expression of CRLF2 was demonstrated, together with constitutive tyrosine phosphorylation of JAK2 and STAT5, the signaling intermediates of the classical JAK-STAT pathway. Further evidence of increased STAT5 activation came from retroviral infection of normal mouse fetal liver cells with human CRLF2, which sustained the proliferation of B-cell precursors in vitro. Conversely, initial studies of CRLF2 knockdown in the cell lines using shRNA demonstrated decreased proliferation. Together, these results show for the first time, the involvement of both sex chromosomes in an IGH@ translocation in leukemia, resulting in deregulated expression of CRLF2 and constitutive JAK-STAT activation. These data suggest CRLF2 as a potential therapeutic target in this subset of patients. Laboratories of MJSD, RS and CJH contributed equally to this work.