Defective mitochondria remodelling in B cells leads to an aged immune response.

The B cell response in the germinal centre (GC) reaction requires a unique bioenergetic supply. Although mitochondria are remodelled upon antigen-mediated B cell receptor stimulation, mitochondrial function in B cells is still poorly understood. To gain a better understanding of the role of mitochondria in B cell function, here we generate mice with B cell-specific deficiency in Tfam, a transcription factor necessary for mitochondrial biogenesis. Tfam conditional knock-out (KO) mice display a blockage of the GC reaction and a bias of B cell differentiation towards memory B cells and aged-related B cells, hallmarks of an aged immune response. Unexpectedly, blocked GC reaction in Tfam KO mice is not caused by defects in the bioenergetic supply but is associated with a defect in the remodelling of the lysosomal compartment in B cells. Our results may thus describe a mitochondrial function for lysosome regulation and the downstream antigen presentation in B cells during the GC reaction, the dysruption of which is manifested as an aged immune response.

Physiological and therapeutic relevance of T cell receptor-mediated antigen trogocytosis.

Trogocytosis is an active process whereby fragments of plasma membrane proteins and cytoplasm are transferred from one cell to another in a cell-cell contact-dependent manner. T cells trogocytose pieces of the cells presenting antigen to them at the site of the immunological synapse. Fragments of the antigen-presenting cell membrane rich in antigen/major histocompatibility (MHC) complexes are internalized by the T cell. Those complexes are redirected to the plasma membrane of the T cell, which becomes an antigen-presenting cell to other T cells. Removing antigen/MHC complexes from professional and tumoral cells has consequences for the intensity and duration of the immune response. However, the acquired capacity of T cells to present the acquired cognate antigen/MHC complexes also affects the properties of the antigen-presenting trogocytotic T cells. Acting as antigen-presenting cells, trogocytotic CD4 T cells influence the differentiation of cytotoxic T cells and the differentiation of other CD4 T cells into pro-inflammatory effector T cells. Furthermore, trogocytosis of antigen/MHC complexes promotes the differentiation of the trogocytotic CD4 T cells towards regulatory T cells and Th2 effector cells. Trogoctyosis is, therefore, a parallel mechanism to signal transduction by membrane receptors, including the T cell antigen receptor, at the plane of the plasma membrane.

Growth hormone remodels the 3D-structure of the mitochondria of inflammatory macrophages and promotes metabolic reprogramming.

Introduction: Macrophages are a heterogeneous population of innate immune cells that support tissue homeostasis through their involvement in tissue development and repair, and pathogen defense. Emerging data reveal that metabolism may control macrophage polarization and function and, conversely, phenotypic polarization may drive metabolic reprogramming. Methods: Here we use biochemical analysis, correlative cryogenic fluorescence microscopy and cryo-focused ion-beam scanning electron microscopy. Results: We demonstrate that growth hormone (GH) reprograms inflammatory GM-CSF-primed monocyte-derived macrophages (GM-MØ) by functioning as a metabolic modulator. We found that exogenous treatment of GM-MØ with recombinant human GH reduced glycolysis and lactate production to levels similar to those found in anti-inflammatory M-MØ. Moreover, GH treatment of GM-MØ augmented mitochondrial volume and altered mitochondrial dynamics, including the remodeling of the inner membrane to increase the density of cristae. Conclusions: Our data demonstrate that GH likely serves a modulatory role in the metabolism of inflammatory macrophages and suggest that metabolic reprogramming of macrophages should be considered as a new target to intervene in inflammatory diseases.

Quantification of Intracellular ATP Content in Ex Vivo GC B Cells.

The study of immunometabolism is an important and emerging field in immunology. B-cell activation upon antigen recognition induces profound metabolic changes in the cell, leading to an increase in ATP production to sustain cell proliferation and differentiation. Current methods available to determine the amount of ATP are time-consuming, require extensive sample processing, and need a large amount of starting material. We set up an easy follow-up protocol to determine the relative amount of ATP in living cells, combining cell surface staining with quinacrine. This acridine dye emits a green fluorescent signal in the presence of intracellular ATP. This protocol allows us to determine ATP in small populations of cells using flow cytometry, such as the germinal center.

Bacteria-instructed B cells cross-prime naïve CD8+ T cells triggering effective cytotoxic responses.

In addition to triggering humoral responses, conventional B cells have been described in vitro to cross-present exogenous antigens activating naïve CD8+ T cells. Nevertheless, the way B cells capture these exogenous antigens and the physiological roles of B cell-mediated cross-presentation remain poorly explored. Here, we show that B cells capture bacteria by trans-phagocytosis from previously infected dendritic cells (DC) when they are in close contact. Bacterial encounter "instructs" the B cells to acquire antigen cross-presentation abilities, in a process that involves autophagy. Bacteria-instructed B cells, henceforth referred to as BacB cells, rapidly degrade phagocytosed bacteria, process bacterial antigens and cross-prime naïve CD8+ T cells which differentiate into specific cytotoxic cells that efficiently control bacterial infections. Moreover, a proof-of-concept experiment shows that BacB cells that have captured bacteria expressing tumor antigens could be useful as novel cellular immunotherapies against cancer.

Oncogenic Rag GTPase signaling enhances B cell activation and drives follicular lymphoma sensitive to pharmacological inhibition of mTOR.

The humoral immune response demands that B cells undergo a sudden anabolic shift and high cellular nutrient levels which are required to sustain the subsequent proliferative burst. Follicular lymphoma (FL) originates from B cells that have participated in the humoral response, and 15% of FL samples harbor point, activating mutations in RRAGC, an essential activator of mTORC1 downstream of the sensing of cellular nutrients. The impact of recurrent RRAGC mutations in B cell function and lymphoma is unexplored. RRAGC mutations, targeted to the endogenous locus in mice, confer a partial insensitivity to nutrient deprivation, but strongly exacerbate B cell responses and accelerate lymphomagenesis, while creating a selective vulnerability to pharmacological inhibition of mTORC1. This moderate increase in nutrient signaling synergizes with paracrine cues from the supportive T cell microenvironment that activates B cells via the PI3K-Akt-mTORC1 axis. Hence, Rragc mutations sustain induced germinal centers and murine and human FL in the presence of decreased T cell help. Our results support a model in which activating mutations in the nutrient signaling pathway foster lymphomagenesis by corrupting a nutrient-dependent control over paracrine signals from the T cell microenvironment.

Dynamic reorganisation of intermediate filaments coordinates early B-cell activation.

During B-cell activation, the dynamic reorganisation of the cytoskeleton is crucial for multiple cellular responses, such as receptor signalling, cell spreading, antigen internalisation, intracellular trafficking, and antigen presentation. However, the role of intermediate filaments (IFs), which represent a major component of the mammalian cytoskeleton, is not well defined. Here, by using multiple super-resolution microscopy techniques, including direct stochastic optical reconstruction microscopy, we show that IFs in B cells undergo drastic reorganisation immediately upon antigen stimulation and that this reorganisation requires actin and microtubules. Although the loss of vimentin in B cells did not impair B-cell development, receptor signalling, and differentiation, vimentin-deficient B cells exhibit altered positioning of antigen-containing and lysosomal associated membrane protein 1 (LAMP1+) compartments, implying that vimentin may play a role in the fine-tuning of intracellular trafficking. Indeed, vimentin-deficient B cells exhibit impaired antigen presentation and delayed antibody responses in vivo. Thus, our study presents a new perspective on the role of IFs in B-cell activation.

Antigen phagocytosis by B cells is required for a potent humoral response.

Successful vaccines rely on activating a functional humoral response that results from promoting a proper germinal center (GC) reaction. Key in this process is the activation of follicular B cells that need to acquire antigens and to present them to cognate CD4 T cells. Here, we report that follicular B cells can phagocytose large antigen-coated particles, a process thought to be exclusive of specialized antigen-presenting cells such as macrophages and dendritic cells. We show that antigen phagocytosis by B cells is BCR-driven and mechanistically dependent on the GTPase RhoG. Using Rhog-/- mice, we show that phagocytosis of antigen by B cells is important for the development of a strong GC response and the generation of high-affinity class-switched antibodies. Importantly, we show that the potentiation effect of alum, a common vaccine adjuvant, requires direct phagocytosis of alum-antigen complexes by B cells. These data suggest a new avenue for vaccination approaches by aiming to deliver 1-3 µm size antigen particles to follicular B cells.

Protein Kinase C-ß Dictates B Cell Fate by Regulating Mitochondrial Remodeling, Metabolic Reprogramming, and Heme Biosynthesis.

PKCß-null (Prkcb-/-) mice are severely immunodeficient. Here we show that mice whose B cells lack PKCß failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection. At the cellular level, we have shown that Prkcb-/- B cells exhibited defective antigen polarization and mTORC1 signaling. While altered antigen polarization impaired antigen presentation and likely restricted the potential of GC development, defective mTORC1 signaling impaired metabolic reprogramming, mitochondrial remodeling, and heme biosynthesis in these cells, which altogether overwhelmingly opposed plasma cell differentiation. Taken together, our study reveals mechanistic insights into the function of PKCß as a key regulator of B cell polarity and metabolic reprogramming that instructs B cell fate.

R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes.

Upon antigen recognition within peripheral lymphoid organs, B cells interact with T cells and other immune cells to transiently form morphological structures called germinal centers (GCs), which are required for B cell clonal expansion, immunoglobulin class switching, and affinity maturation. This process, known as the GC response, is an energetically demanding process that requires the metabolic reprogramming of B cells. We showed that the Ras-related guanosine triphosphate hydrolase (GTPase) R-Ras2 (also known as TC21) plays an essential, nonredundant, and B cell-intrinsic role in the GC response. Both the conversion of B cells into GC B cells and their expansion were impaired in mice lacking R-Ras2, but not in those lacking a highly related R-Ras subfamily member or both the classic H-Ras and N-Ras GTPases. In the absence of R-Ras2, activated B cells did not exhibit increased oxidative phosphorylation or aerobic glycolysis. We showed that R-Ras2 was an effector of both the B cell receptor (BCR) and CD40 and that, in its absence, B cells exhibited impaired activation of the PI3K-Akt-mTORC1 pathway, reduced mitochondrial DNA replication, and decreased expression of genes involved in glucose metabolism. Because most human B cell lymphomas originate from GC B cells or B cells that have undergone the GC response, our data suggest that R-Ras2 may also regulate metabolism in B cell malignancies.

El tratamiento grupal de adolescentes con trastornos graves de la personalidad: Una aproximación contextual / Group therapy for adolescents with severe personality disorders: a contextual approach

El presente trabajo se centra en los trastornos mentales graves en la adolescencia, y más específicamente en los trastornos de personalidad. La Terapia Dialéctico Conductual (DBT) es uno de los tratamientos con más evidencia para adolescentes con este tipo de trastornos. Entre las terapias contextuales, la DBT se centra en la reducción de los trastornos de conducta graves, haciendo énfasis en el entrenamiento de habilidades. La relación terapéutica es uno de los componentes clave para tratar la patología de personalidad en adolescentes. Asimismo, la combinación con otras terapias contextuales, como la Psicoterapia Analítica Funcional (PAF), permite un mayor efecto de la DBT. El objetivo de este artículo es mostrar cómo la PAF, combinada con estrategias de otras psicoterapias contextuales, supone una mejora clínica en adolescentes con trastornos de personalidad graves. En este artículo se describe la intervención clínica ambulatoria realizada con cuatro adolescentes de entre 13 y 17 años de edad, diagnosticadas de enfermedad mental grave y con criterios de trastornos de personalidad. Los principales síntomas evaluados fueron la impulsividad, los intentos de suicidio, las autolesiones, la auto y heteroagresividad, los síntomas psicóticos y los problemas del yo. El instrumento de medida utilizado fue el Inventario Clínico para Adolescentes de Millon (MACI). Tras una intervención de cuatro meses, se observó un descenso en las escalas clínicas significativas del MACI. También se encontró una mejoría en cognición, tendencias suicidas, regulación emocional y experiencia del yo (AU)

The present work focuses on severe mental disorders in adolescence; and more specifically on personality disorders. Dialectical Behavioral Therapy (DBT) has proven effective in teenagers with these disorders. Among contextual therapies, DBT aims to reduce serious behaviour disorders, emphasizing skills training. The therapeutic relationship is one of the key components of successful treatment of personality pathology in adolescents. DBT has a greater effect combined with other contextual therapies, such as Functional Analytic Psychotherapy (FAP). The purpose of this paper is to show how FAP, in combination with the latest technical advances in other contextual therapies, brings about clinical improvement in adolescents with severe personality disorders. Four teenagers, between 13 and 17 years old, diagnosed with a severe mental disorder and meeting some criteria for personality disorders, received treatment in an outpatient setting. The main symptoms evaluated were: high impulsivity, suicide attempts, self-injury, self and heteroagressivity, psychotic symptoms, and problems of the self. Patients were assessed with the Millon Adolescent Clinical Inventory (MACI). After a four-month intervention, a decrease in the MACI Clinical scales was observed. We also found an improvement in cognition, suicidal tendencies, emotional regulation, and experience of self (AU)

A switch from canonical to noncanonical autophagy shapes B cell responses.

Autophagy is important in a variety of cellular and pathophysiological situations; however, its role in immune responses remains elusive. Here, we show that among B cells, germinal center (GC) cells exhibited the highest rate of autophagy during viral infection. In contrast to mechanistic target of rapamycin complex 1-dependent canonical autophagy, GC B cell autophagy occurred predominantly through a noncanonical pathway. B cell stimulation was sufficient to down-regulate canonical autophagy transiently while triggering noncanonical autophagy. Genetic ablation of WD repeat domain, phosphoinositide-interacting protein 2 in B cells alone enhanced this noncanonical autophagy, resulting in changes of mitochondrial homeostasis and alterations in GC and antibody-secreting cells. Thus, B cell activation prompts a temporal switch from canonical to noncanonical autophagy that is important in controlling B cell differentiation and fate.

The Analysis of Cell Cycle, Proliferation, and Asymmetric Cell Division by Imaging Flow Cytometry.

Measuring cellular DNA content by conventional flow cytometry (CFC) and fluorescent DNA-binding dyes is a highly robust method for analysing cell cycle distributions within heterogeneous populations. However, any conclusions drawn from single-parameter DNA analysis alone can often be confounded by the asynchronous nature of cell proliferation. We have shown that by combining fluorescent DNA stains with proliferation tracking dyes and antigenic staining for mitotic cells one can elucidate the division history and cell cycle position of any cell within an asynchronously dividing population. Furthermore if one applies this panel to an imaging flow cytometry (IFC) system then the spatial information allows resolution of the four main mitotic phases and the ability to study molecular distributions within these populations. We have employed such an approach to study the prevalence of asymmetric cell division (ACD) within activated immune cells by measuring the distribution of key fate determining molecules across the plane of cytokinesis in a high-throughput, objective, and internally controlled manner. Moreover the ability to perform high-resolution, temporal dissection of the cell division process lends itself perfectly to investigating the influence chemotherapeutic agents exert on the proliferative capacity of transformed cell lines. Here we describe the method in detail and its application to both ACD and general cell cycle analysis.

Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity.

The small Rho GTPase Cdc42, known to interact with Wiskott-Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell-intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.

Stimulant and atypical antipsychotic medications for children placed in foster homes.

OBJECTIVES: The purpose of this study is to examine the use of prescribed psychoactive medications in a prospective cohort of children shortly after they entered foster homes; and to identify demographics, maltreatment history, psychiatric diagnoses including ADHD comorbidity, and level of aggression that contribute to prescribed use of stimulant and atypical antipsychotic medication over time. METHODS: The sample included N = 252 children (nested in 95 sibling groups) followed for three years up to 4 yearly waves. RESULTS: Nearly all (89%) met criteria for at least one of eight psychiatric diagnoses and 31% (75/252) used one or more prescribed psychoactive medications. Over half (55%) were diagnosed with Attention Deficit Hyperactivity Disorder (ADHD); of these 38% used stimulants and 36% used atypical antipsychotics. Of the 75 medicated children, 19% received ≥3 different classes of drugs over the course of the study. Stimulants (69%) and atypical antipsychotics (65%) were the most frequently used drugs among medicated children. Adjusted odds ratios (AOR) showed that male gender (AOR = 3.2; 95% CI = 1.5-9.3), African American vs Latino ethnicity (AOR = 5.4; 95% CI = 2.1-14.2), ADHD regardless of Oppositional Defiant (ODD) or Conduct (CD) comorbidity (AOR = 6.0, 95% CI = 1.3-27.5), ODD or CD (AOR = 11.1, 95% CI = 2.1-58.6), and Separation Anxiety (AOR = 2.0, 95% CI = 1.0-4.0) psychiatric disorders were associated with the use of prescribed stimulants; while male gender (AOR = 3.8, 95% CI = 1.5-9.3), African American vs Latino (AOR = 5.1, 95% CI = 1.2-9.2) or Mixed/Other ethnicity (AOR = 3.3, 95% CI = 1.9-13.7), ADHD regardless of ODD or CD comorbidity (AOR = 5.8, 95% CI = 1.2-28.7), ODD or CD (AOR = 13.9, 95% CI = 3.3-58.5), Major Depression/Dysthymia (AOR = 2.8, 95% CI = 1.1-6.7) psychiatric disorders, and history of sexual abuse (AOR = 4.6, 95% CI = 1.3-18.4) were associated with the use of prescribed atypical antipsychotics. CONCLUSION: The aggressive use of atypical antipsychotics, which has unknown metabolic risks, suggests that the efficacy and safety of such treatment strategies for psychiatrically ill children in foster care should be monitored.

RRas2, RhoG and T-cell phagocytosis.

Activating mutations and overexpression of classical Ras subfamily members (K-Ras, N-Ras and H-Ras) have been widely investigated as key events in the development of human cancers. The role in cancer of its closest relatives, the Ras-related (RRas) subfamily members, has been less studied despite the fact that one of its members (TC21 or RRas2) is strongly transforming in vitro. Nevertheless, and in spite the paucity of publications, several studies have shown that wild type TC21 is overexpressed in different types of carcinomas and lymphomas. If the study of RRas members in cancer is still in its infancy, their role in physiological functions is even behind. For instance, T and B cell immunologists still use the vague term "Ras activation" without indication of what Ras family molecule is indeed intervening. In this view, we discuss the participation of TC21 in the specific process of T cell antigen receptor internalization from the immunological synapse and acquisition of membrane fragments from the antigen presenting cells by phagocytosis.

Asymmetric segregation of polarized antigen on B cell division shapes presentation capacity.

During the activation of humoral immune responses, B cells acquire antigen for subsequent presentation to cognate T cells. Here we show that after mouse B cells accumulate antigen, it is maintained in a polarized distribution for extended periods in vivo. Using high-throughput imaging flow cytometry, we observed that this polarization is preserved during B cell division, promoting asymmetric antigen segregation among progeny. Antigen inheritance correlates with the ability of progeny to activate T cells: Daughter cells receiving larger antigen stores exhibit a prolonged capacity to present antigen, which renders them more effective in competing for T cell help. The generation of progeny with differential capacities for antigen presentation may have implications for somatic hypermutation and class switching during affinity maturation and as B cells commit to effector cell fates.

Metabolomics of the effect of AMPK activation by AICAR on human umbilical vein endothelial cells.

AMP-activated protein kinase (AMPK) is a metabolic master switch expressed in a great number of cells and tissues. AMPK is thought to modulate the cellular response to different stresses that increase cellular AMP concentration. The adenosine analog, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) is an AMPK activator used in many studies to assess the effects of AMPK activation on cellular metabolism and function. However, the effect of AICAR on cell metabolism reaches many different pathways and metabolites, some of which do not seem to be fully related to AMPK activation. We have now for the first time used NMR metabolomics on human umbilical vein endothelial cells (HUVEC) for the study of the global metabolic impact of AMPK activation by AICAR. In our study, incubation with AICAR activates AMPK and is associated with, among others, broad metabolic alterations in energy metabolism and phospholipid biosynthesis. Using NMR spectroscopy and metabolic network tools, we analyzed the connections between the different metabolic switches activated by AICAR. Our approach reveals a strong interconnection between different phospholipid precursors and oxidation by-products. Metabolomics profiling is a useful tool for detecting major metabolic alterations, generating new hypotheses and provides some insight about the different molecular correlations in a complex system. The present study shows that AICAR induces metabolic effects in cell metabolism well beyond energy production pathways.

Self-perceived needs are related to violent behavior among schizophrenia outpatients.

This study assessed the relationship between self-perceived clinical and social needs and aggressive behavior in outpatients with schizophrenia. A total of 895 outpatients with schizophrenia were enrolled. The presence of aggressive episodes was assessed using the Modified Overt Aggression Scale. Self-perceived needs were assessed using the Camberwell Assessment of Need in six areas of needs (food, household skills, self-care, daytime activities, psychotic symptoms, satisfaction with treatment, and company). The most common areas of needs were "psychotic symptoms" (81.6%), "daytime activities" (60.6%), and "household skills" (57.5%). More needs were expressed by patients who had more severe illnesses (p < 0.001) and more aggressive behavior (p < 0.001). Multivariate analysis showed that, in schizophrenia outpatients, self-perceived needs were associated with aggressive behavior (adjusted odds ratio, 11.43; 95% confidence interval, 5.11 to 25.56). Appropriate compliance with antipsychotic treatment was related with lower aggressive behavior (p < 0.001).