A molecular signature of human regulatory B cells in health and disease

Les lymphocytes B régulateurs (LBreg) chez l’Homme correspondent à des sous-populations de LB avec une grande variété de phénotypes (Mauri et al., 2015) et de mécanismes (Floudas et al., 2016). Cette diversité rend leur caractérisation et leur suivi difficile. L’objectif de ce travail était donc d’identifier et d’étudier une signature moléculaire des LB humains ayant une fonction régulatrice. Nous avons développé un modèle in vitro de polarisation des LB en LBreg et en LB avec une fonction inflammatoire (LBinf). Par la suite, un RNA-seq de ces 2 types de LB a été effectué et les gènes différentiellement exprimés (DEG) entre les LBreg et LBinf ont été identifiés. Enfin, une méta-analyse de cette signature a été faite pour définir les potentiels éléments régulateurs de cette fonction. Puis, des études in vitro ont été faites pour valider ces observations. À partir de l’analyse RNA-seq, 225 DEG ont été identifiés entre les LBreg et les LBinf. Parmi eux, le facteur de transcription (FT) c-MAF est le FT le plus augmenté dans les LBreg (FC = 16,2). De plus, des comparaisons avec des CHIP-seq publiques de c-MAF, confirment un enrichissement significatif des cibles de c-MAF dans la signature des LBreg. Parmi eux, le gène IL10 est présent. Par la suite, les analyses in vitro ont permis de montrer que l’expression d’IL10 est restreinte aux LB c-MAFhi. Ceux-ci ont également une forte expression de CD27, CD38 et BLIMP1, suggérant ainsi un stade de différenciation de type plasmablaste (PB) / plasmocyte (PC). Les analyses phénotypiques et la comparaison de la signature des LBreg avec une signature des PB humains permettent également d’associer les LBreg à des PB/PC. Enfin, un siRNA MAF montre une perturbation de la balance BLIMP1/T-BET, avec une diminution de BLIMP1 et une augmentation de T-BET. Ces résultats suggèrent que le FT c-MAF pourrait être essentiel à la régulation issue des LB, comme observé dans d’autres types de cellules immunitaires. En effet, c- MAF est un FT important pour la régulation via les LTreg (Xu et al., 2018) et des macrophages M2 (Kang et al., 2017). De plus, le lien entre c-MAF et BLIMP1, ainsi que les résultats du siRNA MAF, suggèrent un potentiel rôle de c-MAF dans la différenciation des LB. En effet, BLIMP1 et T-BET sont respectivement associés aux PB/PC et aux LB mémoires (Shaffer et al., 2002 ; Tellier et al., 2016; Cancro et al., 2017; Kenderes et al., 2018).Regulatory B cells (Breg) in human are included in a large group of B-cell subsets encompassing a high heterogeneity of phenotypes (Mauri et al., 2015) and suppressor mechanisms (Floudas et al., 2016). This variability leads to a high difficulty to characterize and monitor human Breg. One aim of our work was to establish and study a molecular signature of B cells with regulatory properties. We developed an in vitro model to polarize peripheral B cells in Breg and inflammatory B cell (Binf). Then, we performed RNA-sequencing on these two functional subsets. A meta-analysis on differentially expressed genes (DEG) has led to the definition of critical factors involved in regulatory function. In vitro studies were used for data validation. From the RNA-seq analysis, we obtained 225 DEG between Breg and Binf. Among them, the c-MAF transcription factor (TF) was the most upregulated TF (FC = 16.2). Also, comparisons with public c-MAF CHIP-seq data confirmed a significant enrichment of c-MAF target-genes in Breg signature. We thus established that c-MAF could be induced in human blood B cells after TLR and BCR stimulation. Besides, we observed that IL-10 production was restricted to c-MAFhi expressing B cells and is associated with the expression of CD27, CD38, and BLIMP1, suggesting a differentiation state close to plasmablast (PB) / plasma cells (PC). Phenotype analysis and comparison of homemade human PB signature with Breg signature also linked Breg with PB state. Moreover, siRNA MAF impairs BLIMP1/T-BET balance in B cells with a decrease and increase of BLIMP1 and T-BET respectively. These results suggest that the c-MAF TF could be an essential factor in the regulatory function of B cells, as observed in other immune cells. Indeed, c-MAF is a significant TF involved in several regulatory immune cells, such as regulatory T cells (Xu et al., 2018) or M2 macrophages (Kang et al., 2017). Moreover, the link between c-MAF and BLIMP1, as well as siRNA MAF results, suggest a potential role of c-MAF in B cell fate. Indeed, BLIMP1 and T-BET are associated with PB/PC and memory B cells respectively (Shaffer et al., 2002 ; Tellier et al., 2016; Cancro 2017; Kenderes et al., 2018)

Signature moléculaire des lymphocytes B régulateurs en physiologie et en pathologie

Regulatory B cells (Breg) in human are included in a large group of B-cell subsets encompassing a high heterogeneity of phenotypes (Mauri et al., 2015) and suppressor mechanisms (Floudas et al., 2016). This variability leads to a high difficulty to characterize and monitor human Breg. One aim of our work was to establish and study a molecular signature of B cells with regulatory properties. We developed an in vitro model to polarize peripheral B cells in Breg and inflammatory B cell (Binf). Then, we performed RNA-sequencing on these two functional subsets. A meta-analysis on differentially expressed genes (DEG) has led to the definition of critical factors involved in regulatory function. In vitro studies were used for data validation. From the RNA-seq analysis, we obtained 225 DEG between Breg and Binf. Among them, the c-MAF transcription factor (TF) was the most upregulated TF (FC = 16.2). Also, comparisons with public c-MAF CHIP-seq data confirmed a significant enrichment of c-MAF target-genes in Breg signature. We thus established that c-MAF could be induced in human blood B cells after TLR and BCR stimulation. Besides, we observed that IL-10 production was restricted to c-MAFhi expressing B cells and is associated with the expression of CD27, CD38, and BLIMP1, suggesting a differentiation state close to plasmablast (PB) / plasma cells (PC). Phenotype analysis and comparison of homemade human PB signature with Breg signature also linked Breg with PB state. Moreover, siRNA MAF impairs BLIMP1/T-BET balance in B cells with a decrease and increase of BLIMP1 and T-BET respectively. These results suggest that the c-MAF TF could be an essential factor in the regulatory function of B cells, as observed in other immune cells. Indeed, c-MAF is a significant TF involved in several regulatory immune cells, such as regulatory T cells (Xu et al., 2018) or M2 macrophages (Kang et al., 2017). Moreover, the link between c-MAF and BLIMP1, as well as siRNA MAF results, suggest a potential role of c-MAF in B cell fate. Indeed, BLIMP1 and T-BET are associated with PB/PC and memory B cells respectively (Shaffer et al., 2002 ; Tellier et al., 2016; Cancro 2017; Kenderes et al., 2018).Les lymphocytes B régulateurs (LBreg) chez l’Homme correspondent à des sous-populations de LB avec une grande variété de phénotypes (Mauri et al., 2015) et de mécanismes (Floudas et al., 2016). Cette diversité rend leur caractérisation et leur suivi difficile. L’objectif de ce travail était donc d’identifier et d’étudier une signature moléculaire des LB humains ayant une fonction régulatrice. Nous avons développé un modèle in vitro de polarisation des LB en LBreg et en LB avec une fonction inflammatoire (LBinf). Par la suite, un RNA-seq de ces 2 types de LB a été effectué et les gènes différentiellement exprimés (DEG) entre les LBreg et LBinf ont été identifiés. Enfin, une méta-analyse de cette signature a été faite pour définir les potentiels éléments régulateurs de cette fonction. Puis, des études in vitro ont été faites pour valider ces observations. À partir de l’analyse RNA-seq, 225 DEG ont été identifiés entre les LBreg et les LBinf. Parmi eux, le facteur de transcription (FT) c-MAF est le FT le plus augmenté dans les LBreg (FC = 16,2). De plus, des comparaisons avec des CHIP-seq publiques de c-MAF, confirment un enrichissement significatif des cibles de c-MAF dans la signature des LBreg. Parmi eux, le gène IL10 est présent. Par la suite, les analyses in vitro ont permis de montrer que l’expression d’IL10 est restreinte aux LB c-MAFhi. Ceux-ci ont également une forte expression de CD27, CD38 et BLIMP1, suggérant ainsi un stade de différenciation de type plasmablaste (PB) / plasmocyte (PC). Les analyses phénotypiques et la comparaison de la signature des LBreg avec une signature des PB humains permettent également d’associer les LBreg à des PB/PC. Enfin, un siRNA MAF montre une perturbation de la balance BLIMP1/T-BET, avec une diminution de BLIMP1 et une augmentation de T-BET. Ces résultats suggèrent que le FT c-MAF pourrait être essentiel à la régulation issue des LB, comme observé dans d’autres types de cellules immunitaires. En effet, c- MAF est un FT important pour la régulation via les LTreg (Xu et al., 2018) et des macrophages M2 (Kang et al., 2017). De plus, le lien entre c-MAF et BLIMP1, ainsi que les résultats du siRNA MAF, suggèrent un potentiel rôle de c-MAF dans la différenciation des LB. En effet, BLIMP1 et T-BET sont respectivement associés aux PB/PC et aux LB mémoires (Shaffer et al., 2002 ; Tellier et al., 2016; Cancro et al., 2017; Kenderes et al., 2018)

A molecular signature of human regulatory B cells

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B cell receptor activation: molecular mechanisms and therapeutic targets

National audienceThe B cell receptor (BCR) is a membrane receptor specific to B cells and involved in the selection, activation, maturation, survival and proliferation of these cells. Its activation involves a large number of proteins, triggering a series of signalling pathways which, if they become constitutive, particularly via mutations, may be at the origin of and/or be key in many B cancers. The BCR associates with various co-receptors such as CD19, CD20 or CD22, thus positively or negatively regulating the signalling cascades induced by its activation. These include mitogen-activated protein kinases (MAPK), nuclear factor-.B (NF-.B), nuclear factor of activated T-cells (NF-AT) and protein kinase B (AKT); their activation is particularly dependent on BCR-related proteins such as Bruton's agammaglobulinemia tyrosine kinase (BTK) or phospho-inositide 3-kinase (PI3K). Targeting these proteins with inhibitors such as ibrutinib or idelasib has revolutionised the treatment of certain B cancers for which constitutive BCR hyperactivation could not, until now, be effectively attenuated. A great deal of research, both basic and clinical, is underway to identify other therapeutics that can specifically target (i) the different signalling pathways mentioned above, (ii) proteins directly linked to the BCR, or (iii) co-receptors associated with it. This review presents all existing therapies to date, innovative treatments currently in clinical trials and innovative treatments proposed by fundamental research. These include therapies using the most innovative technologies, such as conjugated monoclonal antibodies, chimeric antigen receptor T cells (CAR-T cells) and inhibitors capable of reversible covalent binding. In addition, this review accurately describes the therapeutic effects of alternative targets in the context of resistance to prolonged treatments. Such resistance, particularly to ibrutinib, represents a major public health issue and needs to be studied regularly in order to keep pace with the development of cancers in society

Study of functional orientation of B cells in physiology and autoimmunity

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Assessing the robustness of clinical trials by estimating Jadad’s score using artificial intelligence approaches

International audienceBACKGROUND: Clinical trials are essential in medical science and are currently the most robust strategy for evaluating the effectiveness of a treatment. However, some of these studies are less reliable than others due to flaws in their design. Assessing the robustness of a clinical trial can be a very complex and time-consuming task, with factors such as randomization, masking and the description of withdrawals needing to be considered. METHOD: We built a program based on artificial intelligence (AI) approaches, designed to assess the robustness of a clinical trial by estimating its Jadad’s score. The program is composed of five Recursive Neural Networks (RNN), each of them trained to spot one specific item constituting the Jadad’s scale. After training, the algorithm was tested on two different validation sets (one from the original database: 35% of this database was used for validation and 65% for training; one composed of 10 articles, out of the original database, for which the Jadad’s score has been computed by each contributor of this study). RESULT: After training, the algorithm achieved a mean accuracy of 96,2% (ranging from 93% to 98%) and a mean area under the curve (AUC) of 96% (ranging from 95% to 97%) on the first validation dataset. These results indicate good feature detection capacity for each of the five RNN. On the second validation dataset the algorithm extracted 100% of the item to retrieve for 70% of the articles and between 66% and 75% for 30% of the articles. Overall 85% of the items present in the second validation dataset were correctly extracted. None of the extracted items was misclassified. CONCLUSION: We developed a program that can automatically estimate the Jadad’s score of a clinical trial with a good accuracy. Automating the assessment of this metric could be very useful in a systematic review of the literature and will probably save clinicians time

Study of functional orientation of B cells in physiology and autoimmunity

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Study of functional orientation of B cells in physiology and Sjögren’s syndrome.

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Study of functional orientation of B cells in physiology and Sjögren’s syndrome.

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Innate B Cells: the Archetype of Protective Immune Cells

International audienceThe innate B cell (IBC) population is heterogeneous and involved in the primary immune response. IBC functions include a high ability to produce natural antibodies with IgM isotype, the elimination of apoptotic cells, and a capacity to be cognate help to T cells. Among IBC subsets, B-1 cells and marginal zone B cells are the main producers of IgM, act as rapid immune responders that may relocate to follicular lymphoid and differentiate to cytokine and antibody-secreting cells shortly after infection. IBCs functions are highly dependent on their localization site and the nature of their B cell receptor repertoire, suggesting a high plasticity range of different immune responses. In this review, we will describe the nature and functions of the different innate-like B cell subsets, first in mice and then in humans. Besides this, we will emphasize the strong ability of these cells to undertake different protective functions from the first line of defense against pathogens to the regulatory role of the broader immune response