RESUMO
B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunidade Inata , Influenza Humana/imunologia , Interleucina-4/genética , Células Matadoras Naturais/imunologia , Infecção por Zika virus/imunologia , Animais , Galinhas , Cães , Centro Germinativo/citologia , Humanos , Interleucina-4/metabolismo , Macaca , Macrófagos/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Lung-resident memory B cells (MBCs) provide localized protection against reinfection in respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. We found that two main transcriptionally distinct subsets of MBCs colonized the lung peribronchial niche after infection. These subsets arose from different progenitors and were both class switched, somatically mutated, and intrinsically biased in their differentiation fate toward plasma cells. Combined analysis of antigen specificity and B cell receptor repertoire segregated these subsets into "bona fide" virus-specific MBCs and "bystander" MBCs with no apparent specificity for eliciting viruses generated through an alternative permissive process. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.
Assuntos
COVID-19 , Memória Imunológica , Animais , Linfócitos B , Pulmão , Células B de Memória , Camundongos , Reinfecção , SARS-CoV-2RESUMO
Lymphatic transport of molecules and migration of myeloid cells to lymph nodes (LNs) continuously inform lymphocytes on changes in drained tissues. Here, using LN transplantation, single-cell RNA-seq, spectral flow cytometry, and a transgenic mouse model for photolabeling, we showed that tissue-derived unconventional T cells (UTCs) migrate via the lymphatic route to locally draining LNs. As each tissue harbored a distinct spectrum of UTCs with locally adapted differentiation states and distinct T cell receptor repertoires, every draining LN was thus populated by a distinctive tissue-determined mix of these lymphocytes. By making use of single UTC lineage-deficient mouse models, we found that UTCs functionally cooperated in interconnected units and generated and shaped characteristic innate and adaptive immune responses that differed between LNs that drained distinct tissues. Lymphatic migration of UTCs is, therefore, a key determinant of site-specific immunity initiated in distinct LNs with potential implications for vaccination strategies and immunotherapeutic approaches.
Assuntos
Linfonodos , Linfócitos T , Animais , Modelos Animais de Doenças , Imunidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos TRESUMO
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.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Ativação Linfocitária/imunologia , Plasmócitos/imunologia , Proteína Quinase C beta/imunologia , Animais , Heme/biossíntese , Camundongos , Camundongos Knockout , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Plasmócitos/citologiaRESUMO
The adaptor Nck links receptor signaling to cytoskeleton regulation. Here we found that Nck also controlled the phosphatidylinositol-3-OH kinase (PI(3)K)-kinase Akt pathway by recruiting the adaptor BCAP after activation of B cells. Nck bound directly to the B cell antigen receptor (BCR) via the non-immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated tyrosine residue at position 204 in the tail of the immunoglobulin-α component. Genetic ablation of Nck resulted in defective BCR signaling, which led to hampered survival and proliferation of B cells in vivo. Indeed, antibody responses in Nck-deficient mice were also considerably impaired. Thus, we demonstrate a previously unknown adaptor function for Nck in recruiting BCAP to sites of BCR signaling and thereby modulating the PI(3)K-Akt pathway in B cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/metabolismo , Proteínas Oncogênicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/imunologia , Feminino , Cadeias alfa de Imunoglobulina/química , Cadeias alfa de Imunoglobulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Fosforilação , Ligação Proteica , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tirosina/metabolismoRESUMO
Intestinal homeostasis relies on a continuous dialogue between the commensal bacteria and the immune system. Natural killer T (NKT) cells, which recognize CD1d-restricted microbial lipids and self-lipids, contribute to the regulation of mucosal immunity, yet the mechanisms underlying their functions remain poorly understood. Here, we demonstrate that NKT cells respond to intestinal lipids and CD11c+ cells (including dendritic cells (DCs) and macrophages) are essential to mediate lipid presentation within the gut ultimately controlling intestinal NKT cell homeostasis and activation. Conversely, CD1d and NKT cells participate in the control of the intestinal bacteria composition and compartmentalization, in the regulation of the IgA repertoire and in the induction of regulatory T cells within the gut. These changes in intestinal homeostasis require CD1d expression on DC/macrophage populations as mice with conditional deletion of CD1d on CD11c+ cells exhibit dysbiosis and altered immune homeostasis. These results unveil the importance of CD11c+ cells in controlling lipid-dependent immunity in the intestinal compartment and reveal an NKT cell-DC crosstalk as a key mechanism for the regulation of gut homeostasis.
Assuntos
Mucosa Intestinal/imunologia , Lipídeos de Membrana/imunologia , Células T Matadoras Naturais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD1d/biossíntese , Antígenos CD1d/genética , Antígenos CD1d/imunologia , Antígeno CD11c/metabolismo , Células Dendríticas/imunologia , Disbiose/genética , Microbioma Gastrointestinal/imunologia , Imunoglobulina A/imunologia , Interleucina-4/imunologia , Mucosa Intestinal/microbiologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: Obesity is a serious health problem all over the world, and inhibition of adipogenesis constitutes one of the therapeutic strategies for its treatment. Carnosic acid (CA), the main bioactive compound of Rosmarinus officinalis extract, inhibits 3T3-L1 preadipocytes differentiation. However, very little is known about the molecular mechanism responsible for its antiadipogenic effect. METHODS: We evaluated the effect of CA on the differentiation of 3T3-L1 preadipocytes analyzing the process of mitotic clonal expansion, the level of adipogenic markers, and the subcellular distribution of C/EBPß. RESULTS: CA treatment only during the first day of 3T3-L1 differentiation process was enough to inhibit adipogenesis. This inhibition was accompanied by a blockade of mitotic clonal expansion. CA did not interfere with C/EBPß and C/EBPδ mRNA levels but blocked PPARγ, and FABP4 expression. C/EBPß has different forms known as LIP and LAP. CA induced an increase in the level of LIP within 24h of differentiation, leading to an increment in LIP/LAP ratio. Importantly, overexpression of LAP restored the capacity of 3T3-L1 preadipocytes to differentiate in the presence of CA. Finally, CA promoted subnuclear de-localization of C/EBPß. CONCLUSIONS: CA exerts its anti-adipogenic effect in a multifactorial manner by interfering mitotic clonal expansion, altering the ratio of the different C/EBPß forms, inducing the loss of C/EBPß proper subnuclear distribution, and blocking the expression of C/EBPα and PPARγ. GENERAL SIGNIFICANCE: Understanding the molecular mechanism by which CA blocks adipogenesis is relevant because CA could be new a food additive beneficial for the prevention and/or treatment of obesity.
Assuntos
Abietanos/farmacologia , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Antioxidantes/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , PPAR gama/metabolismo , Extratos Vegetais/farmacologia , Rosmarinus/química , Transdução de Sinais/efeitos dos fármacos , Células 3T3-L1 , Abietanos/química , Adipócitos/citologia , Animais , Antioxidantes/química , Diferenciação Celular/fisiologia , Proteínas de Ligação a Ácido Graxo/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Camundongos , PPAR gama/genética , Extratos Vegetais/química , Transdução de Sinais/fisiologiaRESUMO
Mice with a loss-of-function mutation in the LAT adaptor (LatY136F) develop an autoimmune and type 2 inflammatory disorder called defective LAT signalosome pathology (DLSP). We analyzed via single-cell omics the trajectory leading to LatY136F DLSP and the underlying CD4+ T cell diversification. T follicular helper cells, CD4+ cytotoxic T cells, activated B cells, and plasma cells were found in LatY136F spleen and lung. Such cell constellation entailed all the cell types causative of human IgG4-related disease (IgG4-RD), an autoimmune and inflammatory condition with LatY136F DLSP-like histopathological manifestations. Most previously described T cell-mediated autoimmune manifestations require persistent TCR input. In contrast, following their first engagement by self-antigens, the autoreactive TCR expressed by LatY136F CD4+ T cells hand over their central role in T cell activation to CD28 costimulatory molecules. As a result, all subsequent LatY136F DLSP manifestations, including the production of autoantibodies, solely rely on CD28 engagement. Our findings elucidate the etiology of the LatY136F DLSP and qualify it as a model of IgG4-RD.
Assuntos
Doença Relacionada a Imunoglobulina G4 , Humanos , Animais , Camundongos , Antígenos CD28 , Autoanticorpos , Autoantígenos , Receptores de Antígenos de Linfócitos TRESUMO
Nonclassical T cells are a heterogeneous group of T lymphocytes that are activated during the early stages of infection and act as a bridge between the innate and adaptive immune system. Among them, Natural Killer T (NKT) cells have been extensively studied in the last two decades due to their unique ability to recognize foreign/self-lipid antigens in the context of CD1d, a nonclassical major histocompatibility complex molecule. In this chapter, we describe our protocols to track murine NKT cells in lymph nodes by flow cytometry and confocal microscopy.
Assuntos
Células T Matadoras Naturais , Animais , Antígenos CD1d , Citometria de Fluxo , Contagem de Linfócitos , CamundongosRESUMO
Wiskott-Aldrich syndrome (WAS) is an immune pathology associated with mutations in WAS protein (WASp) or in WASp interacting protein (WIP). Together with the small GTPase Cdc42 and other effectors, these proteins participate in the remodelling of the actin network downstream of BCR engagement. Here we show that mice lacking the adaptor protein ITSN2, a G-nucleotide exchange factor (GEF) for Cdc42 that also interacts with WASp and WIP, exhibited increased mortality during primary infection, incomplete protection after Flu vaccination, reduced germinal centre formation and impaired antibody responses to vaccination. These defects were found, at least in part, to be intrinsic to the B cell compartment. In vivo, ITSN2 deficient B cells show a reduction in the expression of SLAM, CD84 or ICOSL that correlates with a diminished ability to form long term conjugates with T cells, to proliferate in vivo, and to differentiate into germinal centre cells. In conclusion, our study not only revealed a key role for ITSN2 as an important regulator of adaptive immune-response during vaccination and viral infection but it is also likely to contribute to a better understanding of human immune pathologies.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Linfócitos B/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/patologia , Orthomyxoviridae/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Adesão Celular , Proliferação de Células , Vacinas contra Influenza/administração & dosagem , Camundongos , Análise de SobrevidaRESUMO
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.
Assuntos
Autofagia/imunologia , Linfócitos B/imunologia , Linfócitos B/virologia , Viroses/imunologia , Animais , Regulação para Baixo , Centro Germinativo/imunologia , Centro Germinativo/virologia , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Repetições WD40/genéticaRESUMO
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.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Imunidade Humoral/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteína cdc42 de Ligação ao GTP/imunologia , Animais , Formação de Anticorpos/imunologia , Linfócitos B/metabolismo , Linfócitos B/ultraestrutura , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Citometria de Fluxo , Expressão Gênica/imunologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Imunidade Humoral/genética , Vírus da Influenza A/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
The layer of macrophages at the subcapsular sinus (SCS) captures pathogens entering the lymph node, preventing their global dissemination and triggering an immune response. However, how infection affects SCS macrophages remains largely unexplored. Here we show that infection and inflammation disrupt the organization of SCS macrophages in a manner that involves the migration of mature dendritic cells to the lymph node. This disrupted organization reduces the capacity of SCS macrophages to retain and present antigen in a subsequent secondary infection, resulting in diminished B cell responses. Thus, the SCS macrophage layer may act as a sensor or valve during infection to temporarily shut down the lymph node to further antigenic challenge. This shutdown may increase an organism's susceptibility to secondary infections.