RESUMEN
Melioidosis is a potentially fatal bacterial disease caused by Burkholderia pseudomallei and is estimated to cause 89,000 deaths per year in endemic areas of Southeast Asia and Northern Australia. People with diabetes mellitus are most at risk of melioidosis, with a 12-fold increased susceptibility for severe disease. Interferon gamma (IFN-γ) responses from CD4 and CD8 T cells, but also from natural killer (NK) and natural killer T (NKT) cells, are necessary to eliminate the pathogen. We previously reported that immunization with B. pseudomallei OmpW (BpOmpW antigen) protected mice from lethal B. pseudomallei challenge for up to 81 days. Elucidating the immune correlates of protection of the protective BpOmpW vaccine is an essential step prior to clinical trials. Thus, we immunized either non-insulin-resistant C57BL/6J mice or an insulin-resistant C57BL/6J mouse model of type 2 diabetes (T2D) with a single dose of BpOmpW. BpOmpW induced strong antibody responses, stimulated effector CD4+ and CD8+ T cells and CD4+ CD25+ Foxp3+ regulatory T cells, and produced higher IFN-γ responses in CD4+, CD8+, NK, and NKT cells in non-insulin-resistant mice. The T-cell responses of insulin-resistant mice to BpOmpW were comparable to those of non-insulin-resistant mice. In addition, as a precursor to its evaluation in human studies, humanized HLA-DR and HLA-DQ (human leukocyte antigen DR and DQ isotypes, respectively) transgenic mice elicited IFN-γ recall responses in an enzyme-linked immune absorbent spot (ELISpot)-based study. Moreover, human donor peripheral blood mononuclear cells (PBMCs) exposed to BpOmpW for 7 days showed T-cell proliferation. Finally, plasma from melioidosis survivors with diabetes recognized our BpOmpW vaccine antigen. Overall, the range of approaches used strongly indicated that BpOmpW elicits the necessary immune responses to combat melioidosis and bring this vaccine closer to clinical trials.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Burkholderia pseudomallei/inmunología , Melioidosis/inmunología , Linfocitos T/inmunología , Animales , Vacunas Bacterianas/administración & dosificación , Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/fisiología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/microbiología , Células Cultivadas , Diabetes Mellitus Tipo 2/inmunología , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/microbiología , Masculino , Melioidosis/microbiología , Melioidosis/prevención & control , Ratones Endogámicos C57BL , Ratones Transgénicos , Linfocitos T/metabolismo , Linfocitos T/microbiología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/microbiologíaRESUMEN
Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Epítopos/inmunología , Vacunas contra la Malaria/inmunología , Malaria/inmunología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/inmunología , Vacunas de ADN/inmunología , Traslado Adoptivo , Animales , Anticuerpos Antiprotozoarios/metabolismo , Modelos Animales de Enfermedad , Epítopos/genética , Ingeniería Genética , Humanos , Evasión Inmune , Inmunogenicidad Vacunal , Ratones , Ratones SCID , Proteínas Protozoarias/genética , Relación Estructura-Actividad , VacunaciónRESUMEN
Transitional B cells must actively undergo selection for self-tolerance before maturing into their resting follicular B cell successors. We found that metabolic quiescence was acquired at the follicular B cell stage in both humans and mice. In follicular B cells, the expression of genes involved in ribosome biogenesis, aerobic respiration, and mammalian target of rapamycin complex 1 (mTORC1) signaling was reduced when compared to that in transitional B cells. Functional metabolism studies, profiling of whole-cell metabolites, and analysis of cell surface proteins in human B cells suggested that this transition was also associated with increased extracellular adenosine salvage. Follicular B cells increased the abundance of the cell surface ectonucleotidase CD73, which coincided with adenosine 5'-monophosphate-activated protein kinase (AMPK) activation. Differentiation to the follicular B cell stage in vitro correlated with surface acquisition of CD73 on human transitional B cells and was augmented with the AMPK agonist, AICAR. Last, individuals with gain-of-function PIK3CD (PI3Kδ) mutations and increased pS6 activation exhibited a near absence of circulating follicular B cells. Together, our data suggest that mTORC1 attenuation may be necessary for human follicular B cell development. These data identify a distinct metabolic switch during human B cell development at the transitional to follicular stages, which is characterized by an induction of extracellular adenosine salvage, AMPK activation, and the acquisition of metabolic quiescence.
Asunto(s)
Linfocitos B/metabolismo , 5'-Nucleotidasa/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Linfocitos B/citología , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Proteínas Ligadas a GPI/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ribonucleótidos/farmacologíaRESUMEN
The involvement of macroautophagy/autophagy proteins in B-cell receptor (BCR) trafficking, although suspected, is not well understood. We show that ATG5 (autophagy related 5) contributes to BCR polarization after stimulation and internalization into LAMP1 (lysosomal-associated membrane protein 1)+ and major histocompatibility complex class II (MHC-II)+ compartments. BCR polarization is crucial in the context of immobilized antigen processing. Moreover, antigen presentation to cognate T cells is decreased in the absence of ATG5 when the model antigen OVAL/ovalbumin is provided in an immobilized form in contrast to the normal presentation of soluble OVAL. We further show that ATG5 is required for centrosome polarization and actin nucleation in the immune synapse area. This event is accompanied by an increased interaction between ATG16L1 (autophagy related 16-like 1 [S. cerevisiae]) and the microtubule-organizing center-associated protein PCM1 (pericentriolar material 1). In the human B cell line BJAB, PCM1 is required for BCR polarization after stimulation. We thus propose that the ATG12 (autophagy related 12)-ATG5-ATG16L1 complex under BCR stimulation allows its interaction with PCM1 and consequently facilitates centrosome relocalization to the immune synapse, optimizing the presentation of particulate antigens. Abbreviations: ACTB: actin beta; ACTR2/3: ARP2/3 actin-related protein 2/3; APC: antigen-presenting cells; ATG: autophagy-related; BCR: B cell receptor; BECN1/Beclin 1: beclin 1, autophagy related; CDC42: cell division cycle 42; Cr2: complement receptor 2; CSFE: carboxyfluorescein succinimidyl ester; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; EEA1: early endosome antigen 1; ELISA: enzyme-linked immunosorbent assay; FITC: fluorescein isothyocyanate; GC: germinal center; GJA1/CX3: gap junction protein, alpha 1; Ig: immunoglobulin; LAMP1: lysosomal-associated membrane protein 1; LAP: LC3-associated phagocytosis; LM: littermate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen activated protein kinase; MHC-II: major histocompatibility complex class II; MIIC: MHC class II compartment; OVAL: ovalbumin; PBS: phosphate-buffered saline; PCM1: pericentriolar material 1; PtdIns3K: phosphatidylinositol 3-kinase; PTPRC/CD45RB/B220; Protein tyrosine phosphatase, receptor type, C; SYK: spleen tyrosine kinase; TBS: Tris-buffered saline; TCR: T cell receptor; ULK1: unc-51 like kinase 1.
Asunto(s)
Presentación de Antígeno , Proteína 5 Relacionada con la Autofagia/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Polaridad Celular , Material Particulado/metabolismo , Animales , Autoantígenos/metabolismo , Autofagia , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Citoesqueleto/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Sinapsis Inmunológicas/metabolismo , Lisosomas/metabolismo , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Transporte de Proteínas , Receptores de Antígenos de Linfocitos B/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
Here, we describe a one-step, in vivo CRISPR/Cas9 nuclease-mediated strategy to generate knock-in mice. We produced knock-in (KI) mice wherein a 1.9-kb DNA fragment bearing a pre-arranged human B-cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease-induced double-stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double-stranded breaks are subsequently repaired via homology-directed repair by a plasmid-borne template containing the pre-arranged human immunoglobulin heavy chain. To validate our knock-in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B-cell development and performing single B-cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30-50%). In the future, we envision that such knock-in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases.
Asunto(s)
Linfocitos B/inmunología , Sistemas CRISPR-Cas , Técnicas de Sustitución del Gen/métodos , Cadenas Pesadas de Inmunoglobulina , Animales , Linfocitos B/citología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Ratones , Ratones TransgénicosRESUMEN
Studies of mice deficient for autophagy in T cells since thymic development, concluded that autophagy is integral to mature T cell homeostasis. Basal survival and functional impairments in vivo, limited the use of these models to delineate the role of autophagy during the immune response. We generated Atg5 f/f distal Lck (dLck)-cre mice, with deletion of autophagy only at a mature stage. In this model, autophagy deficiency impacts CD8+ T cell survival but has no influence on CD4+ T cell number and short-term activation. Moreover, autophagy in T cells is dispensable during early humoral response but critical for long-term antibody production. Autophagy in CD4+ T cells is required to transfer humoral memory as shown by injection of antigen-experienced cells in naive mice. We also observed a selection of autophagy-competent cells in the CD4+ T cell memory compartment. We performed in vitro differentiation of memory CD4+ T cells, to better characterize autophagy-deficient memory cells. We identified mitochondrial and lipid load defects in differentiated memory CD4+ T cells, together with a compromised survival, without any collapse of energy production. We then propose that memory CD4+ T cells rely on autophagy for their survival to regulate toxic effects of mitochondrial activity and lipid overload.
Asunto(s)
Autofagia/inmunología , Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica/inmunología , Animales , Anticuerpos/inmunología , Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Supervivencia Celular/inmunología , Homeostasis/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/inmunología , Mitocondrias/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Linfocitos B/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/patología , Orthomyxoviridae/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Animales , Adhesión Celular , Proliferación Celular , Vacunas contra la Influenza/administración & dosificación , Ratones , Análisis de SupervivenciaRESUMEN
Macroautophagy often abbreviated by "autophagy" is an intracellular degradation mechanism linked to lysosomal activity. Autophagy is conserved from yeast to mammals and plays a role in the response to energetic stress and in organelle homeostasis. Autophagy is also involved in the regulation of immunity, in particular in the adaptive immune response, which involves B and T lymphocytes. It was indeed shown that autophagy impacts the development of B and T cells as well as the education of T cells in the thymus. Autophagy also modulates activation, survival and polarization of T cells. It plays a role in antigen presentation by B cells, and in their TLR-mediated activation, and thus likely in their initial activation. Finally, autophagy is required for the survival of memory lymphocytes and effector cells like antibody-producing plasma cells. Interestingly, autophagy is deregulated in several autoimmune pathologies. The modulation of this phenomenon could possibly lead to new treatments aiming at limiting lymphocyte activation driving these pathologies.
Asunto(s)
Autofagia/fisiología , Linfocitos B/fisiología , Homeostasis/inmunología , Linfocitos T/fisiología , Animales , Humanos , Inmunidad Innata/fisiologíaRESUMEN
UNLABELLED: Antiviral immunity in the model organism Drosophila melanogaster involves the broadly active intrinsic mechanism of RNA interference (RNAi) and virus-specific inducible responses. Here, using a panel of six viruses, we investigated the role of hemocytes and autophagy in the control of viral infections. Injection of latex beads to saturate phagocytosis, or genetic depletion of hemocytes, resulted in decreased survival and increased viral titers following infection with Cricket paralysis virus (CrPV), Flock House virus (FHV), and vesicular stomatitis virus (VSV) but had no impact on Drosophila C virus (DCV), Sindbis virus (SINV), and Invertebrate iridescent virus 6 (IIV6) infection. In the cases of CrPV and FHV, apoptosis was induced in infected cells, which were phagocytosed by hemocytes. In contrast, VSV did not trigger any significant apoptosis but we confirmed that the autophagy gene Atg7 was required for full virus resistance, suggesting that hemocytes use autophagy to recognize the virus. However, this recognition does not depend on the Toll-7 receptor. Autophagy had no impact on DCV, CrPV, SINV, or IIV6 infection and was required for replication of the sixth virus, FHV. Even in the case of VSV, the increases in titers were modest in Atg7 mutant flies, suggesting that autophagy does not play a major role in antiviral immunity in Drosophila Altogether, our results indicate that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in insects. IMPORTANCE: Phagocytosis and autophagy are two cellular processes that involve lysosomal degradation and participate in Drosophila immunity. Using a panel of RNA and DNA viruses, we have addressed the contribution of phagocytosis and autophagy in the control of viral infections in this model organism. We show that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in Drosophila This work brings to the front a novel facet of antiviral host defense in insects, which may have relevance in the control of virus transmission by vector insects or in the resistance of beneficial insects to viral pathogens.
Asunto(s)
Autofagia , Virus ADN/inmunología , Drosophila/inmunología , Drosophila/virología , Hemocitos/inmunología , Fagocitosis , Virus ARN/inmunología , Animales , Apoptosis , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Línea Celular , Drosophila/citología , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Interferencia de ARN , Virus Sindbis/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Replicación ViralRESUMEN
Macroautophagy was recently shown to regulate both lymphocyte biology and innate immunity. In this study we sought to determine whether a deregulation of autophagy was linked to the development of autoimmunity. Genome-wide association studies have pointed out nucleotide polymorphisms that can be associated with systemic lupus erythematosus, but the potential role of autophagy in the initiation and/or development of this syndrome is still unknown. Here, we provide first clues of macroautophagy deregulation in lupus. By the use of LC3 conversion assays and electron microscopy experiments, we observed that T cells from two distinct lupus-prone mouse models, i.e., MRL (lpr/lpr) and (NZB/NZW)F1, exhibit high loads of autophagic compartments compared with nonpathologic control CBA/J and BALB/c mice. Unlike normal mice, autophagy increases with age in murine lupus. In vivo lipopolysaccharide stimulation in CBA/J control mice efficiently activates T lymphocytes but fails to upregulate formation of autophagic compartments in these cells. This argues against a deregulation of autophagy in lupus T cells solely resulting from an acute inflammation injury. Autophagic vacuoles quantified by electron microscopy are also found to be significantly more frequent in T cells from lupus patients compared with healthy controls and patients with non-lupus autoimmune diseases. This elevated number of autophagic structures is not distributed homogeneously and appears to be more pronounced in certain T cells. These results suggest that autophagy could regulate the survival of autoreactive T cell during lupus, and could thus lead to design new therapeutic options for lupus.