RESUMEN
Germinal centers (GCs) are microanatomical sites of B cell clonal expansion and antibody affinity maturation. Therein, B cells undergo the Darwinian process of somatic diversification and affinity-driven selection of immunoglobulins that produces the high-affinity antibodies essential for effective humoral immunity. Here, we review recent developments in the field of GC biology, primarily as it pertains to GCs induced by infection or immunization. First, we summarize the phenotype and function of the different cell types that compose the GC, focusing on GC B cells. Then, we review the cellular and molecular bases of affinity-dependent selection within the GC and the export of memory and plasma cells. Finally, we present an overview of the emerging field of GC clonal dynamics, focusing on how GC and post-GC selection shapes the diversity of antibodies secreted into serum.
Asunto(s)
Linfocitos B , Centro Germinal , Animales , Anticuerpos , Afinidad de Anticuerpos , Humanos , Inmunidad HumoralRESUMEN
The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.
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Serina Endopeptidasas , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/química , Humanos , Cristalografía por Rayos X , Coronavirus/metabolismo , Coronavirus/química , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Modelos Moleculares , Unión Proteica , Células HEK293 , Animales , Activación Enzimática , Internalización del VirusRESUMEN
Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma.
Asunto(s)
Citidina Desaminasa , Hipermutación Somática de Inmunoglobulina , Animales , Ratones , Anticuerpos/genética , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , ADN/genética , ADN de Cadena Simple , Mutación , Evolución Molecular , Regiones Determinantes de Complementariedad/genética , Motivos de NucleótidosRESUMEN
Germinal centers (GCs) form in lymph nodes after immunization or infection to facilitate antibody affinity maturation and memory and plasma cell (PC) development. PC differentiation is thought to involve stringent selection for GC B cells expressing the highest-affinity antigen receptors, but how this plays out during complex polyclonal responses is unclear. We combine temporal lineage tracing with antibody characterization to gain a snapshot of PCs developing during influenza infection. GCs co-mature B cell clones with antibody affinities spanning multiple orders of magnitude; however, each generates PCs with similar efficiencies, including weak binders. Within lineages, PC selection is not restricted to variants with the highest-affinity antibodies. Differentiation is commonly associated with proliferative expansion to produce "nodes" of identical PCs. Immunization-induced GCs generate fewer PCs but still of low- and high-antibody affinities. We propose that generating low-affinity antibody PCs reflects an evolutionary compromise to facilitate diverse serum antibody responses.
Asunto(s)
Afinidad de Anticuerpos , Linfocitos B , Centro Germinal , Células Plasmáticas , Formación de Anticuerpos , Linfocitos B/citología , Linfocitos B/inmunología , Ganglios Linfáticos , Línea Celular , Humanos , Animales , Ratones , Cricetinae , Virus de la Influenza A/inmunología , Diferenciación CelularRESUMEN
Photosynthesis is central to food production and the Earth's biogeochemistry, yet the molecular basis for its regulation remains poorly understood. Here, using high-throughput genetics in the model eukaryotic alga Chlamydomonas reinhardtii, we identify with high confidence (false discovery rate [FDR] < 0.11) 70 poorly characterized genes required for photosynthesis. We then enable the functional characterization of these genes by providing a resource of proteomes of mutant strains, each lacking one of these genes. The data allow assignment of 34 genes to the biogenesis or regulation of one or more specific photosynthetic complexes. Further analysis uncovers biogenesis/regulatory roles for at least seven proteins, including five photosystem I mRNA maturation factors, the chloroplast translation factor MTF1, and the master regulator PMR1, which regulates chloroplast genes via nuclear-expressed factors. Our work provides a rich resource identifying regulatory and functional genes and placing them into pathways, thereby opening the door to a system-level understanding of photosynthesis.
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Chlamydomonas reinhardtii , Fotosíntesis , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Fotosíntesis/genética , Regulación de la Expresión Génica , Proteínas/genética , Proteínas/metabolismo , Mutación , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genéticaRESUMEN
The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.
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Células-Madre Neurales , Ratones , Animales , Humanos , Células-Madre Neurales/metabolismo , Neuronas , Diferenciación Celular/fisiología , Neuroglía/metabolismo , Encéfalo , AstrocitosRESUMEN
Herman Eisen and Sondra Schlesinger spent several days together in September 2007 in Woods Hole, Massachusetts, talking about immunology, focusing on his remembrances of the field over the more than 60 years of his involvement. This article is an abridged version of those discussions (the full version is available on the Annual Reviews website). It is both an oral history and a written memory of some important but selected areas of immunology.
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Alergia e Inmunología , Alergia e Inmunología/historia , Alergia e Inmunología/organización & administración , Alergia e Inmunología/tendencias , Animales , Anticuerpos , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Sistema Inmunológico , Linfocitos TRESUMEN
Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.
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Reno , Cicatrización de Heridas , Adulto , Animales , Humanos , Cicatriz/patología , Fibroblastos/patología , Trasplante de Piel , Piel/patología , Feto/patologíaRESUMEN
Many individuals mount nearly identical antibody responses to SARS-CoV-2. To gain insight into how the viral spike (S) protein receptor-binding domain (RBD) might evolve in response to common antibody responses, we studied mutations occurring during virus evolution in a persistently infected immunocompromised individual. We use antibody Fab/RBD structures to predict, and pseudotypes to confirm, that mutations found in late-stage evolved S variants confer resistance to a common class of SARS-CoV-2 neutralizing antibodies we isolated from a healthy COVID-19 convalescent donor. Resistance extends to the polyclonal serum immunoglobulins of four out of four healthy convalescent donors we tested and to monoclonal antibodies in clinical use. We further show that affinity maturation is unimportant for wild-type virus neutralization but is critical to neutralization breadth. Because the mutations we studied foreshadowed emerging variants that are now circulating across the globe, our results have implications to the long-term efficacy of S-directed countermeasures.
Asunto(s)
Anticuerpos Antivirales/inmunología , COVID-19 , Evolución Molecular , Evasión Inmune/inmunología , Huésped Inmunocomprometido , Fragmentos Fab de Inmunoglobulinas/inmunología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Neutralizantes , COVID-19/genética , COVID-19/inmunología , Femenino , Células HEK293 , Humanos , Masculino , Dominios Proteicos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Repeated exposure to pathogens or their antigens triggers anamnestic antibody responses that are higher in magnitude and affinity than the primary response. These involve reengagement of memory B cell (MBC) clones, the diversity and specificity of which determine the breadth and effectiveness of the ensuing antibody response. Using prime-boost models in mice, we find that secondary responses are characterized by a clonality bottleneck that restricts the engagement of the large diversity of MBC clones generated by priming. Rediversification of mutated MBCs is infrequent within secondary germinal centers (GCs), which instead consist predominantly of B cells without prior GC experience or detectable clonal expansion. Few MBC clones, generally derived from higher-affinity germline precursors, account for the majority of secondary antibody responses, while most primary-derived clonal diversity is not reengaged detectably by boosting. Understanding how to counter this bottleneck may improve our ability to elicit antibodies to non-immunodominant epitopes by vaccination.
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Linfocitos B/inmunología , Centro Germinal/inmunología , Memoria Inmunológica/inmunología , Inmunidad Adaptativa/inmunología , Animales , Formación de Anticuerpos/inmunología , Formación de Anticuerpos/fisiología , Antígenos/inmunología , Linfocitos B/metabolismo , Células CHO , Línea Celular , Cricetulus , Femenino , Centro Germinal/metabolismo , Humanos , Memoria Inmunológica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos AnimalesRESUMEN
All proteins interact with other cellular components to fulfill their function. While tremendous progress has been made in the identification of protein complexes, their assembly and dynamics remain difficult to characterize. Here, we present a high-throughput strategy to analyze the native assembly kinetics of protein complexes. We apply our approach to characterize the co-assembly for 320 pairs of nucleoporins (NUPs) constituting the ≈50 MDa nuclear pore complex (NPC) in yeast. Some NUPs co-assemble fast via rapid exchange whereas others require lengthy maturation steps. This reveals a hierarchical principle of NPC biogenesis where individual subcomplexes form on a minute timescale and then co-assemble from center to periphery in a â¼1 h-long maturation process. Intriguingly, the NUP Mlp1 stands out as joining very late and associating preferentially with aged NPCs. Our approach is readily applicable beyond the NPC, making it possible to analyze the intracellular dynamics of a variety of multiprotein assemblies.
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Sustancias Macromoleculares/metabolismo , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Coloración y Etiquetado , Bioensayo , Cinética , Modelos Biológicos , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de TiempoRESUMEN
Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.
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Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Centro Germinal/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Inmunización Pasiva , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linfocitos B/patología , Femenino , Centro Germinal/patología , Centro Germinal/virología , Macaca mulatta , Masculino , Linfocitos T Colaboradores-Inductores/patología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Induction of commensal-specific immunity contributes to tissue homeostasis, yet the mechanisms underlying induction of commensal-specific B cells remain poorly understood in part due to a lack of tools to identify these cells. Using phage display, we identified segmented filamentous bacteria (SFB) antigens targeted by serum and intestinal antibodies and generated B cell tetramers to track SFB-specific B cells in gut-associated lymphoid tissues. We revealed a compartmentalized response in SFB-specific B cell activation, with a gradient of immunoglobulin A (IgA), IgG1, and IgG2b isotype production along Peyer's patches contrasted by selective production of IgG2b within mesenteric lymph nodes. V(D)J sequencing and monoclonal antibody generation identified somatic hypermutation driven affinity maturation to SFB antigens under homeostatic conditions. Combining phage display and B cell tetramers will enable investigation of the ontogeny and function of commensal-specific B cell responses in tissue immunity, inflammation, and repair.
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Linfocitos B , Animales , Linfocitos B/inmunología , Ratones , Ratones Endogámicos C57BL , Ganglios Linfáticos Agregados/inmunología , Activación de Linfocitos/inmunología , Antígenos Bacterianos/inmunología , Hipermutación Somática de Inmunoglobulina , Biblioteca de Péptidos , Ganglios Linfáticos/inmunología , Técnicas de Visualización de Superficie Celular , Simbiosis/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina A/inmunologíaRESUMEN
Memory B cells (MBCs) formed over the individual's lifetime constitute nearly half of the circulating B cell repertoire in humans. These pre-existing MBCs dominate recall responses to their cognate antigens, but how they respond to recognition of novel antigens is not well understood. Here, we tracked the origin and followed the differentiation paths of MBCs in the early anti-spike (S) response to mRNA vaccination in SARS-CoV-2-naive individuals on single-cell and monoclonal antibody levels. Pre-existing, highly mutated MBCs showed no signs of germinal center re-entry and rapidly developed into mature antibody-secreting cells (ASCs). By contrast, and despite similar levels of S reactivity, naive B cells showed strong signs of antibody affinity maturation before differentiating into MBCs and ASCs. Thus, pre-existing human MBCs differentiate into ASCs in response to novel antigens, but the quality of the humoral and cellular anti-S response improved through the clonal selection and affinity maturation of naive precursors.
RESUMEN
Long-lived plasma cells (PCs) secrete antibodies that can provide sustained immunity against infection. High-affinity cells are proposed to preferentially select into this compartment, potentiating the immune response. We used single-cell RNA-seq to track the germinal center (GC) development of Ighg2A10 B cells, specific for the Plasmodium falciparum circumsporozoite protein (PfCSP). Following immunization with Plasmodium sporozoites, we identified 3 populations of cells in the GC light zone (LZ). One LZ population expressed a gene signature associated with the initiation of PC differentiation and readily formed PCs in vitro. The estimated affinity of these pre-PC B cells was indistinguishable from that of LZ cells that remained in the GC. This remained true when high- or low-avidity recombinant PfCSP proteins were used as immunogens. These findings suggest that the initiation of PC development occurs via an affinity-independent process.
Asunto(s)
Linfocitos B , Centro Germinal , Células Plasmáticas , Diferenciación Celular , Células Precursoras de Linfocitos BRESUMEN
Embryonic cell fates are defined by transcription factors that are rapidly deployed, yet attempts to visualize these factors in vivo often fail because of slow fluorescent protein maturation. Here, we pioneer a protein tag, LlamaTag, which circumvents this maturation limit by binding mature fluorescent proteins, making it possible to visualize transcription factor concentration dynamics in live embryos. Implementing this approach in the fruit fly Drosophila melanogaster, we discovered stochastic bursts in the concentration of transcription factors that are correlated with bursts in transcription. We further used LlamaTags to show that the concentration of protein in a given nucleus heavily depends on transcription of that gene in neighboring nuclei; we speculate that this inter-nuclear signaling is an important mechanism for coordinating gene expression to delineate straight and sharp boundaries of gene expression. Thus, LlamaTags now make it possible to visualize the flow of information along the central dogma in live embryos.
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Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Edición Génica/métodos , Factores de Transcripción/genética , Animales , Núcleo Celular/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Microscopía Confocal , Factores de Transcripción/metabolismoRESUMEN
Heterogeneity is a hallmark feature of the adaptive immune system in vertebrates. Following infection, naive T cells differentiate into various subsets of effector and memory T cells, which help to eliminate pathogens and maintain long-term immunity. The current model suggests there is a single lineage of naive T cells that give rise to different populations of effector and memory T cells depending on the type and amounts of stimulation they encounter during infection. Here, we have discovered that multiple sub-populations of cells exist in the naive CD8+ T cell pool that are distinguished by their developmental origin, unique transcriptional profiles, distinct chromatin landscapes, and different kinetics and phenotypes after microbial challenge. These data demonstrate that the naive CD8+ T cell pool is not as homogeneous as previously thought and offers a new framework for explaining the remarkable heterogeneity in the effector and memory T cell subsets that arise after infection.
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Linfocitos T CD8-positivos/inmunología , Genes del Desarrollo , Listeria monocytogenes/patogenicidad , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Cromatina/metabolismo , Citocinas/farmacología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/metabolismo , Memoria Inmunológica , Interferón gamma/metabolismo , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Listeria monocytogenes/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis de Componente Principal , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Timo/trasplante , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Germinal centers (GCs) are sites of B cell clonal expansion, diversification, and antibody affinity selection. This process is limited and directed by T follicular helper cells that provide helper signals to B cells that endocytose, process, and present cognate antigens in proportion to their B cell receptor (BCR) affinity. Under this model, the BCR functions as an endocytic receptor for antigen capture. How signaling through the BCR contributes to selection is not well understood. To investigate the role of BCR signaling in GC selection, we developed a tracker for antigen binding and presentation and a Bruton's tyrosine kinase drug-resistant-mutant mouse model. We showed that BCR signaling per se is necessary for the survival and priming of light zone B cells to receive T cell help. Our findings provide insight into how high-affinity antibodies are selected within GCs and are fundamental to our understanding of adaptive immunity and vaccine development.
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Linfocitos B , Centro Germinal , Ratones , Animales , Receptores de Antígenos de Linfocitos B/metabolismo , Antígenos , Transducción de SeñalRESUMEN
V2-glycan/apex broadly neutralizing antibodies (bnAbs) recognize a closed quaternary epitope of the HIV-1 envelope glycoprotein (Env). This closed structure is necessary to elicit apex antibodies and useful to guide the maturation of other bnAb classes. To compare antigens designed to maintain this conformation, we evaluated apex-specific responses in mice engrafted with a diverse repertoire of B cells expressing the HCDR3 of the apex bnAb VRC26.25. Engineered B cells affinity matured, guiding the improvement of VRC26.25 itself. We found that soluble Env (SOSIP) variants differed significantly in their ability to raise anti-apex responses. A transmembrane SOSIP (SOSIP-TM) delivered as an mRNA-lipid nanoparticle elicited more potent neutralizing responses than multimerized SOSIP proteins. Importantly, SOSIP-TM elicited neutralizing sera from B cells engineered with the predicted VRC26.25-HCDR3 progenitor, which also affinity matured. Our data show that HCDR3-edited B cells facilitate efficient in vivo comparisons of Env antigens and highlight the potential of an HCDR3-focused vaccine approach.
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Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Vacunas , Animales , Ratones , Anticuerpos Anti-VIH , Anticuerpos Neutralizantes , Anticuerpos ampliamente neutralizantes , Antígenos Virales , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
Germinal centers (GCs), transient structures within B cell follicles and central to affinity maturation, require the coordinated behavior of T and B cells. IL-21, a pleiotropic T cell-derived cytokine, is key to GC biology through incompletely understood mechanisms. By genetically restricting production and receipt of IL-21 in vivo, we reveal how its independent actions on T and B cells combine to regulate the GC. IL-21 established the magnitude of the GC B cell response by promoting CD4+ T cell expansion and differentiation in a dose-dependent manner and with paracrine activity. Within GC, IL-21 specifically promoted B cell centroblast identity and, when bioavailability was high, plasma cell differentiation. Critically, these actions may occur irrespective of cognate T-B interactions, making IL-21 a general promoter of growth as distinct to a mediator of affinity-driven selection via synaptic delivery. This promiscuous activity of IL-21 explains the consequences of IL-21 deficiency on antibody-based immunity.