RESUMEN
Highly effective vaccines elicit specific, robust, and durable adaptive immune responses. To advance informed vaccine design, it is critical that we understand the cellular dynamics underlying responses to different antigen formats. Here, we sought to understand how antigen-specific B and T cells were activated and participated in adaptive immune responses within the mucosal site. Using a human tonsil organoid model, we tracked the differentiation and kinetics of the adaptive immune response to influenza vaccine and virus modalities. Each antigen format elicited distinct B and T cell responses, including differences in their magnitude, diversity, phenotype, function, and breadth. These differences culminated in substantial changes in the corresponding antibody response. A major source of antigen format-related variability was the ability to recruit naive vs. memory B and T cells to the response. These findings have important implications for vaccine design and the generation of protective immune responses in the upper respiratory tract.
Asunto(s)
Vacunas contra la Influenza , Gripe Humana , Humanos , Formación de Anticuerpos , Anticuerpos Antivirales , Linfocitos T , Antígenos , OrganoidesRESUMEN
B cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. A consistent strategy for classifying human B cells by using surface molecules is essential to harness this functional diversity for clinical translation. We developed a highly multiplexed screen to quantify the co-expression of 351 surface molecules on millions of human B cells. We identified differentially expressed molecules and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these analyses, we propose a classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets, including a CD45RB+CD27- early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation. This classification framework and underlying datasets provide a resource for further investigations of human B cell identity and function.
Asunto(s)
Subgrupos de Linfocitos B/clasificación , Subgrupos de Linfocitos B/inmunología , Isotipos de Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , 5'-Nucleotidasa/metabolismo , Apirasa/metabolismo , Antígeno CD11c/metabolismo , Femenino , Proteínas Ligadas a GPI/metabolismo , Humanos , Memoria Inmunológica/inmunología , Antígenos Comunes de Leucocito/metabolismo , Persona de Mediana Edad , Transducción de Señal/inmunología , Receptor fas/metabolismoRESUMEN
Current influenza A and B virus (IABV) vaccines provide suboptimal protection and efforts are underway to develop a universal IABV vaccine. Blood neutralizing antibodies are the current gold standard for protection, but many processes that regulate human IABV-specific immunity occur in mucosal and lymphoid tissues. We need an improved mechanistic understanding of how immune cells respond within these tissues to advance our current (slow and expensive) vaccine testing model. We posit that advanced in vitro models of human adaptive immunity can bridge some of the gaps between vaccine design, animal models, and human clinical trials. Here, we highlight how they can be integrated into current practices and play a role in reverse translating the defined features of protective vaccines to rationally design new candidates.
Asunto(s)
Vacunas contra la Influenza , Gripe Humana , Animales , Humanos , Anticuerpos Antivirales , Anticuerpos Neutralizantes , Inmunidad Adaptativa , Organoides , Gripe Humana/prevención & controlRESUMEN
Given the limited efficacy of clinical approaches that rely on ex vivo generated dendritic cells (DCs), it is imperative to design strategies that harness specialized DC subsets in situ. This requires delineating the expression of surface markers by DC subsets among individuals and tissues. Here, we performed a multiparametric phenotypic characterization and unbiased analysis of human DC subsets in blood, tonsil, spleen, and skin. We uncovered previously unreported phenotypic heterogeneity of human cDC2s among individuals, including variable expression of functional receptors such as CD172a. We found marked differences in DC subsets localized in blood and lymphoid tissues versus skin, and a striking absence of the newly discovered Axl+ DCs in the skin. Finally, we evaluated the capacity of anti-receptor monoclonal antibodies to deliver vaccine components to skin DC subsets. These results offer a promising path for developing DC subset-specific immunotherapies that cannot be provided by transcriptomic analysis alone.
Asunto(s)
Antígenos de Diferenciación/inmunología , Variación Biológica Individual , Células Dendríticas/inmunología , Fenotipo , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Inmunológicos/inmunología , Piel/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacocinética , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación/genética , Biomarcadores/análisis , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/biosíntesis , Citofotometría/métodos , Células Dendríticas/citología , Femenino , Expresión Génica , Humanos , Inmunofenotipificación , Inmunoterapia , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Terapia Molecular Dirigida , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/terapia , Especificidad de Órganos , Tonsila Palatina/citología , Tonsila Palatina/inmunología , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Inmunológicos/genética , Piel/citología , Bazo/citología , Bazo/inmunología , Tirosina Quinasa del Receptor AxlRESUMEN
The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.
Asunto(s)
COVID-19/virología , Pulmón/citología , Modelos Biológicos , Organoides/citología , Organoides/virología , SARS-CoV-2/fisiología , Técnicas de Cultivo de Tejidos , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , COVID-19/metabolismo , COVID-19/patología , Diferenciación Celular , División Celular , Células Clonales/citología , Células Clonales/metabolismo , Células Clonales/virología , Humanos , Técnicas In Vitro , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Integrina alfa6/análisis , Integrina beta4/análisis , Queratina-5/análisis , Organoides/metabolismo , Neumonía Viral/metabolismo , Neumonía Viral/patología , Neumonía Viral/virología , SARS-CoV-2/crecimiento & desarrollo , Análisis de la Célula Individual , Receptor de TWEAK/análisisRESUMEN
The ideal vaccine against viruses such as influenza and SARS-CoV-2 must provide a robust, durable and broad immune protection against multiple viral variants. However, antibody responses to current vaccines often lack robust cross-reactivity. Here we describe a polymeric Toll-like receptor 7 agonist nanoparticle (TLR7-NP) adjuvant, which enhances lymph node targeting, and leads to persistent activation of immune cells and broad immune responses. When mixed with alum-adsorbed antigens, this TLR7-NP adjuvant elicits cross-reactive antibodies for both dominant and subdominant epitopes and antigen-specific CD8+ T-cell responses in mice. This TLR7-NP-adjuvanted influenza subunit vaccine successfully protects mice against viral challenge of a different strain. This strategy also enhances the antibody response to a SARS-CoV-2 subunit vaccine against multiple viral variants that have emerged. Moreover, this TLR7-NP augments antigen-specific responses in human tonsil organoids. Overall, we describe a nanoparticle adjuvant to improve immune responses to viral antigens, with promising implications for developing broadly protective vaccines.
Asunto(s)
COVID-19 , Vacunas contra la Influenza , Gripe Humana , Nanopartículas , Animales , Ratones , Humanos , Gripe Humana/prevención & control , Receptor Toll-Like 7/genética , SARS-CoV-2/genética , COVID-19/prevención & control , Adyuvantes Inmunológicos/farmacología , Inmunidad , Vacunas de SubunidadRESUMEN
T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRß chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.
Asunto(s)
Algoritmos , Antígenos HLA/química , Antígenos HLA/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Adolescente , Secuencia de Aminoácidos , Cristalografía por Rayos X , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Humanos , Ligandos , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/inmunología , Especificidad por SustratoRESUMEN
Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Región Variable de Inmunoglobulina/inmunología , Linfoma de Células del Manto/inmunología , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Linfocitos T CD4-Positivos/inmunología , Citotoxicidad Inmunológica , Análisis Mutacional de ADN , Epítopos de Linfocito T/inmunología , Exoma/genética , Genómica , Antígenos HLA-D/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Inmunoterapia/tendencias , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , Linfoma de Células del Manto/terapia , Mutación , ProteómicaRESUMEN
Chronically undernourished children become stunted during their first 2 years and thereafter bear burdens of ill health for the rest of their lives. Contributors to stunting include poor nutrition and exposure to pathogens, and parental history may also play a role. However, the epigenetic impact of a poor environment on young children is largely unknown. Here we show the unfolding pattern of histone H3 lysine 4 trimethylation (H3K4me3) in children and mothers living in an urban slum in Dhaka, Bangladesh. A pattern of chromatin modification in blood cells of stunted children emerges over time and involves a global decrease in methylation at canonical locations near gene start sites and increased methylation at ectopic sites throughout the genome. This redistribution occurs at metabolic and immune genes and was specific for H3K4me3, as it was not observed for histone H3 lysine 27 acetylation in the same samples. Methylation changes in stunting globally resemble changes that occur in vitro in response to altered methylation capacity, suggesting that reduced levels of one-carbon nutrients in the diet play a key role in stunting in this population. A network of differentially expressed genes in stunted children reveals effects on chromatin modification machinery, including turnover of H3K4me3, as well as posttranscriptional gene regulation affecting immune response pathways and lipid metabolism. Consistent with these changes, reduced expression of the endocytic receptor gene LDL receptor 1 (LRP1) is a driver of stunting in a mouse model, suggesting a target for intervention.
Asunto(s)
Histonas/genética , Desnutrición/genética , Animales , Epigénesis Genética , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Desnutrición/metabolismo , Metilación , RatonesRESUMEN
IL-7 therapy has been evaluated in patients who do not regain normal CD4 T cell counts after virologically successful antiretroviral therapy. IL-7 increases total circulating CD4 and CD8 T cell counts; however, its effect on HIV-specific CD8 T cells has not been fully examined. TRAF1, a prosurvival signaling adaptor required for 4-1BB-mediated costimulation, is lost from chronically stimulated virus-specific CD8 T cells with progression of HIV infection in humans and during chronic lymphocytic choriomeningitis infection in mice. Previous results showed that IL-7 can restore TRAF1 expression in virus-specific CD8 T cells in mice, rendering them sensitive to anti-4-1BB agonist therapy. In this article, we show that IL-7 therapy in humans increases the number of circulating HIV-specific CD8 T cells. For a subset of patients, we also observed an increased frequency of TRAF1+ HIV-specific CD8 T cells 10 wk after completion of IL-7 treatment. IL-7 treatment increased levels of phospho-ribosomal protein S6 in HIV-specific CD8 T cells, suggesting increased activation of the metabolic checkpoint kinase mTORC1. Thus, IL-7 therapy in antiretroviral therapy-treated patients induces sustained changes in the number and phenotype of HIV-specific T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/inmunología , Proteína S6 Ribosómica/metabolismo , Factor 1 Asociado a Receptor de TNF/metabolismo , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Citocinas/biosíntesis , Expresión Génica , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Humanos , Interleucina-7/farmacología , Interleucina-7/uso terapéutico , Recuento de Linfocitos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Unión Proteica , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Proteína S6 Ribosómica/genética , Factor 1 Asociado a Receptor de TNF/genética , Carga ViralRESUMEN
Vaccination with attenuated live varicella zoster virus (VZV) can prevent zoster reactivation, but protection is incomplete especially in an older population. To decipher the molecular mechanisms underlying variable vaccine responses, T- and B-cell responses to VZV vaccination were examined in individuals of different ages including identical twin pairs. Contrary to the induction of VZV-specific antibodies, antigen-specific T cell responses were significantly influenced by inherited factors. Diminished generation of long-lived memory T cells in older individuals was mainly caused by increased T cell loss after the peak response while the expansion of antigen-specific T cells was not affected by age. Gene expression in activated CD4 T cells at the time of the peak response identified gene modules related to cell cycle regulation and DNA repair that correlated with the contraction phase of the T cell response and consequently the generation of long-lived memory cells. These data identify cell cycle regulatory mechanisms as targets to reduce T cell attrition in a vaccine response and to improve the generation of antigen-specific T cell memory, in particular in an older population.
Asunto(s)
Vacuna contra el Herpes Zóster/inmunología , Herpes Zóster/inmunología , Memoria Inmunológica/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Anciano , Diferenciación Celular/inmunología , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Femenino , Herpes Zóster/prevención & control , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Mutations in mitochondrial (mt) DNA accumulate with age and can result in the generation of neopeptides. Immune surveillance of such neopeptides may allow suboptimal mitochondria to be eliminated, thereby avoiding mt-related diseases, but may also contribute to autoimmunity in susceptible individuals. To date, the direct recognition of neo-mtpeptides by the adaptive immune system has not been demonstrated. In this study we used bioinformatics approaches to predict MHC binding of neopeptides identified from known deletions in mtDNA. Six such peptides were confirmed experimentally to bind to HLA-A*02. Pre-existing human CD4(+) and CD8(+) T cells from healthy donors were shown to recognize and respond to these neopeptides. One remarkably promiscuous immunodominant peptide (P9) could be presented by diverse MHC molecules to CD4(+) and/or CD8(+) T cells from 75% of the healthy donors tested. The common soil microbe, Bacillus pumilus, encodes a 9-mer that differs by one amino acid from P9. Similarly, the ATP synthase F0 subunit 6 from normal human mitochondria encodes a 9-mer with a single amino acid difference from P9 with 89% homology to P9. T cells expanded from human PBMCs using the B. pumilus or self-mt peptide bound to P9/HLA-A2 tetramers, arguing for cross-reactivity between T cells with specificity for self and foreign homologs of the altered mt peptide. These findings provide proof of principal that the immune system can recognize peptides arising from spontaneous somatic mutations and that such responses might be primed by foreign peptides and/or be cross-reactive with self.
Asunto(s)
Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , ADN Mitocondrial/inmunología , Proteínas Mitocondriales/inmunología , Oligopéptidos/inmunología , Eliminación de Secuencia , Adulto , Anciano , Bacillus/inmunología , Reacciones Cruzadas , ADN Mitocondrial/genética , Femenino , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/genéticaRESUMEN
Tools to study memory B cell (MBC) development and function are needed to understand their role in supporting sustained protection against recurrent infections. While human MBCs are traditionally measured using blood, there is a growing interest in elucidating their behavior within lymphoid tissues, which are the main sites where adaptive immune responses are orchestrated. In this chapter, we introduce a high-throughput organoid system that is derived from primary human lymphoid tissues. The approach can recapitulate many hallmarks of successful adaptive immune responses and capture inter-individual variation in response to a variety of stimuli. Lymphoid tissue organoids enable characterization of pre-existing antigen-specific MBCs within an entirely human system and can provide valuable insights into MBC dynamics.
Asunto(s)
Linfocitos B , Memoria Inmunológica , Organoides , Tonsila Palatina , Humanos , Organoides/citología , Organoides/inmunología , Tonsila Palatina/citología , Tonsila Palatina/inmunología , Linfocitos B/inmunología , Linfocitos B/citología , Técnicas de Cultivo de Célula/métodos , Células CultivadasRESUMEN
Sex-based differences in immune cell composition and function can contribute to distinct adaptive immune responses. Prior work has quantified these differences in peripheral blood, but little is known about sex differences within human lymphoid tissues. Here, we characterized the composition and phenotypes of adaptive immune cells from male and female ex vivo tonsils and evaluated their responses to influenza antigens using an immune organoid approach. In a pediatric cohort, female tonsils had more memory B cells compared to male tonsils direct ex vivo and after stimulation with live-attenuated but not inactivated vaccine, produced higher influenza-specific antibody responses. Sex biases were also observed in adult tonsils but were different from those measured in children. Analysis of peripheral blood immune cells from in vivo vaccinated adults also showed higher frequencies of tissue homing CD4 T cells in female participants. Together, our data demonstrate that distinct memory B and T cell profiles are present in male vs. female lymphoid tissues and peripheral blood respectively and suggest that these differences may in part explain sex biases in response to vaccines and viruses.
Asunto(s)
Tonsila Palatina , Humanos , Femenino , Masculino , Niño , Tonsila Palatina/inmunología , Adulto , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Caracteres Sexuales , Preescolar , Adolescente , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Células B de Memoria/inmunología , Especificidad de Órganos/inmunología , Adulto Joven , Factores Sexuales , Linfocitos T CD4-Positivos/inmunología , Linfocitos B/inmunología , Memoria InmunológicaRESUMEN
Heterogeneity in the tumor microenvironment (TME) of follicular lymphomas (FLs) can affect clinical outcomes. Current immunotherapeutic strategies, including antibody- and cell-based therapies, variably overcome pro-tumorigenic mechanisms for sustained disease control. Modeling the intact FL TME, with its native, syngeneic tumor-infiltrating leukocytes, is a major challenge. Here, we describe an organoid culture method for cultivating patient-derived lymphoma organoids (PDLOs), which include cells from the native FL TME. We define the robustness of this method by successfully culturing cryopreserved FL specimens from diverse patients and demonstrate the stability of TME cellular composition, tumor somatic mutations, gene expression profiles, and B/T cell receptor dynamics over 3 weeks. PDLOs treated with CD3:CD19 and CD3:CD20 therapeutic bispecific antibodies showed B cell killing and T cell activation. This stable system offers a robust platform for advancing precision medicine efforts in FL through patient-specific modeling, high-throughput screening, TME signature identification, and treatment response evaluation.
Asunto(s)
Linfoma Folicular , Humanos , Linfoma Folicular/terapia , Linfoma Folicular/diagnóstico , Linfoma Folicular/genética , Microambiente Tumoral , Linfocitos B , Receptores de Antígenos de Linfocitos T , OrganoidesRESUMEN
The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell typespecific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology (IL21, IL21R, IL4R, and BCL6) and transcription factors regulating B cell differentiation (POU2AF1 and HHEX). Together, these analyses provide a powerful new cell typeresolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.
Asunto(s)
Autoinmunidad/inmunología , Centro Germinal/inmunología , Proteínas de Homeodominio/inmunología , Interleucinas/inmunología , Análisis de la Célula Individual , Transactivadores/inmunología , Factores de Transcripción/inmunología , Diferenciación Celular/inmunología , Epigenómica , Proteínas de Homeodominio/genética , Humanos , Interleucinas/genética , Tonsila Palatina/inmunología , Análisis de Secuencia de ARN , Transactivadores/genética , Factores de Transcripción/genética , TranscriptomaRESUMEN
Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.
Asunto(s)
Vacunas contra la Influenza/inmunología , Tonsila Palatina/inmunología , Adyuvantes Inmunológicos , Linfocitos B/citología , Linfocitos B/inmunología , Vacunas contra la COVID-19/inmunología , Centro Germinal/citología , Glicoproteínas Hemaglutininas del Virus de la Influenza , Humanos , Técnicas In Vitro , Tejido Linfoide/inmunología , Vacuna contra el Sarampión-Parotiditis-Rubéola/inmunología , Organoides/citología , Organoides/inmunología , Vacunas Antirrábicas/inmunología , Linfocitos T/inmunologíaRESUMEN
Antibodies are typically thought to mediate antiviral immunity by blocking host-cell infection. However, immunoglobulin G (IgG) can also stimulate antiviral responses through its constant region. A recent study in Nature (Bournazos et al., 2020) shows that modifying Fc binding affinities to better target FcɣRIIa enhances the influenza CD8 T cell response.
Asunto(s)
Gripe Humana , Orthomyxoviridae , Anticuerpos Antivirales , Linfocitos T CD8-positivos , Humanos , Fragmentos Fc de InmunoglobulinasRESUMEN
The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5 + basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5 + cells contained a distinct ITGA6 + ITGB4 + mitotic population whose proliferation segregated to a TNFRSF12A hi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12A hi subset of FACS-purified ITGA6 + ITGB4 + basal cells from human lung or derivative organoids. In vivo, TNFRSF12A + cells comprised ~10% of KRT5 + basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 apical-out organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.
RESUMEN
In mass cytometry, sample loss is of considerable concern due to the relative inefficiency of cell event collection compared to similar techniques such as flow cytometry. Cell stimulation and the harsh conditions required in the later stages of certain sample preparations also contribute to cell loss. Low starting cell numbers are especially susceptible to these effects, potentially limiting the ability to use mass cytometry. Here is presented a live cell barcoding scheme and additional efficiency methods to improve recovery and achieve consistent staining for small samples.