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BACKGROUND: Cytometry is an experimental technique used to measure molecules expressed by cells at a single cell resolution. Recently, several technological improvements have made possible to increase greatly the number of cell markers that can be simultaneously measured. Many computational methods have been proposed to identify clusters of cells having similar phenotypes. Nevertheless, only a limited number of computational methods permits to compare the phenotypes of the cell clusters identified by different clustering approaches. These phenotypic comparisons are necessary to choose the appropriate clustering methods and settings. Because of this lack of tools, comparisons of cell cluster phenotypes are often performed manually, a highly biased and time-consuming process. RESULTS: We designed CytoCompare, an R package that performs comparisons between the phenotypes of cell clusters with the purpose of identifying similar and different ones, based on the distribution of marker expressions. For each phenotype comparison of two cell clusters, CytoCompare provides a distance measure as well as a p-value asserting the statistical significance of the difference. CytoCompare can import clustering results from various algorithms including SPADE, viSNE/ACCENSE, and Citrus, the most current widely used algorithms. Additionally, CytoCompare can generate parallel coordinates, parallel heatmaps, multidimensional scaling or circular graph representations to visualize easily cell cluster phenotypes and the comparison results. CONCLUSIONS: CytoCompare is a flexible analysis pipeline for comparing the phenotypes of cell clusters identified by automatic gating algorithms in high-dimensional cytometry data. This R package is ideal for benchmarking different clustering algorithms and associated parameters. CytoCompare is freely distributed under the GPL-3 license and is available on https://github.com/tchitchek-lab/CytoCompare.
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Citometria de Fluxo/métodos , Software , Algoritmos , Biomarcadores , Análise por Conglomerados , Biologia Computacional , Gráficos por Computador , Humanos , Análise Multivariada , FenótipoRESUMO
Motivation: Flow, hyperspectral and mass cytometry are experimental techniques measuring cell marker expressions at the single cell level. The recent increase of the number of markers simultaneously measurable has led to the development of new automatic gating algorithms. Especially, the SPADE algorithm has been proposed as a novel way to identify clusters of cells having similar phenotypes in high-dimensional cytometry data. While SPADE or other cell clustering algorithms are powerful approaches, complementary analysis features are needed to better characterize the identified cell clusters. Results: We have developed SPADEVizR, an R package designed for the visualization, analysis and integration of cell clustering results. The available statistical methods allow highlighting cell clusters with relevant biological behaviors or integrating them with additional biological variables. Moreover, several visualization methods are available to better characterize the cell clusters, such as volcano plots, streamgraphs, parallel coordinates, heatmaps, or distograms. SPADEVizR can also generate linear, Cox or random forest models to predict biological outcomes, based on the cell cluster abundances. Additionally, SPADEVizR has several features allowing to quantify and to visualize the quality of the cell clustering results. These analysis features are essential to better interpret the behaviors and phenotypes of the identiï¬ed cell clusters. Importantly, SPADEVizR can handle clustering results from other algorithms than SPADE. Availability and Implementation: SPADEVizR is distributed under the GPL-3 license and is available at https://github.com/tchitchek-lab/SPADEVizR . Contact: nicolas.tchitchek@gmail.com. Supplementary information: Supplementary data are available at Bioinformatics online.
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Citometria de Fluxo/métodos , Software , Algoritmos , Análise por ConglomeradosRESUMO
Broadening our understanding of the abundance and phenotype of B cell subsets that are induced or perturbed by exogenous Ags will improve the vaccine evaluation process. Mass cytometry (CyTOF) is being used to increase the number of markers that can be investigated in single cells, and therefore characterize cell phenotype at an unprecedented level. We designed a panel of CyTOF Abs to compare the B cell response in cynomolgus macaques at baseline, and 8 and 28 d after the second homologous immunization with modified vaccinia virus Ankara. The spanning-tree progression analysis of density-normalized events (SPADE) algorithm was used to identify clusters of CD20(+) B cells. Our data revealed the phenotypic complexity and diversity of circulating B cells at steady-state and significant vaccine-induced changes in the proportions of some B cell clusters. All SPADE clusters, including those altered quantitatively by vaccination, were characterized phenotypically and compared using double hierarchical clustering. Vaccine-altered clusters composed of previously described subsets including CD27(hi)CD21(lo) activated memory and CD27(+)CD21(+) resting memory B cells, and subphenotypes with novel patterns of marker coexpression. The expansion, followed by the contraction, of a single memory B cell SPADE cluster was positively correlated with serum anti-vaccine Ab titers. Similar results were generated by a different algorithm, automatic classification of cellular expression by nonlinear stochastic embedding. In conclusion, we present an in-depth characterization of B cell subphenotypes and proportions, before and after vaccination, using a two-step clustering analysis of CyTOF data, which is suitable for longitudinal studies and B cell subsets and biomarkers discovery.
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Linfócitos B/imunologia , Citometria de Fluxo , Vacinas/imunologia , Animais , Análise por Conglomerados , Macaca fascicularis , Masculino , FenótipoRESUMO
Comparative immune-profiling of innate responses in humans and non-human primates is important to understand the pathogenesis of infectious and chronic inflammatory diseases as well as for the preclinical development of vaccines and immune therapies. However, direct comparisons of the two species are rare and were never performed using mass cytometry. Here, whole-blood-derived leukocytes from healthy humans and cynomolgus macaques were analyzed with mass cytometry. Two similar panels of around 30 monoclonal antibodies targeting human markers associated with innate myeloid cells to stain fixed human and macaque leukocytes were constructed. To compare the circulating innate cells from the two primate species, an analysis pipeline combining a clustering analysis by the Spanning-tree Progression Analysis of Density-normalized Events (SPADE) algorithm with a two-step hierarchical clustering of cells nodes and markers was used. Identical SPADE settings were applied to both datasets, except for the 20 clustering markers which slightly differed. A correlation analysis designed to compare the phenotypes of human and macaque cell nodes and based on 16 markers, including 15 shared clustering markers and CD19 for humans or CD20 for macaques, revealed similarities and differences between staining patterns. This study unique by the number of individuals (26 humans and 5 macaques) and the use of mass cytometry certainly contributes to better assess the advantages and limits of the use of non-human primates in preclinical research. © 2017 International Society for Advancement of Cytometry.
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Imunidade Inata/imunologia , Leucócitos/citologia , Leucócitos/imunologia , Células Mieloides/citologia , Células Mieloides/imunologia , Adulto , Animais , Biomarcadores/metabolismo , Análise por Conglomerados , Feminino , Citometria de Fluxo , Humanos , Leucócitos/metabolismo , Macaca , Masculino , Células Mieloides/metabolismo , FenótipoRESUMO
Pathogens that persist in their host induce immune dysfunctions even in the absence of detectable replication. To better understand the phenotypic and functional changes that persistent infections induce in sentinel innate immune cells, we developed human PBMC-based HIV models of persistent infection. Autologous nonactivated PBMCs were cocultured with chronically infected, acutely infected, or uninfected cells and were then analyzed by unsupervised high-dimensional flow cytometry. Using this approach, we identified prevalent patterns of innate immune dysfunctions associated with persistent HIV infections that at least in part mirror immune dysfunctions observed in patients. In one or more models of chronic infection, bystander CD16+ NK cells expressing markers of activation, such as CD94, CD45RO, CD62L, CD69, CD25, and immune checkpoints PD1, Tim3, TIGIT, NKG2A and Lag3, were significantly reduced. Conversely, helper ILC subsets expressing PDL1/PDL2 were significantly enriched in chronic infection compared with either uninfected or acute infection, suggesting that chronic HIV-1 infection was associated with an inhibitory environment for bystander ILC and NK subsets. The cell-based models of persistent infection that we describe here provide versatile tools to explore the molecular mechanisms of these immune dysfunctions and unveil the contribution of innate immunity in sustaining pathogen persistence.
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Infecções por HIV , Humanos , Infecção Persistente , Imunidade Inata , Leucócitos Mononucleares , Células Matadoras NaturaisRESUMO
Autoimmune disorders have been well characterized over the years and many pathways-but not all of them-have been found to explain their pathophysiology. Autoinflammatory disorders, on the other hand, are still hiding most of their molecular and cellular mechanisms. During the past few years, a newcomer has challenged the idea that only adaptive immunity could display memory response. Trained immunity is defined by innate immune responses that are faster and stronger to a second stimulus than to the first one, being the same or not. In response to the trained immunity inducer, and through metabolic and epigenetic changes of hematopoietic stem and progenitor cells in the bone marrow that are transmitted to their cellular progeny (peripheral trained immunity), or directly of tissue-resident cells (local innate immunity), innate cells responsiveness and functions upon stimulation are improved in the long-term. Innate immunity can be beneficial, but it could also be detrimental when maladaptive. Here, we discuss how trained immunity could contribute to the physiopathology of autoimmune and autoinflammatory diseases.
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Given the time and resources invested in clinical trials, innovative prediction methods are needed to decrease late-stage failure in vaccine development. We identify combinations of early innate responses that predict neutralizing antibody (nAb) responses induced in HIV-Env SOSIP immunized cynomolgus macaques using various routes of vaccine injection and adjuvants. We analyze blood myeloid cells before and 24 h after each immunization by mass cytometry using a three-step clustering, and we discriminate unique vaccine signatures based on HLA-DR, CD39, CD86, CD11b, CD45, CD64, CD14, CD32, CD11c, CD123, CD4, CD16, and CADM1 surface expression. Various combinations of these markers characterize cell families positively associated with nAb production, whereas CADM1-expressing cells are negatively associated (p < 0.05). Our results demonstrate that monitoring immune signatures during early vaccine development could assist in identifying biomarkers that predict vaccine immunogenicity.
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HIV-1 , Animais , Macaca , Subunidade alfa de Receptor de Interleucina-3 , Anticorpos Anti-HIV , Anticorpos NeutralizantesRESUMO
A vaccine is required to effectively control the COVID-19 pandemic in the mid and long term. The development of vaccines against SARS-CoV-2 was initiated as soon as the genetic sequence of the virus was published, and has evolved at an unprecedented speed, with a first clinical trial launched in March 2020. One year later, more than a dozen of vaccines based on different concepts, with some having been evaluated only in clinical trials so far, are authorized under emergency procedures. Here, we review these vaccines, compare their properties and discuss the challenges they face, including the emergence of viral variants of concern.
TITLE: COVID-19, des vaccins à la vitesse de l'éclair. ABSTRACT: Un vaccin est nécessaire pour endiguer efficacement, à moyen et long terme, une pandémie comme celle de la COVID-19 (coronavirus disease 2019). Le développement de vaccins contre le virus responsable de la maladie, le SARS-CoV-2 (severe acute respiratory syndrome-coronavirus 2), a été débuté dès la publication de la séquence du génome viral. Ce développement a progressé à une vitesse sans précédent, avec un premier essai clinique réalisé peu de temps après, en mars 2020. Un an plus tard, une dizaine de vaccins reposant sur des concepts différents, dont certains n'avaient été testés que dans des essais cliniques, sont autorisés dans le cadre de procédures d'urgence. Dans cet article, nous passons en revue ces différents vaccins, nous comparons leurs propriétés et nous discutons les défis auxquels ils sont confrontés, en particulier l'émergence de nouveaux variants viraux.
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Vacinas contra COVID-19/uso terapêutico , COVID-19/prevenção & controle , Desenvolvimento de Medicamentos , SARS-CoV-2/imunologia , Aceleração , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , COVID-19/epidemiologia , Desenvolvimento de Medicamentos/métodos , Desenvolvimento de Medicamentos/organização & administração , Desenvolvimento de Medicamentos/normas , Emergências , História do Século XXI , Humanos , Pandemias/prevenção & controle , Saúde Pública/métodos , Saúde Pública/tendências , Vacinação/métodos , Vacinação/estatística & dados numéricosRESUMO
Most vaccines require multiple doses to induce long-lasting protective immunity in a high frequency of vaccines, and to ensure strong both individual and herd immunity. Repetitive immunogenic stimulations not only increase the intensity and durability of adaptive immunity, but also influence its quality. Several vaccine parameters are known to influence adaptive immune responses, including notably the number of immunizations, the delay between them, and the delivery sequence of different recombinant vaccine vectors. Furthermore, the initial effector innate immune response is key to activate and modulate B and T cell responses. Optimization of homologous and heterologous prime/boost vaccination strategies requires a thorough understanding of how vaccination history affects memory B and T cell characteristics. This requires deeper knowledge of how innate cells respond to multiple vaccine encounters. Here, we review how innate cells, more particularly those of the myeloid lineage, sense and respond differently to a 1st and a 2nd vaccine dose, both in an extrinsic and intrinsic manner. On one hand, the presence of primary specific antibodies and memory T cells, whose critical properties change with time after priming, provides a distinct environment for innate cells at the time of re-vaccination. On the other hand, innate cells themselves can exert enhanced intrinsic antimicrobial functions, long after initial stimulation, which is referred to as trained immunity. We discuss the potential of trained innate cells to be game-changers in prime/boost vaccine strategies. Their increased functionality in antigen uptake, antigen presentation, migration, and as cytokine producers, could indeed improve the restimulation of primary memory B and T cells and their differentiation into potent secondary memory cells in response to the boost. A better understanding of trained immunity mechanisms will be highly valuable for harnessing the full potential of trained innate cells, to optimize immunization strategies.
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Imunidade Adaptativa , Imunização Secundária , Vacinação , Vacinas/imunologia , Animais , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Humanos , Imunidade Humoral , Imunidade Inata , Esquemas de Imunização , Imunização Secundária/métodos , Memória Imunológica , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinação/métodos , Vacinas/administração & dosagemRESUMO
Innate immunity modulates adaptive immunity and defines the magnitude, quality, and longevity of antigen-specific T- and B- cell immune memory. Various vaccine and administration factors influence the immune response to vaccination, including the route of vaccine delivery. We studied the dynamics of innate cell responses in blood using a preclinical model of non-human primates immunized with a live attenuated vaccinia virus, a recombinant Modified vaccinia virus Ankara (MVA) expressing a gag-pol-nef fusion of HIV-1, and mass cytometry. We previously showed that it induces a strong, early, and transient innate response, but also late phenotypic modifications of blood myeloid cells after two months when injected subcutaneously. Here, we show that the early innate effector cell responses and plasma inflammatory cytokine profiles differ between subcutaneous and intradermal vaccine injection. Additionally, we show that the intradermal administration fails to induce more highly activated/mature neutrophils long after immunization, in contrast to subcutaneous administration. Different batches of antibodies, staining protocols and generations of mass cytometers were used to generate the two datasets. Mass cytometry data were analyzed in parallel using the same analytical pipeline based on three successive clustering steps, including SPADE, and categorical heatmaps were compared using the Manhattan distance to measure the similarity between cell cluster phenotypes. Overall, we show that the vaccine per se is not sufficient for the late phenotypic modifications of innate myeloid cells, which are evocative of innate immune training. Its route of administration is also crucial, likely by influencing the early innate response, and systemic inflammation, and vaccine biodistribution.
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Vacinas contra a AIDS , HIV-1 , Neutrófilos/imunologia , Vaccinia virus , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Citocinas/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/genética , HIV-1/imunologia , Macaca fascicularis , Masculino , Vaccinia virus/genética , Vaccinia virus/imunologiaRESUMO
Vaccination is one of the most efficient public healthcare measures to fight infectious diseases. Nevertheless, the immune mechanisms induced in vivo by vaccination are still unclear. The route of administration, an important vaccination parameter, can substantially modify the quality of the response. How the route of administration affects the generation and profile of immune responses is of major interest. Here, we aimed to extensively characterize the profiles of the innate and adaptive response to vaccination induced after intradermal, subcutaneous, or intramuscular administration with a modified vaccinia virus Ankara model vaccine in non-human primates. The adaptive response following subcutaneous immunization was clearly different from that following intradermal or intramuscular immunization. The subcutaneous route induced a higher level of neutralizing antibodies than the intradermal and intramuscular vaccination routes. In contrast, polyfunctional CD8+ T-cell responses were preferentially induced after intradermal or intramuscular injection. We observed the same dichotomy when analyzing the early molecular and cellular immune events, highlighting the recruitment of cell populations, such as CD8+ T lymphocytes and myeloid-derived suppressive cells, and the activation of key immunomodulatory gene pathways. These results demonstrate that the quality of the vaccine response induced by an attenuated vaccine is shaped by early and subtle modifications of the innate immune response. In this immunization context, the route of administration must be tailored to the desired type of protective immune response. This will be achieved through systems vaccinology and mathematical modeling, which will be critical for predicting the efficacy of the vaccination route for personalized medicine.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Supressoras Mieloides/imunologia , Vacinação , Vaccinia virus/imunologia , Vacínia/imunologia , Vacinas Virais/farmacologia , Animais , Injeções Intradérmicas , Injeções Intramusculares , Macaca fascicularis , Masculino , Vacinas Atenuadas/farmacologiaRESUMO
CD4 T cell responses constitute an important component of adaptive immunity and are critical regulators of anti-microbial protection. CD4+ T cells expressing CD32a have been identified as a target for HIV. CD32a is an Fcγ receptor known to be expressed on myeloid cells, granulocytes, B cells and NK cells. Little is known about the biology of CD32+CD4+ T cells. Our goal was to understand the dynamics of CD32+CD4+ T cells in tissues. We analyzed these cells in the blood, lymph nodes, spleen, ileum, jejunum and liver of two nonhuman primate models frequently used in biomedical research: African green monkeys (AGM) and macaques. We studied them in healthy animals and during viral (SIV) infection. We performed phenotypic and transcriptomic analysis at different stages of infection. In addition, we compared CD32+CD4+ T cells in tissues with well-controlled (spleen) and not efficiently controlled (jejunum) SIV replication in AGM. The CD32+CD4+ T cells more frequently expressed markers associated with T cell activation and HIV infection (CCR5, PD-1, CXCR5, CXCR3) and had higher levels of actively transcribed SIV RNA than CD32-CD4+T cells. Furthermore, CD32+CD4+ T cells from lymphoid tissues strongly expressed B-cell-related transcriptomic signatures, and displayed B cell markers at the cell surface, including immunoglobulins CD32+CD4+ T cells were rare in healthy animals and blood but increased strongly in tissues with ongoing viral replication. CD32+CD4+ T cell levels in tissues correlated with viremia. Our results suggest that the tissue environment induced by SIV replication drives the accumulation of these unusual cells with enhanced susceptibility to viral infection.
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Linfócitos B/virologia , Linfócitos T CD4-Positivos/virologia , Tecido Linfoide/virologia , Receptores de IgG/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Replicação Viral , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Chlorocebus aethiops , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Jejuno/imunologia , Jejuno/metabolismo , Jejuno/virologia , Ativação Linfocitária , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Macaca fascicularis , Fenótipo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Vírus da Imunodeficiência Símia/imunologia , Baço/imunologia , Baço/metabolismo , Baço/virologia , Carga ViralRESUMO
Vaccines represent one of the major advances of modern medicine. Despite the many successes of vaccination, continuous efforts to design new vaccines are needed to fight "old" pandemics, such as tuberculosis and malaria, as well as emerging pathogens, such as Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Vaccination aims at reaching sterilizing immunity, however assessing vaccine efficacy is still challenging and underscores the need for a better understanding of immune protective responses. Identifying reliable predictive markers of immunogenicity can help to select and develop promising vaccine candidates during early preclinical studies and can lead to improved, personalized, vaccination strategies. A systems biology approach is increasingly being adopted to address these major challenges using multiple high-dimensional technologies combined with in silico models. Although the goal is to develop predictive models of vaccine efficacy in humans, applying this approach to animal models empowers basic and translational vaccine research. In this review, we provide an overview of vaccine immune signatures in preclinical models, as well as in target human populations. We also discuss high-throughput technologies used to probe vaccine-induced responses, along with data analysis and computational methodologies applied to the predictive modeling of vaccine efficacy.
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AIDS vaccination has a pressing need for more potent vaccination vectors capable of eliciting strong, diversified, and long-lasting cellular immune responses against human immunodeficiency virus (HIV). Lentiviral vectors have demonstrated efficiency not only as gene delivery vehicles for gene therapy applications but also as vaccination tools. This is likely due to their ability to transduce nondividing cells, including dendritic cells, enabling sustained endogenous antigen presentation and thus the induction of high proportions of specific cytotoxic T cells and long-lasting memory T cells. We show in a first proof-of-concept pilot study that a prime/boost vaccination strategy using lentiviral vectors pseudotyped with a glycoprotein G from two non-cross-reactive vesicular stomatitis virus serotypes elicited robust and broad cellular immune responses against the vector-encoded antigen, simian immunodeficiency virus (SIV) GAG, in cynomolgus macaques. Vaccination conferred strong protection against a massive intrarectal challenge with SIVmac251, as evidenced both by the reduction of viremia at the peak of acute infection (a mean of over 2 log(10) fold reduction) and by the full preservation of the CD28(+) CD95(+) memory CD4(+) T cells during the acute phase, a strong correlate of protection against pathogenesis. Although vaccinees continued to display lower viremia than control macaques during the early chronic phase, these differences were not statistically significant by day 50 postchallenge. A not-optimized SIV GAG antigen was chosen to show the strong potential of the lentiviral vector system for vaccination. Given that a stronger protection can be anticipated from a modern HIV-1 antigen design, gene transfer vectors derived from HIV-1 appear as promising candidates for vaccination against HIV-1 infection.
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Vacinas contra a AIDS/imunologia , Síndrome da Imunodeficiência Adquirida , Ácido Aspártico Endopeptidases , Vetores Genéticos , Lentivirus , Vírus da Imunodeficiência Símia/imunologia , Vacinação , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/imunologia , Produtos do Gene gag/administração & dosagem , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Lentivirus/genética , Lentivirus/imunologia , Macaca/imunologia , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Projetos Piloto , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , ViremiaRESUMO
Comprehending the mechanisms behind the impact of vaccine regimens on immunity is critical for improving vaccines. Indeed, the time-interval between immunizations may influence B and T cells, as well as innate responses. We compared two vaccine schedules using cynomolgus macaques immunized with an attenuated vaccinia virus. Two subcutaneous injections 2 weeks apart led to an impaired secondary antibody response and similar innate myeloid responses to both immunizations. In contrast, a delayed boost (2 months) improved the quality of the antibody response and involved more activated/mature innate cells, induced late after the prime and responding to the recall. The magnitude and quality of the secondary antibody response correlated with the abundance of these neutrophils, monocytes, and dendritic cells that were modified phenotypically and enriched prior to revaccination at 2 months, but not 2 weeks. These late phenotypic modifications were associated with an enhanced ex vivo cytokine production (including IL-12/23 and IL-1ß) by PBMCs short after the second immunization, linking phenotype and functions. This integrated analysis reveals a deep impact of the timing between immunizations, and highlights the importance of early but also late innate responses involving phenotypical changes, in shaping humoral immunity.
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Natural killer (NK) cells play essential roles in immunity to viruses and tumors. Their function is genetically determined but also modulated by environmental factors. The distribution and functional regulation of these cells vary depending on the tissue. NK cell behavior in lymphoid tissues is so far understudied. Non-human primate (NHP) models are essential for the development of therapies and vaccines against human diseases, and access to NHP tissues allows insights into spatial regulations of NK cells. Here, we investigated tissue-specific parameters of NK cells from NHP species, i.e., cynomolgus macaque (Macaca fascicularis), African green monkey (Chlorocebus sabaeus), rhesus macaque (Macaca mulatta), and baboon (Papio anubis). By comprehensive multi-dimensional analysis of NK cells from secondary lymphoid organs, intestinal mucosa, liver, and blood, we identified tissue- and species-specific patterns of NK cell frequencies, phenotypes, and potential activity. Also, we defined the tissue-specific characteristics of NK cells during infection by the simian immunodeficiency virus. Altogether, our results provide a comprehensive anatomic analysis of NK cells in different tissues of primates at steady-state and during a viral infection.
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Síndrome da Imunodeficiência Adquirida/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Células Matadoras Naturais/imunologia , Tecido Linfoide/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Células Cultivadas , Humanos , Imunofenotipagem , Receptor 2 Desencadeador da Citotoxicidade Natural/metabolismo , Especificidade de Órgãos , Primatas , Receptores CXCR5/metabolismo , Especificidade da EspécieRESUMO
Dendritic cells (DC), which are involved in orchestrating early immune responses against pathogens, are dysregulated in their function by HIV infection. This dysregulation likely contributes to tip the balance toward viral persistence. Different DC subpopulations, including classical (cDCs) and plasmacytoid (pDCs) dendritic cells, are subjected to concomitant inflammatory and immunoregulatory events during HIV infection, which hampers the precise characterization of their regulation through classical approaches. Here, we carried out mass cytometry analysis of blood samples from early HIV-infected patients that were longitudinally collected before and after 1 year of effective combination antiretroviral therapy (cART). Blood samples from HIV controller patients who naturally control the infection were also included. Our data revealed that plasma HIV RNA level was positively associated with a loss of cDC and pDC subpopulations that display high expression of LILR immunomodulatory receptors. Conversely, specific monocyte populations co-expressing high levels of HLA-I, 3 immunomodulatory receptors, CD64, LILRA2, and LILRB4, and the restriction factor CD317 (also known as BST2/Tetherin), were more abundant in early HIV-infection. Finally, our analysis revealed that the blood of HIV controller patients contained in a higher abundance a particular subtype of CD1c+ cDCs, characterized by elevated co-expression of CD32b inhibitory receptor and HLA-DR antigen-presentation molecules. Overall, this study unravels the modifications induced in DC and monocyte subpopulations in different HIV+ conditions, and provides a better comprehension of the immune regulation/dysregulation mechanisms induced during this viral infection.
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Células Dendríticas/imunologia , Infecções por HIV/imunologia , Monócitos/imunologia , Adulto , Fármacos Anti-HIV/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Feminino , Citometria de Fluxo , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Receptores Imunológicos/efeitos dos fármacos , Receptores Imunológicos/imunologiaRESUMO
A better understanding of innate responses induced by vaccination is critical for designing optimal vaccines. Here, we studied the diversity and dynamics of the NK cell compartment after prime-boost immunization with the modified vaccinia virus Ankara using cynomolgus macaques as a model. Mass cytometry was used to deeply characterize blood NK cells. The NK cell subphenotype composition was modified by the prime. Certain phenotypic changes induced by the prime were maintained over time and, as a result, the NK cell composition prior to boost differed from that before prime. The key phenotypic signature that distinguished NK cells responding to the boost from those responding to the prime included stronger expression of several cytotoxic, homing, and adhesion molecules, suggesting that NK cells at recall were functionally distinct. Our data reveal potential priming or imprinting of NK cells after the first vaccine injection. This study provides novel insights into prime-boost vaccination protocols that could be used to optimize future vaccines.
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Vacinas contra a AIDS/administração & dosagem , HIV/imunologia , Imunização Secundária/métodos , Células Matadoras Naturais/efeitos dos fármacos , Vaccinia virus/imunologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Biomarcadores/metabolismo , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Expressão Gênica , Heterogeneidade Genética , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Esquemas de Imunização , Imunofenotipagem , Injeções Subcutâneas , Células Matadoras Naturais/classificação , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Macaca fascicularis , Masculino , Vacinas AtenuadasRESUMO
HIV-1 directly activates human plasmacytoid DCs (pDCs) by upregulating the expression of costimulatory and MHC molecules and maturation markers, increasing T cell stimulatory activity, and inducing the production of type I interferons and TNF-alpha. A consequence of this activation is the bystander maturation of myeloid DCs and overall enhancement of antigen-presenting function. However, little is known about the mechanism(s) of pDC activation by HIV-1. Here we demonstrate by in vitro studies that IFN-alpha production by pDC in response to HIV-1 requires at least 2 interactions between the cell and virus. Initially, envelope-CD4 interactions mediate endocytosis of HIV-1, as demonstrated through the use of inhibitors of binding, fusion, endocytosis, and endosomal acidification. Subsequently, endosomally delivered viral nucleic acids, particularly RNA, stimulate pDCs through TLRs, as activation is reproduced with purified genomic RNA but not viral RNA packaging-deficient HIV-1 and blocked with different inhibitory TLR ligands. Finally, by using genetic complementation, we show that TLR7 is the likely primary target. Viral RNA rather than DNA in early retrotranscripts appears to be the active factor in HIV-1 that induces IFN-alpha secretion by pDCs. Since the decline in pDCs in chronic HIV-1 infection is associated with high viral loads and opportunistic infections, exploiting this natural adjuvant activity of HIV-1 RNA might be useful in the development of vaccines for the prevention of AIDS.
Assuntos
Células Dendríticas/imunologia , Endocitose/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , RNA Viral/metabolismo , Receptores Toll-Like/metabolismo , Antígenos CD4/metabolismo , Células Cultivadas , Células Dendríticas/virologia , Proteína gp160 do Envelope de HIV/metabolismo , HumanosRESUMO
CD32a has been proposed as a specific marker of latently HIV-infected CD4+ T cells. However, CD32a was recently found to be expressed on CD4+ T cells of healthy donors, leading to controversy on the relevance of this marker in HIV persistence. Here, we used mass cytometry to characterize the landscape and variation in the abundance of CD32a+ CD4+ T cells during HIV infection. To this end, we analyzed CD32a+ CD4+ T cells in primary HIV infection before and after effective combination antiretroviral therapy (cART) and in healthy donors. We found that CD32a+ CD4+ T cells include heterogeneous subsets that are differentially affected by HIV infection. Our analysis revealed that naive (N), central memory (CM), and effector/memory (Eff/Mem) CD32a+ CD4+ T-cell clusters that co-express LILRA2- and CD64-activating receptors were more abundant in primary HIV infection and cART stages. Conversely, LILRA2- CD32a+ CD4+ T-cell clusters of either the TN, TCM, or TEff/Mem phenotype were more abundant in healthy individuals. Finally, an activated CD32a+ CD4+ TEff/Mem cell cluster co-expressing LILRA2, CD57, and NKG2C was more abundant in all HIV stages, particularly during primary HIV infection. Overall, our data show that multiple abundance modifications of CD32a+ CD4+ T-cell subsets occur in the early phase of HIV infection, and some of which are conserved after effective cART. Our study brings a better comprehension of the relationship between CD32a expression and CD4+ T cells during HIV infection.