RESUMO
Coronavirus disease 2019 (COVID-19) exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, pro-inflammatory cytokines, and high anti-receptor binding domain (RBD) antibody levels. Although anti-RBD immunoglobulin G (IgG) levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting cross-protection from reinfection by either strain. However, SARS-CoV-2 sera generally lacked cross-neutralization to a highly homologous pre-emergent bat coronavirus, WIV1-CoV, which has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.
Assuntos
Anticorpos Neutralizantes/imunologia , Biomarcadores/análise , COVID-19/imunologia , COVID-19/fisiopatologia , Adulto , Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Biomarcadores/sangue , COVID-19/sangue , COVID-19/epidemiologia , Comorbidade , Coronavirus/classificação , Coronavirus/fisiologia , Reações Cruzadas , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina A/análise , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Massachusetts/epidemiologia , Pessoa de Meia-Idade , Domínios Proteicos , SARS-CoV-2/química , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/química , Análise de Sobrevida , Resultado do TratamentoRESUMO
CD4+ T lymphocytes are the principal target of human immunodeficiency virus (HIV), but infected macrophages also contribute to viral pathogenesis. The killing of infected cells by CD8+ cytotoxic T lymphocytes (CTLs) leads to control of viral replication. Here we found that the killing of macrophages by CTLs was impaired relative to the killing of CD4+ T cells by CTLs, and this resulted in inefficient suppression of HIV. The killing of macrophages depended on caspase-3 and granzyme B, whereas the rapid killing of CD4+ T cells was caspase independent and did not require granzyme B. Moreover, the impaired killing of macrophages was associated with prolonged effector cell-target cell contact time and higher expression of interferon-γ by CTLs, which induced macrophage production of pro-inflammatory chemokines that recruited monocytes and T cells. Similar results were obtained when macrophages presented other viral antigens, suggestive of a general mechanism for macrophage persistence as antigen-presenting cells that enhance inflammation and adaptive immunity. Inefficient killing of macrophages by CTLs might contribute to chronic inflammation, a hallmark of chronic disease caused by HIV.
Assuntos
Linfócitos T CD4-Positivos/virologia , Citotoxicidade Imunológica/imunologia , Infecções por HIV/imunologia , Macrófagos/virologia , Linfócitos T Citotóxicos/imunologia , Células Cultivadas , HumanosRESUMO
T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Tolerância Imunológica/imunologia , Proteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD/química , Antígenos CD/imunologia , Autoimunidade/imunologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/imunologia , Linhagem Celular , Neoplasias Colorretais/imunologia , Modelos Animais de Doenças , Feminino , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Inflamação/imunologia , Inflamação/patologia , Ligantes , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mucosa/imunologia , Mucosa/patologia , Conformação Proteica , Multimerização Proteica , Receptores Virais/química , Receptores Virais/imunologiaRESUMO
In chronic diseases, such as HIV infection, plasmacytoid dendritic cells (pDCs) are rendered dysfunctional, as measured by their decreased capacity to produce IFN-α. In this study, we identified elevated levels of T cell Ig and mucin-domain containing molecule-3 (Tim-3)-expressing pDCs in the blood of HIV-infected donors. The frequency of Tim-3-expressing pDCs correlated inversely with CD4 T cell counts and positively with HIV viral loads. A lower frequency of pDCs expressing Tim-3 produced IFN-α or TNF-α in response to the TLR7 agonists imiquimod and Sendai virus and to the TLR9 agonist CpG. Thus, Tim-3 may serve as a biomarker of pDC dysfunction in HIV infection. The source and function of Tim-3 was investigated on enriched pDC populations from donors not infected with HIV. Tim-3 induction was achieved in response to viral and artificial stimuli, as well as exogenous IFN-α, and was PI3K dependent. Potent pDC-activating stimuli, such as CpG, imiquimod, and Sendai virus, induced the most Tim-3 expression and subsequent dysfunction. Small interfering RNA knockdown of Tim-3 increased IFN-α secretion in response to activation. Intracellular Tim-3, as measured by confocal microscopy, was dispersed throughout the cytoplasm prior to activation. Postactivation, Tim-3 accumulated at the plasma membrane and associated with disrupted TLR9 at the submembrane. Tim-3-expressing pDCs had reduced IRF7 levels. Furthermore, intracellular Tim-3 colocalized with p85 and IRF7 within LAMP1+ lysosomes, suggestive of a role in degradation. We conclude that Tim-3 is a biomarker of dysfunctional pDCs and may negatively regulate IFN-α, possibly through interference with TLR signaling and recruitment of IRF7 and p85 into lysosomes, enhancing their degradation.
Assuntos
Biomarcadores/análise , Células Dendríticas/imunologia , Infecções por HIV/imunologia , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Transdução de Sinais/imunologia , Adulto , Separação Celular , Células Dendríticas/metabolismo , Feminino , Infecções por HIV/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Fator Regulador 7 de Interferon/imunologia , Fator Regulador 7 de Interferon/metabolismo , Lisossomos/imunologia , Lisossomos/metabolismo , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo , Adulto JovemRESUMO
HIV infection induces phenotypic and functional changes to CD8+ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Receptores Imunológicos/imunologia , Animais , Antígeno B7-H1/imunologia , Separação Celular , DNA Viral/análise , Progressão da Doença , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Macaca mulatta , RNA Viral/análise , Síndrome de Imunodeficiência Adquirida dos Símios/imunologiaRESUMO
UNLABELLED: Chronic HIV infection results in a loss of HIV-specific CD8(+) T cell effector function, termed "exhaustion," which is mediated, in part, by the membrane coinhibitory receptor T cell immunoglobulin mucin domain-3 (Tim-3). Like many other receptors, a soluble form of this protein has been described in human blood plasma. However, soluble Tim-3 (sTim-3) is poorly characterized, and its role in HIV disease is unknown. Here, we show that Tim-3 is shed from the surface of responding CD8(+) T cells by the matrix metalloproteinase ADAM10, producing a soluble form of the coinhibitory receptor. Despite previous reports in the mouse model, no alternatively spliced, soluble form of Tim-3 was observed in humans. Shed sTim-3 was found in human plasma and was significantly elevated during early and chronic untreated HIV infection, but it was not found differentially modulated in highly active antiretroviral therapy (HAART)-treated HIV-infected subjects or in elite controllers compared to HIV-uninfected subjects. Plasma sTim-3 levels were positively correlated with HIV load and negatively correlated with CD4 counts. Thus, plasma sTim-3 shedding correlated with HIV disease progression. Despite these correlations, we found that shedding Tim-3 did not improve the function of CD8(+) T cells in terms of gamma interferon production or prevent their apoptosis through galectin-9. Further characterization studies of sTim-3 function are needed to understand the contribution of sTim-3 in HIV disease pathogenesis, with implications for novel therapeutic interventions. IMPORTANCE: Despite the overall success of HAART in slowing the progression to AIDS in HIV-infected subjects, chronic immune activation and T cell exhaustion contribute to the eventual deterioration of the immune system. Understanding these processes will aid in the development of interventions and therapeutics to be used in combination with HAART to slow or reverse this deterioration. Here, we show that a soluble form of T cell exhaustion associated coinhibitory molecule 3, sTim-3, is shed from the surface of T cells. Furthermore, sTim-3 is elevated in the plasma of treatment-naive subjects with acute or chronic HIV infection and is associated with markers of disease progression. This is the first study to characterize sTim-3 in human plasma, its source, and mechanism of production. While it is still unclear whether sTim-3 contributes to HIV pathogenesis, sTim-3 may represent a new correlate of HIV disease progression.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Biomarcadores/sangue , Linfócitos T CD8-Positivos/metabolismo , Infecções por HIV/diagnóstico , Infecções por HIV/patologia , Proteínas de Membrana/sangue , Proteínas de Membrana/metabolismo , Plasma/química , Proteína ADAM10 , Contagem de Linfócito CD4 , Estudos de Coortes , Progressão da Doença , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Estudos Prospectivos , Carga ViralRESUMO
CD8(+) CTLs are adept at killing virally infected cells and cancer cells and releasing cytokines (e.g., IFN-γ) to aid this response. However, during cancer and chronic viral infections, such as with HIV, this CTL response is progressively impaired due to a process called T cell exhaustion. Previous work has shown that the glycoprotein T cell Ig and mucin domain-containing protein 3 (Tim-3) plays a functional role in establishing T cell exhaustion. Tim-3 is highly upregulated on virus and tumor Ag-specific CD8(+) T cells, and antagonizing Tim-3 helps restore function of CD8(+) T cells. However, very little is known of how Tim-3 signals in CTLs. In this study, we assessed the role of Tim-3 at the immunological synapse as well as its interaction with proximal TCR signaling molecules in primary human CD8(+) T cells. Tim-3 was found within CD8(+) T cell lipid rafts at the immunological synapse. Blocking Tim-3 resulted in a significantly greater number of stable synapses being formed between Tim-3(hi)CD8(+) T cells and target cells, suggesting that Tim-3 plays a functional role in synapse formation. Further, we confirmed that Tim-3 interacts with Lck, but not the phospho-active form of Lck. Finally, Tim-3 colocalizes with receptor phosphatases CD45 and CD148, an interaction that is enhanced in the presence of the Tim-3 ligand, galectin-9. Thus, Tim-3 interacts with multiple signaling molecules at the immunological synapse, and characterizing these interactions could aid in the development of therapeutics to restore Tim-3-mediated immune dysfunction.
Assuntos
Proteínas de Membrana/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Sinapses/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Galectinas/metabolismo , Infecções por HIV/metabolismo , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Antígenos Comuns de Leucócito/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The T cell Ig- and mucin domain-containing molecule-3 (Tim-3) negative immune checkpoint receptor demarcates functionally exhausted CD8(+) T cells arising from chronic stimulation in viral infections like HIV. Tim-3 blockade leads to improved antiviral CD8(+) T cell responses in vitro and, therefore, represents a novel intervention strategy to restore T cell function in vivo and protect from disease progression. However, the Tim-3 pathway in the physiologically relevant rhesus macaque SIV model of AIDS remains uncharacterized. We report that Tim-3(+)CD8(+) T cell frequencies are significantly increased in lymph nodes, but not in peripheral blood, in SIV-infected animals. Tim-3(+)PD-1(+)CD8(+) T cells are similarly increased during SIV infection and positively correlate with SIV plasma viremia. Tim-3 expression was found primarily on effector memory CD8(+) T cells in all tissues examined. Tim-3(+)CD8(+) T cells have lower Ki-67 content and minimal cytokine responses to SIV compared with Tim-3(-)CD8(+) T cells. During acute-phase SIV replication, Tim-3 expression peaked on SIV-specific CD8(+) T cells by 2 wk postinfection and then rapidly diminished, irrespective of mutational escape of cognate Ag, suggesting non-TCR-driven mechanisms for Tim-3 expression. Thus, rhesus Tim-3 in SIV infection partially mimics human Tim-3 in HIV infection and may serve as a novel model for targeted studies focused on rejuvenating HIV-specific CD8(+) T cell responses.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunoterapia/métodos , Proteínas de Membrana/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Síndrome da Imunodeficiência Adquirida/terapia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/virologia , Proliferação de Células , Células Cultivadas , Citotoxicidade Imunológica , Modelos Animais de Doenças , Regulação da Expressão Gênica , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Memória Imunológica , Macaca mulatta , Proteínas de Membrana/genética , Dados de Sequência Molecular , Terapia de Alvo Molecular , Receptor de Morte Celular Programada 1/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Carga Viral , Replicação ViralRESUMO
PURPOSE OF REVIEW: Despite decades of insights about how CD8 + T cells and natural killer (NK) cells contribute to natural control of infection, additional hurdles (mutational escape from cellular immunity, sequence diversity, and hard-to-access tissue reservoirs) will need to be overcome to develop a cure. In this review, we highlight recent findings of novel mechanisms of antiviral cellular immunity and discuss current strategies for therapeutic deisgn. RECENT FINDINGS: Of note are the apparent converging roles of viral antigen-specific MHC-E-restricted CD8 + T cells and NK cells, interleukin (IL)-15 biologics to boost cytotoxicity, and broadly neutralizing antibodies in their native form or as anitbody fragments to neutralize virus and engage cellular immunity, respectively. Finally, renewed interest in myeloid cells as relevant viral reservoirs is an encouraging sign for designing inclusive therapeutic strategies. SUMMARY: Several studies have shown promise in many preclinical models of disease, including simian immunodeficiency virus (SIV)/SHIV infection in nonhuman primates and HIV infection in humanized mice. However, each model comes with its own limitations and may not fully predict human responses. We eagerly await the results of clinical trails assessing the efficacy of these strategies to achieve reductions in viral reservoirs, delay viral rebound, or ultimately elicit immune based control of infection without combination antiretroviral therapy (cART).
Assuntos
Infecções por HIV , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Humanos , Camundongos , Vírus da Imunodeficiência Símia/fisiologia , Macaca mulatta , Modelos Animais de Doenças , Carga ViralRESUMO
Introduction: Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined. Methods: We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches. Results: We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 - ULBP1 complex indicates some similarities to the m152-RAE1γ complex.
Assuntos
Proteínas Ligadas por GPI , Herpesvirus Humano 6 , Células Matadoras Naturais , Ativação Linfocitária , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Herpesvirus Humano 6/imunologia , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Ativação Linfocitária/imunologia , Ligação Proteica , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Durable cellular immunity against pathogens is dependent upon a coordinated recall response to antigen by memory CD8+ T cells, involving their proliferation and the generation of secondary cytotoxic effector cells. Conventional assays measuring ex vivo cytotoxicity fail to capture this secondary cytolytic potential, especially in settings where cells have not been recently exposed to their cognate antigen in vivo. Here we describe the expanded antigen-specific elimination assay (EASEA), a flow cytometric endpoint assay to measure the capacity of human CD8+ T cells to expand in vitro upon antigen re-exposure and generate secondary effector cells capable of selectively eliminating autologous antigen-pulsed target cells across a range of effector-to-target ratios. Unlike alternative assays, EASEA avoids the hazards of radioactive labeling and viral infection and can be used to study responses to individual or pooled antigens of interest. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Expanded antigen-specific elimination assay.
Assuntos
Linfócitos T CD8-Positivos , Citometria de Fluxo , Humanos , Linfócitos T CD8-Positivos/imunologia , Citometria de Fluxo/métodos , Antígenos/imunologia , Citotoxicidade ImunológicaRESUMO
Natural killer (NK) cells are innate cytolytic effectors that target HIV-infected CD4+ T cells. In conjunction with antibodies recognizing the HIV envelope, NK cells also eliminate HIV-infected targets through antibody-dependent cellular cytotoxicity (ADCC). However, how these NK cell functions impact infected macrophages is less understood. We show that HIV-infected macrophages resist NK cell-mediated killing. Compared with HIV-infected CD4+ T cells, initial innate NK cell interactions with HIV-infected macrophages skew the response toward cytokine production, rather than release of cytolytic contents, causing inefficient elimination of infected macrophages. Studies with chimeric antigen receptor (CAR) T cells demonstrate that the viral envelope is equally accessible on CD4+ T cells and macrophages. Nonetheless, ADCC against macrophages is muted compared with ADCC against CD4+ T cells. Thus, HIV-infected macrophages employ mechanisms to evade immediate cytolytic NK cell function while preserving inflammatory cytokine responses. These findings emphasize the importance of eliminating infected macrophages for HIV cure efforts.
Assuntos
Citocinas/metabolismo , Infecções por HIV/imunologia , Células Matadoras Naturais/imunologia , Macrófagos/virologia , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1 , HumanosRESUMO
Quantification of cell-secreted molecules, e.g., cytokines, is fundamental to the characterization of immune responses. Cytokine capture assays that use engineered antibodies to anchor the secreted molecules to the secreting cells are widely used to characterize immune responses because they allow both sensitive identification and recovery of viable responding cells. However, if the cytokines diffuse away from the secreting cells, non-secreting cells will also be identified as responding cells. Here we encapsulate immune cells in microfluidic droplets and perform in-droplet cytokine capture assays to limit the diffusion of the secreted cytokines. We use microfluidic devices to rapidly encapsulate single natural killer NK-92 MI cells and their target K562 cells into microfluidic droplets. We perform in-droplet IFN-γ capture assays and demonstrate that NK-92 MI cells recognize target cells within droplets and become activated to secrete IFN-γ. Droplet encapsulation prevents diffusion of secreted products to neighboring cells and dramatically reduces both false positives and false negatives, relative to assays performed without droplets. In a sample containing 1% true positives, encapsulation reduces, from 94% to 2%, the number of true-positive cells appearing as negatives; in a sample containing 50% true positives, the number of non-stimulated cells appearing as positives is reduced from 98% to 1%. After cells are released from the droplets, secreted cytokine remains captured onto secreting immune cells, enabling FACS-isolation of populations highly enriched for activated effector immune cells. Droplet encapsulation can be used to reduce background and improve detection of any single-cell secretion assay.
Assuntos
Dispositivos Lab-On-A-Chip , Microfluídica , Citocinas , Células Matadoras NaturaisRESUMO
COVID-19 exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, and high anti-RBD antibody levels. While anti-RBD IgG levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting protection from reinfection by this strain. However, SARS-CoV-2 sera was unable to cross-neutralize a highly-homologous pre-emergent bat coronavirus, WIV1-CoV, that has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.
RESUMO
Understanding and reshaping cellular behaviors with synthetic gene networks requires the ability to sense and respond to changes in the intracellular environment. Intracellular proteins are involved in almost all cellular processes, and thus can provide important information about changes in cellular conditions such as infections, mutations, or disease states. Here we report the design of a modular platform for intrabody-based protein sensing-actuation devices with transcriptional output triggered by detection of intracellular proteins in mammalian cells. We demonstrate reporter activation response (fluorescence, apoptotic gene) to proteins involved in hepatitis C virus (HCV) infection, human immunodeficiency virus (HIV) infection, and Huntington's disease, and show sensor-based interference with HIV-1 downregulation of HLA-I in infected T cells. Our method provides a means to link varying cellular conditions with robust control of cellular behavior for scientific and therapeutic applications.
Assuntos
Proteínas Reguladoras de Apoptose/análise , Técnicas Biossensoriais , Redes Reguladoras de Genes , Engenharia Genética/métodos , Hepacivirus/genética , Proteína Huntingtina/análise , Anticorpos/química , Anticorpos/metabolismo , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endopeptidases/química , Genes Reporter , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Antígenos HLA/genética , Antígenos HLA/imunologia , Hepacivirus/metabolismo , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Células Jurkat , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Imagem Óptica/métodos , Plasmídeos/química , Plasmídeos/metabolismoRESUMO
Although CD4+ T cells represent the major reservoir of persistent HIV and SIV infection, accumulating evidence suggests that macrophages also contribute. However, investigations of the role of macrophages are often underrepresented at HIV pathogenesis and cure meetings. This was the impetus for a scientific workshop dedicated to this area of study, held in Cambridge, MA in January 2017. The workshop brought together experts in the fields of HIV/SIV immunology and virology, macrophage biology and immunology, and animal models of HIV/SIV infection to discuss the role of macrophages as a physiologically relevant viral reservoir, and the implications of macrophage infection for HIV pathogenesis and strategies for cure. While still controversial, there is an emerging theory that infected macrophages likely persist in the setting of combination antiretroviral therapy. These macrophages could then drive persistent inflammation and contribute to the viral reservoir, which indicates the importance of addressing macrophages as well as CD4+ T cells with future therapeutic strategies.