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1.
Cell ; 184(17): 4401-4413.e10, 2021 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-34265281

RESUMEN

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape convalescent and vaccine-induced antibody responses has renewed focus on the development of broadly protective T-cell-based vaccines. Here, we apply structure-based network analysis and assessments of HLA class I peptide stability to define mutationally constrained CD8+ T cell epitopes across the SARS-CoV-2 proteome. Highly networked residues are conserved temporally among circulating variants and sarbecoviruses and disproportionately impair spike pseudotyped lentivirus infectivity when mutated. Evaluation of HLA class I stabilizing activity for 18 globally prevalent alleles identifies CD8+ T cell epitopes within highly networked regions with limited mutational frequencies in circulating SARS-CoV-2 variants and deep-sequenced primary isolates. Moreover, these epitopes elicit demonstrable CD8+ T cell reactivity in convalescent individuals but reduced recognition in recipients of mRNA-based vaccines. These data thereby elucidate key mutationally constrained regions and immunogenic epitopes in the SARS-CoV-2 proteome for a global T-cell-based vaccine against emerging variants and SARS-like coronaviruses.


Asunto(s)
Vacunas contra la COVID-19/inmunología , Epítopos de Linfocito T , Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Vacunas contra la COVID-19/química , Antígenos HLA/inmunología , Humanos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo
2.
Cell ; 183(1): 143-157.e13, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32877699

RESUMEN

Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.


Asunto(s)
Infecciones por Coronavirus/inmunología , Centro Germinal/inmunología , Neumonía Viral/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Anciano , Anciano de 80 o más Años , Linfocitos B/inmunología , COVID-19 , Femenino , Centro Germinal/patología , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Bazo/inmunología , Bazo/patología , Factor de Necrosis Tumoral alfa/metabolismo
3.
Immunity ; 54(12): 2859-2876.e7, 2021 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-34788599

RESUMEN

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Epítopos/inmunología , Vacunas contra la Malaria/inmunología , Malaria/inmunología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/inmunología , Vacunas de ADN/inmunología , Traslado Adoptivo , Animales , Anticuerpos Antiprotozoarios/metabolismo , Modelos Animales de Enfermedad , Epítopos/genética , Ingeniería Genética , Humanos , Evasión Inmune , Inmunogenicidad Vacunal , Ratones , Ratones SCID , Proteínas Protozoarias/genética , Relación Estructura-Actividad , Vacunación
4.
Immunity ; 47(4): 648-663.e8, 2017 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-29045899

RESUMEN

Distinct molecular pathways govern the differentiation of CD8+ effector T cells into memory or exhausted T cells during acute and chronic viral infection, but these are not well studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus-specific CD8+ T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that was linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex, and the presence of HCV-specific CD4+ T cell populations. These early changes in HCV-specific CD8+ T cell transcription preceded the overt establishment of T cell exhaustion, making this signature a prime target in the search for the regulatory origins of T cell dysfunction in chronic viral infection.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Hepatitis C Crónica/inmunología , Transcripción Genética/inmunología , Enfermedad Aguda , Inmunidad Adaptativa/genética , Inmunidad Adaptativa/inmunología , Adulto , Anciano , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/inmunología , Variación Genética/inmunología , Hepacivirus/fisiología , Hepatitis C Crónica/genética , Hepatitis C Crónica/virología , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Factores de Tiempo , Adulto Joven
5.
Immunity ; 41(6): 1001-12, 2014 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-25526311

RESUMEN

Decreased HIV-specific CD8(+) T cell proliferation is a hallmark of chronic infection, but the mechanisms of decline are unclear. We analyzed gene expression profiles from antigen-stimulated HIV-specific CD8(+) T cells from patients with controlled and uncontrolled infection and identified caspase-8 as a correlate of dysfunctional CD8(+) T cell proliferation. Caspase-8 activity was upregulated in HIV-specific CD8(+) T cells from progressors and correlated positively with disease progression and programmed cell death-1 (PD-1) expression, but negatively with proliferation. In addition, progressor cells displayed a decreased ability to upregulate membrane-associated caspase-8 activity and increased necrotic cell death following antigenic stimulation, implicating the programmed cell death pathway necroptosis. In vitro necroptosis blockade rescued HIV-specific CD8(+) T cell proliferation in progressors, as did silencing of necroptosis mediator RIPK3. Thus, chronic stimulation leading to upregulated caspase-8 activity contributes to dysfunctional HIV-specific CD8(+) T cell proliferation through activation of necroptosis and increased cell death.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Caspasa 8/metabolismo , Infecciones por VIH/inmunología , VIH/fisiología , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T CD8-positivos/virología , Proliferación Celular/genética , Células Cultivadas , Progresión de la Enfermedad , Activación Enzimática , Regulación de la Expresión Génica , Proteína p24 del Núcleo del VIH/inmunología , Humanos , Necrosis , Fragmentos de Péptidos/inmunología , Receptor de Muerte Celular Programada 1/genética , ARN Interferente Pequeño/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transcriptoma , Carga Viral
6.
Clin Immunol ; 237: 108991, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35364330

RESUMEN

Many studies have been performed in severe COVID-19 on immune cells in the circulation and on cells obtained by bronchoalveolar lavage. Most studies have tended to provide relative information rather than a quantitative view, and it is a combination of approaches by various groups that is helping the field build a picture of the mechanisms that drive severe lung disease. Approaches employed to date have not revealed information on lung parenchymal T cell subsets in severe COVID-19. Therefore, we sought to examine early and late T cell subset alterations in the lungs and draining lymph nodes in severe COVID-19 using a rapid autopsy protocol and quantitative imaging approaches. Here, we have established that cytotoxic CD4+ T cells (CD4 + CTLs) increase in the lungs, draining lymph nodes and blood as COVID-19 progresses. CD4 + CTLs are prominently expanded in the lung parenchyma in severe COVID-19. In contrast CD8+ T cells are not prominent, exhibit increased PD-1 expression, and no obvious increase is seen in the number of Granzyme B+ CD8+ T cells in the lung parenchyma in severe COVID-19. Based on quantitative evidence for re-activation in the lung milieu, CD4 + CTLs may be as likely to drive viral clearance as CD8+ T cells and may also be contributors to lung inflammation and eventually to fibrosis in severe COVID-19.


Asunto(s)
Linfocitos T CD4-Positivos , COVID-19 , Linfocitos T CD8-positivos , Humanos , Pulmón , Subgrupos de Linfocitos T , Linfocitos T Citotóxicos
7.
Nature ; 487(7407): 325-9, 2012 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-22763456

RESUMEN

During progression of atherosclerosis, myeloid cells destabilize lipid-rich plaques in the arterial wall and cause their rupture, thus triggering myocardial infarction and stroke. Survivors of acute coronary syndromes have a high risk of recurrent events for unknown reasons. Here we show that the systemic response to ischaemic injury aggravates chronic atherosclerosis. After myocardial infarction or stroke, Apoe-/- mice developed larger atherosclerotic lesions with a more advanced morphology. This disease acceleration persisted over many weeks and was associated with markedly increased monocyte recruitment. Seeking the source of surplus monocytes in plaques, we found that myocardial infarction liberated haematopoietic stem and progenitor cells from bone marrow niches via sympathetic nervous system signalling. The progenitors then seeded the spleen, yielding a sustained boost in monocyte production. These observations provide new mechanistic insight into atherogenesis and provide a novel therapeutic opportunity to mitigate disease progression.


Asunto(s)
Aterosclerosis/etiología , Aterosclerosis/patología , Infarto del Miocardio/complicaciones , Infarto del Miocardio/patología , Animales , Apolipoproteínas E/genética , Células Madre Hematopoyéticas/citología , Inflamación/complicaciones , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Bazo/citología , Células Madre/citología
8.
Cytometry A ; 87(8): 784-9, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26012776

RESUMEN

Understanding aerosols produced by cell sorting is critical to biosafety risk assessment and validation of containment efficiency. In this study an Optical Airborne Particle Counter was used to analyze aerosols produced by the BD FACSAria and to assess the effectiveness of its aerosol containment. The suitability of using this device to validate containment was directly compared to the Glo-Germ method put forth by the International Society for Advancement of Cytometry (ISAC) as a standard for testing. It was found that high concentrations of aerosols ranging from 0.3 µm to 10 µm can be detected in failure mode, with most less than 5 µm. In most cases, while numerous aerosols smaller than 5 µm were detected by the Optical Airborne Particle Counter, no Glo-Germ particles were detected, indicating that small aerosols are under-evaluated by the Glo-Germ method. The results demonstrate that the Optical Airborne Particle Counter offers a rapid, economic, and quantitative analysis of cell sorter aerosols and represents an improved method over Glo-Germ for the task of routine validation and monitoring of aerosol containment for cell sorting.


Asunto(s)
Aerosoles/análisis , Contención de Riesgos Biológicos/instrumentación , Diseño de Equipo/instrumentación , Citometría de Flujo/instrumentación , Sustancias Peligrosas/análisis , Contaminación de Equipos/prevención & control
9.
Nature ; 443(7110): 421-6, 2006 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-16957735

RESUMEN

Stem-cell ageing is thought to contribute to altered tissue maintenance and repair. Older humans experience increased bone marrow failure and poorer haematologic tolerance of cytotoxic injury. Haematopoietic stem cells (HSCs) in older mice have decreased per-cell repopulating activity, self-renewal and homing abilities, myeloid skewing of differentiation, and increased apoptosis with stress. Here we report that the cyclin-dependent kinase inhibitor p16INK4a, the level of which was previously noted to increase in other cell types with age, accumulates and modulates specific age-associated HSC functions. Notably, in the absence of p16INK4a, HSC repopulating defects and apoptosis were mitigated, improving the stress tolerance of cells and the survival of animals in successive transplants, a stem-cell-autonomous tissue regeneration model. Inhibition of p16INK4a may ameliorate the physiological impact of ageing on stem cells and thereby improve injury repair in aged tissue.


Asunto(s)
Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Envejecimiento , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Trasplante de Médula Ósea , Recuento de Células , Proliferación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Factor de Transcripción HES-1
10.
Blood ; 114(2): 346-56, 2009 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-19365081

RESUMEN

Murine models indicate that interleukin-10 (IL-10) can suppress viral clearance, and interventional blockade of IL-10 activity has been proposed to enhance immunity in chronic viral infections. Increased IL-10 levels have been observed during HIV infection and IL-10 blockade has been shown to enhance T-cell function in some HIV-infected subjects. However, the categories of individuals in whom the IL-10 pathway is up-regulated are poorly defined, and the cellular sources of IL-10 in these subjects remain to be determined. Here we report that blockade of the IL-10 pathway augmented in vitro proliferative capacity of HIV-specific CD4 and CD8 T cells in individuals with ongoing viral replication. IL-10 blockade also increased cytokine secretion by HIV-specific CD4 T cells. Spontaneous IL-10 expression, measured as either plasma IL-10 protein or IL-10 mRNA in peripheral blood mononuclear cells (PBMCs), correlated positively with viral load and diminished after successful antiretroviral therapy. IL-10 mRNA levels were up-regulated in multiple PBMC subsets in HIV-infected subjects compared with HIV-negative controls, particularly in T, B, and natural killer (NK) cells, whereas monocytes were a major source of IL-10 mRNA in HIV-infected and -uninfected individuals. These data indicate that multiple cell types contribute to IL-10-mediated immune suppression in the presence of uncontrolled HIV viremia.


Asunto(s)
Infecciones por VIH/inmunología , Interleucina-10/inmunología , Linfocitos T/inmunología , Regulación hacia Arriba/inmunología , Viremia/inmunología , Proliferación Celular , Células Cultivadas , Enfermedad , Regulación de la Expresión Génica , Infecciones por VIH/complicaciones , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , ARN Mensajero/genética , Receptores de Interleucina-10/inmunología , Transducción de Señal/inmunología , Linfocitos T/citología , Linfocitos T/metabolismo , Viremia/complicaciones , Viremia/genética , Viremia/metabolismo
11.
medRxiv ; 2021 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-33791730

RESUMEN

The contributions of T cells infiltrating the lungs to SARS-CoV-2 clearance and disease progression are poorly understood. Although studies of CD8+ T cells in bronchoalveolar lavage and blood have suggested that these cells are exhausted in severe COVID-19, CD4+ T cells have not been systematically interrogated within the lung parenchyma. We establish here that cytotoxic CD4+ T cells (CD4+CTLs) are prominently expanded in the COVID-19 lung infiltrate. CD4+CTL numbers in the lung increase with disease severity and progression is accompanied by widespread HLA-DR expression on lung epithelial and endothelial cells, increased apoptosis of epithelial cells and tissue remodeling. Based on quantitative evidence for re-activation in the lung milieu, CD4+ CTLs are as likely to drive viral clearance as CD8+ T cells and may also be contributors to lung inflammation and eventually to fibrosis in severe COVID-19. IN BRIEF: In severe COVID-19 cytotoxic CD4+ T cells accumulate in draining lymph nodes and in the lungs during the resolving phase of the disease. Re-activated cytotoxic CD4+ T cells and cytotoxic CD8+ T cells are present in roughly equivalent numbers in the lungs at this stage and these cells likely collaborate to eliminate virally infected cells and potentially induce fibrosis. A large fraction of epithelial and endothelial cells in the lung express HLA class II in COVID-19 and there is temporal convergence between CD4+CTL accumulation and apoptosis in the lung. HIGHLIGHTS: In severe COVID-19, activated CD4+ CTLs accumulate in the lungs late in diseaseThese cells likely participate in SARS-CoV-2 clearance, collaborating with CD8+ T cells many of which exhibit an exhausted phenotypeT cells likely contribute to the late exacerbation of inflammationCD4+CTLs have been linked to fibrosis in many disorders and could also be responsible for the eventual induction of fibrosis in a subset of COVID-19 patients. SUMMARY: The contributions of T cells infiltrating the lungs to SARS-CoV-2 clearance and disease progression are poorly understood. Although studies of CD8+ T cells in bronchoalveolar lavage and blood have suggested that these cells are exhausted in severe COVID-19, CD4+ T cells have not been systematically interrogated within the lung parenchyma. We establish here that cytotoxic CD4+ T cells (CD4+CTLs) are prominently expanded in the COVID-19 lung infiltrate. CD4+CTL numbers in the lung increase with disease severity and progression is accompanied by widespread HLA-DR expression on lung epithelial and endothelial cells, increased apoptosis of epithelial cells and tissue remodeling. Based on quantitative evidence for re-activation in the lung milieu, CD4+ CTLs are as likely to drive viral clearance as CD8+ T cells and may also be contributors to lung inflammation and eventually to fibrosis in severe COVID-19.

12.
Cell Rep ; 36(2): 109378, 2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34260940

RESUMEN

Defining factors that govern CD8+ T cell immunodominance is critical for the rational design of vaccines for viral pathogens. Here, we assess the contribution of human leukocyte antigen (HLA) class-I-peptide stability for 186 optimal HIV epitopes across 18 HLA alleles using transporter associated with antigen processing (TAP)-deficient mono-allelic HLA-expressing cell lines. We find that immunodominant HIV epitopes increase surface stabilization of HLA class-I molecules in comparison to subdominant epitopes. HLA class-I-peptide stability is also strongly correlated with overall immunodominance hierarchies, particularly for epitopes from high-abundance proteins (e.g., Gag). Moreover, HLA alleles associated with HIV protection are preferentially stabilized by epitopes derived from topologically important viral regions at a greater frequency than neutral and risk alleles. These findings indicate that relative stabilization of HLA class-I is a key factor for CD8+ T cell epitope immunodominance hierarchies, with implications for HIV control and the design of T-cell-based vaccines.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Epítopos Inmunodominantes/inmunología , Péptidos/inmunología , Alelos , Femenino , Células HEK293 , Humanos , Desnaturalización Proteica , Estabilidad Proteica , Propiedades de Superficie
13.
Blood ; 112(9): 3679-87, 2008 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-18728248

RESUMEN

Exhaustion of virus-specific T cells may play an important role in the pathophysiology of chronic viral infections. Here, we analyzed telomere length and telomerase activity in HIV-1-specific CD8+ T cells from progressors or controllers to determine underlying molecular pathways of T-cell exhaustion and senescence. Telomere lengths of HIV-1-specific CD8+ T cells from progressors were significantly shorter compared with autologous cytomegalovirus (CMV)/Epstein-Barr virus (EBV)-specific CD8+ T cells or bulk CD8+ T cells, while telomere lengths from controllers significantly exceeded those of autologous bulk CD8+ T cells and reached a similar level as HIV-1-specific CD8+ T cells collected during primary HIV-1 infection. Telomere length stabilization in controllers corresponded to high levels of constitutive telomerase activity, which was associated with preservation of cytotoxic and proliferative properties. Conversely, limited constitutive telomerase activity was observed in HIV-1-specific CD8+ T cells from progressors, although an increase in both telomere length and telomerase activity was achieved in antigenic-peptide-stimulated cells from progressors after blocking the PD-1/PD ligand 1 (PD-L1) pathway. Collectively, these data suggest a causal role of telomere shortening for the functional deficiencies of HIV-1-specific CD8+ T cells in chronic progressive infection, while high constitutive telomerase activities appears to contribute to maintenance of polyfunctional HIV-1-specific CD8+ T cells from HIV-1 controllers.


Asunto(s)
Linfocitos T CD8-positivos/enzimología , Infecciones por VIH/enzimología , Sobrevivientes de VIH a Largo Plazo , VIH-1 , Telomerasa/metabolismo , Adulto , Antígenos CD/metabolismo , Apoptosis , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Proliferación Celular , Citotoxicidad Inmunológica , Infecciones por VIH/inmunología , Infecciones por VIH/patología , VIH-1/inmunología , Humanos , Técnicas In Vitro , Ligandos , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1 , Telómero/patología
14.
SSRN ; : 3652322, 2020 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-32742244

RESUMEN

Humoral responses in COVID-19 disease are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined postmortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers, a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+TFH cell differentiation together with an increase in T-bet+TH1 cells and aberrant extra-follicular TNF-a accumulation.  Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections and suggest that achieving herd immunity through natural infection may be difficult. Funding: This work was supported by NIH U19 AI110495 to SP, NIH R01 AI146779 to AGS, NIH R01AI137057 and DP2DA042422 to DL, BMH was supported by NIGMS T32 GM007753, TMC was supported by T32 AI007245. Funding for these studies from the Massachusetts Consortium of Pathogen Readiness, the Mark and Lisa Schwartz Foundation and Enid Schwartz is also acknowledged. Conflict of Interest: None. Ethical Approval: This study was performed with the approval of the Institutional Review Boards at the Massachusetts General Hospital and the Brigham and Women's Hospital.

15.
J Immunol Methods ; 333(1-2): 115-25, 2008 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-18314131

RESUMEN

T helper (Th) 17 cells are a distinct lineage of CD4+ T cells mediating tissue inflammation through the secretion of IL-17. In addition, it has been shown that the expression of the transcriptional factor RORgammat is responsible for the induction and maintenance of this cell line. Th17 cells are believed to be involved in a variety of autoimmune disorders, but may also play an important role in host defense. Here we describe a novel technique to reproducibly isolate viable Th17 cells based on their IL-17 secreting ability. We confirmed Th17 cell enrichment by quantitative PCR analysis and demonstrate that positively selected cells using this technique express significantly increased mRNA levels of RORgammat, IL-23 receptor and CCR4 when compared to negatively selected cells. Furthermore, we show that purified Th17 cells can be maintained in long-term culture and expand in vitro. In conclusion, this technique will allow for the first time the direct, ex vivo analysis of phenotypic and functional properties of Th17 cells.


Asunto(s)
Interleucina-17/inmunología , Linfocitos T Colaboradores-Inductores/citología , Anticuerpos/química , Anticuerpos/inmunología , Técnicas de Cultivo de Célula/métodos , Citometría de Flujo/métodos , Perfilación de la Expresión Génica , Humanos , Separación Inmunomagnética/métodos , Interleucina-17/biosíntesis , Interleucina-17/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores CCR4/biosíntesis , Receptores CCR4/genética , Receptores de Interleucina/biosíntesis , Receptores de Interleucina/genética , Receptores de Ácido Retinoico/biosíntesis , Receptores de Ácido Retinoico/genética , Receptores de Hormona Tiroidea/biosíntesis , Receptores de Hormona Tiroidea/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Colaboradores-Inductores/inmunología
16.
Science ; 335(6068): 597-601, 2012 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-22245738

RESUMEN

Recognition and clearance of a bacterial infection are a fundamental properties of innate immunity. Here, we describe an effector B cell population that protects against microbial sepsis. Innate response activator (IRA) B cells are phenotypically and functionally distinct, develop and diverge from B1a B cells, depend on pattern-recognition receptors, and produce granulocyte-macrophage colony-stimulating factor. Specific deletion of IRA B cell activity impairs bacterial clearance, elicits a cytokine storm, and precipitates septic shock. These observations enrich our understanding of innate immunity, position IRA B cells as gatekeepers of bacterial infection, and identify new treatment avenues for infectious diseases.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Infecciones por Escherichia coli/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Inmunidad Innata , Peritonitis/inmunología , Sepsis/inmunología , Animales , Subgrupos de Linfocitos B/metabolismo , Linaje de la Célula , Separación Celular , Femenino , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Inmunoglobulina M/metabolismo , Inmunofenotipificación , Integrina alfa4beta1/inmunología , Integrina alfa4beta1/metabolismo , Lipopolisacáridos , Activación de Linfocitos , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Parabiosis , Choque Séptico/inmunología , Bazo/inmunología , Receptor Toll-Like 4/inmunología
17.
J Virol ; 81(8): 4199-214, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17287271

RESUMEN

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells in early infection are associated with the dramatic decline of peak viremia, whereas their antiviral activity in chronic infection is less apparent. The functional properties accounting for the antiviral activity of HIV-1-specific CD8+ T cells during early infection are unclear. Using cytokine secretion and tetramer decay assays, we demonstrated in intraindividual comparisons that the functional avidity of HIV-1-specific CD8+ T cells was consistently higher in early infection than in chronic infection in the presence of high-level viral replication. This change of HIV-1-specific CD8+ T-cell avidity between early and chronic infections was linked to a substantial switch in the clonotypic composition of epitope-specific CD8+ T cells, resulting from the preferential loss of high-avidity CD8+ T-cell clones. In contrast, the maintenance of the initially recruited clonotypic pattern of HIV-1-specific CD8+ T cells was associated with low-level set point HIV-1 viremia. These data suggest that high-avidity HIV-1-specific CD8+ T-cell clones are recruited during early infection but are subsequently lost in the presence of persistent high-level viral replication.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Adulto , Degranulación de la Célula , Células Cultivadas , Citocinas/biosíntesis , Femenino , Citometría de Flujo , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunología , Viremia
18.
Nat Immunol ; 8(11): 1246-54, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17906628

RESUMEN

In progressive viral infection, antiviral T cell function is impaired by poorly understood mechanisms. Here we report that the inhibitory immunoregulatory receptor CTLA-4 was selectively upregulated in human immunodeficiency virus (HIV)-specific CD4(+) T cells but not CD8(+) T cells in all categories of HIV-infected subjects evaluated, with the exception of rare people able to control viremia in the absence of antiretroviral therapy. CTLA-4 expression correlated positively with disease progression and negatively with the capacity of CD4(+) T cells to produce interleukin 2 in response to viral antigen. Most HIV-specific CD4(+) T cells coexpressed CTLA-4 and another inhibitory immunoregulatory receptor, PD-1. In vitro blockade of CTLA-4 augmented HIV-specific CD4(+) T cell function. These data, indicating a reversible immunoregulatory pathway selectively associated with CD4(+) T cell dysfunction, provide a potential target for immunotherapy in HIV-infected patients.


Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/inmunología , Antígenos CD/inmunología , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Antígeno CTLA-4 , Citocinas/metabolismo , Progresión de la Enfermedad , Infecciones por VIH/metabolismo , Humanos , Reacción en Cadena de la Polimerasa , Receptor de Muerte Celular Programada 1 , Regulación hacia Arriba , Carga Viral
19.
J Immunol ; 175(7): 4355-62, 2005 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-16177076

RESUMEN

The characterization of antiviral CTL responses has largely been limited to assessing Ag-specific immune responses in the peripheral blood. Consequently, there is an incomplete understanding of the cellular immune responses at mucosal sites where many viruses enter and initially replicate and how the Ag specificity and activation status of CTL derived from these mucosal sites may differ from that of blood-derived CTL. In this study, we show that EBV-specific CTL responses in the tonsils are of comparable specificity and breadth but of a significantly higher magnitude compared with responses in the peripheral blood. EBV-specific, tonsil-resident, but not PBMC-derived, T cells expressed the integrin/activation marker CD103 (alphaEbeta7), consistent with the detection of its ligand, E-cadherin, on tonsillar squamous cells. These CD8-positive, CD103-positive, tonsil-derived CTL were largely CCR7- and CD45RA- negative effector-memory cells and responded to lower Ag concentrations in in vitro assays than their CD103-negative PBMC-derived counterparts. Thus, EBV-specific CTL in the tonsil, a crucial site for EBV entry and replication, are of greater magnitude and phenotypically distinct from CTL in the peripheral blood and may be important for effective control of this orally transmitted virus.


Asunto(s)
Memoria Inmunológica/inmunología , Integrinas/biosíntesis , Tonsila Palatina/inmunología , Linfocitos T Citotóxicos/inmunología , Antígenos Virales/inmunología , Cadherinas/biosíntesis , Cadherinas/genética , Epítopos de Linfocito T/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Humanos , Inmunohistoquímica , Memoria Inmunológica/fisiología , Integrinas/genética , Ligandos , Tonsila Palatina/citología , Tonsila Palatina/metabolismo , Linfocitos T Citotóxicos/metabolismo
20.
Blood ; 106(10): 3366-9, 2005 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-16002429

RESUMEN

Natural killer (NK) cells are critical in the first-line defense against viral infections. Chronic HIV-1 infection leads to a perturbation in the NK cell compartment, yet the kinetics of this deregulation and the functional consequences are unclear. Here, we characterized changes in the NK cell compartment longitudinally by multiparameter flow cytometry, starting in acute HIV-1 infection. Acute HIV-1 infection was associated with elevated NK cell numbers, with an expansion of CD3(neg)CD56(dim)CD16(pos) NK cells and an early depletion of CD3(neg)CD56(bright)CD16(neg) NK cells. Ongoing viral replication resulted in a depletion of CD3(neg)CD56(dim)CD16(pos) NK cells with a paralleled increase in functionally anergic CD3(neg)CD56(neg)CD16(pos) NK cells, accompanied by reduced functional activity, as measured by CD107a expression and cytokine secretion. Taken together, these studies demonstrate a sequential impairment of NK cell function with persistent viral replication resulting from a progressive deregulation of NK cell subsets with distinct functional properties.


Asunto(s)
Antígenos CD/sangre , Infecciones por VIH/sangre , VIH-1 , Células Asesinas Naturales , Subgrupos Linfocitarios , Enfermedad Aguda , Antígenos CD/inmunología , Estudios de Casos y Controles , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/patología , VIH-1/inmunología , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Células Asesinas Naturales/virología , Recuento de Linfocitos/métodos , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/patología , Subgrupos Linfocitarios/virología , Masculino , Replicación Viral/inmunología
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