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1.
Proc Natl Acad Sci U S A ; 119(34): e2201541119, 2022 08 23.
Artículo en Inglés | MEDLINE | ID: mdl-35943978

RESUMEN

Whereas pathogen-specific T and B cells are a primary focus of interest during infectious disease, we have used COVID-19 to ask whether their emergence comes at a cost of broader B cell and T cell repertoire disruption. We applied a genomic DNA-based approach to concurrently study the immunoglobulin-heavy (IGH) and T cell receptor (TCR) ß and δ chain loci of 95 individuals. Our approach detected anticipated repertoire focusing for the IGH repertoire, including expansions of clusters of related sequences temporally aligned with SARS-CoV-2-specific seroconversion, and enrichment of some shared SARS-CoV-2-associated sequences. No significant age-related or disease severity-related deficiencies were noted for the IGH repertoire. By contrast, whereas focusing occurred at the TCRß and TCRδ loci, including some TCRß sequence-sharing, disruptive repertoire narrowing was almost entirely limited to many patients aged older than 50 y. By temporarily reducing T cell diversity and by risking expansions of nonbeneficial T cells, these traits may constitute an age-related risk factor for COVID-19, including a vulnerability to new variants for which T cells may provide key protection.


Asunto(s)
Inmunidad Adaptativa , COVID-19 , Cadenas Pesadas de Inmunoglobulina , Receptores de Antígenos de Linfocitos T alfa-beta , Receptores de Antígenos de Linfocitos T , SARS-CoV-2 , Inmunidad Adaptativa/genética , Anciano , Linfocitos B/inmunología , COVID-19/genética , COVID-19/inmunología , Sitios Genéticos , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , SARS-CoV-2/inmunología , Seroconversión , Linfocitos T/inmunología
2.
Cytometry A ; 103(2): 117-126, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-34811890

RESUMEN

Here we consider how high-content flow cytometric methodology at appropriate scale and throughput rapidly provided meaningful biological data in our recent studies of COVID-19, which we discuss in the context of other similar investigations. In our work, high-throughput flow cytometry was instrumental to identify a consensus immune signature in COVID-19 patients, and to investigate the impact of SARS-CoV-2 exposure on patients with either solid or hematological cancers. We provide here some examples of our 'holistic' approach, in which flow cytometry data generated by lymphocyte and myelomonocyte panels were integrated with other analytical metrics, including SARS-CoV-2-specific serum antibody titers, plasma cytokine/chemokine levels, and in-depth clinical annotation. We report how selective differences between T cell subsets were revealed by a newly described flow cytometric TDS assay to distinguish actively cycling T cells in the peripheral blood. By such approaches, our and others' high-content flow cytometry studies collectively identified overt abnormalities and subtle but critical changes that discriminate the immuno-signature of COVID-19 patients from those of healthy donors and patients with non-COVID respiratory infections. Thereby, these studies offered several meaningful biomarkers of COVID-19 severity that have the potential to improve the management of patients and of hospital resources. In sum, flow cytometry provides an important means for rapidly obtaining data that can guide clinical decision-making without requiring highly expensive, sophisticated equipment, and/or "-omics" capabilities. We consider how this approach might be further developed.


Asunto(s)
COVID-19 , Humanos , SARS-CoV-2 , Citometría de Flujo , Citocinas , Subgrupos de Linfocitos T
3.
Lancet Oncol ; 22(6): 765-778, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33930323

RESUMEN

BACKGROUND: The efficacy and safety profiles of vaccines against SARS-CoV-2 in patients with cancer is unknown. We aimed to assess the safety and immunogenicity of the BNT162b2 (Pfizer-BioNTech) vaccine in patients with cancer. METHODS: For this prospective observational study, we recruited patients with cancer and healthy controls (mostly health-care workers) from three London hospitals between Dec 8, 2020, and Feb 18, 2021. Participants who were vaccinated between Dec 8 and Dec 29, 2020, received two 30 µg doses of BNT162b2 administered intramuscularly 21 days apart; patients vaccinated after this date received only one 30 µg dose with a planned follow-up boost at 12 weeks. Blood samples were taken before vaccination and at 3 weeks and 5 weeks after the first vaccination. Where possible, serial nasopharyngeal real-time RT-PCR (rRT-PCR) swab tests were done every 10 days or in cases of symptomatic COVID-19. The coprimary endpoints were seroconversion to SARS-CoV-2 spike (S) protein in patients with cancer following the first vaccination with the BNT162b2 vaccine and the effect of vaccine boosting after 21 days on seroconversion. All participants with available data were included in the safety and immunogenicity analyses. Ongoing follow-up is underway for further blood sampling after the delayed (12-week) vaccine boost. This study is registered with the NHS Health Research Authority and Health and Care Research Wales (REC ID 20/HRA/2031). FINDINGS: 151 patients with cancer (95 patients with solid cancer and 56 patients with haematological cancer) and 54 healthy controls were enrolled. For this interim data analysis of the safety and immunogenicity of vaccinated patients with cancer, samples and data obtained up to March 19, 2021, were analysed. After exclusion of 17 patients who had been exposed to SARS-CoV-2 (detected by either antibody seroconversion or a positive rRT-PCR COVID-19 swab test) from the immunogenicity analysis, the proportion of positive anti-S IgG titres at approximately 21 days following a single vaccine inoculum across the three cohorts were 32 (94%; 95% CI 81-98) of 34 healthy controls; 21 (38%; 26-51) of 56 patients with solid cancer, and eight (18%; 10-32) of 44 patients with haematological cancer. 16 healthy controls, 25 patients with solid cancer, and six patients with haematological cancer received a second dose on day 21. Of the patients with available blood samples 2 weeks following a 21-day vaccine boost, and excluding 17 participants with evidence of previous natural SARS-CoV-2 exposure, 18 (95%; 95% CI 75-99) of 19 patients with solid cancer, 12 (100%; 76-100) of 12 healthy controls, and three (60%; 23-88) of five patients with haematological cancers were seropositive, compared with ten (30%; 17-47) of 33, 18 (86%; 65-95) of 21, and four (11%; 4-25) of 36, respectively, who did not receive a boost. The vaccine was well tolerated; no toxicities were reported in 75 (54%) of 140 patients with cancer following the first dose of BNT162b2, and in 22 (71%) of 31 patients with cancer following the second dose. Similarly, no toxicities were reported in 15 (38%) of 40 healthy controls after the first dose and in five (31%) of 16 after the second dose. Injection-site pain within 7 days following the first dose was the most commonly reported local reaction (23 [35%] of 65 patients with cancer; 12 [48%] of 25 healthy controls). No vaccine-related deaths were reported. INTERPRETATION: In patients with cancer, one dose of the BNT162b2 vaccine yields poor efficacy. Immunogenicity increased significantly in patients with solid cancer within 2 weeks of a vaccine boost at day 21 after the first dose. These data support prioritisation of patients with cancer for an early (day 21) second dose of the BNT162b2 vaccine. FUNDING: King's College London, Cancer Research UK, Wellcome Trust, Rosetrees Trust, and Francis Crick Institute.


Asunto(s)
Vacunas contra la COVID-19/uso terapéutico , COVID-19/inmunología , Neoplasias/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Vacuna BNT162 , COVID-19/sangre , COVID-19/complicaciones , COVID-19/virología , Vacunas contra la COVID-19/inmunología , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Inmunogenicidad Vacunal/inmunología , Londres/epidemiología , Masculino , Persona de Mediana Edad , Neoplasias/sangre , Neoplasias/complicaciones , Neoplasias/virología , Estudios Prospectivos , SARS-CoV-2 , Gales
4.
Am J Hum Genet ; 100(2): 364-370, 2017 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-28157540

RESUMEN

SPG23 is an autosomal-recessive neurodegenerative subtype of lower limb spastic paraparesis with additional diffuse skin and hair dyspigmentation at birth followed by further patchy pigment loss during childhood. Previously, genome-wide linkage in an Arab-Israeli pedigree mapped the gene to an approximately 25 cM locus on chromosome 1q24-q32. By using whole-exome sequencing in a further Palestinian-Jordanian SPG23 pedigree, we identified a complex homozygous 4-kb deletion/20-bp insertion in DSTYK (dual serine-threonine and tyrosine protein kinase) in all four affected family members. DSTYK is located within the established linkage region and we also found the same mutation in the previously reported pedigree and another Israeli pedigree (total of ten affected individuals from three different families). The mutation removes the last two exons and part of the 3' UTR of DSTYK. Skin biopsies revealed reduced DSTYK protein levels along with focal loss of melanocytes. Ultrastructurally, swollen mitochondria and cytoplasmic vacuoles were also noted in remaining melanocytes and some keratinocytes and fibroblasts. Cultured keratinocytes and fibroblasts from an affected individual, as well as knockdown of Dstyk in mouse melanocytes, keratinocytes, and fibroblasts, were associated with increased cell death after ultraviolet irradiation. Keratinocytes from an affected individual showed loss of kinase activity upon stimulation with fibroblast growth factor. Previously, dominant mutations in DSTYK were implicated in congenital urological developmental disorders, but our study identifies different phenotypic consequences for a recurrent autosomal-recessive deletion mutation in revealing the genetic basis of SPG23.


Asunto(s)
Trastornos de la Pigmentación/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Eliminación de Secuencia , Paraplejía Espástica Hereditaria/genética , Vitíligo/genética , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Pueblo Asiatico/genética , Cromosomas Humanos Par 1/genética , Exones , Facies , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Ligamiento Genético , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Homocigoto , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Masculino , Melanocitos/citología , Melanocitos/metabolismo , Ratones , Células 3T3 NIH , Linaje , Trastornos de la Pigmentación/diagnóstico , Paraplejía Espástica Hereditaria/diagnóstico , Vitíligo/diagnóstico , Adulto Joven
5.
Mol Ther ; 23(11): 1783-1793, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26216515

RESUMEN

T-cell depletion therapy is used to prevent acute allograft rejection, treat autoimmunity and create space for bone marrow or hematopoietic cell transplantation. The evolved response to T-cell loss is a transient increase in IL-7 that drives compensatory homeostatic proliferation (HP) of mature T cells. Paradoxically, the exaggerated form of this process that occurs following lymphodepletion expands effector T-cells, often causing loss of immunological tolerance that results in rapid graft rejection, autoimmunity, and exacerbated graft-versus-host disease (GVHD). While standard immune suppression is unable to treat these pathologies, growing evidence suggests that manipulating the incipient process of HP increases allograft survival, prevents autoimmunity, and markedly reduces GVHD. Multipotent adult progenitor cells (MAPC) are a clinical grade immunomodulatory cell therapy known to alter γ-chain cytokine responses in T-cells. Herein, we demonstrate that MAPC regulate HP of human T-cells, prevent the expansion of Th1, Th17, and Th22 effectors, and block the development of pathogenic allograft responses. This occurs via IL-1ß-primed secretion of PGE2 and activates T-cell intrinsic regulatory mechanisms (SOCS2, GADD45A). These data provide proof-of-principle that HP of human T-cells can be targeted by cellular and molecular therapies and lays a basis for the development of novel strategies to prevent immunopathology in lymphodepleted patients.


Asunto(s)
Células Madre Adultas/fisiología , Dinoprostona/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Interleucina-7/inmunología , Células Madre Mesenquimatosas/fisiología , Células Madre Multipotentes/fisiología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adulto , Células Madre Adultas/inmunología , Autoinmunidad , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Rechazo de Injerto , Humanos , Tolerancia Inmunológica , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Interleucina-7/metabolismo , Depleción Linfocítica/efectos adversos , Masculino , Células Madre Mesenquimatosas/inmunología , Células Madre Multipotentes/inmunología , Proteínas Nucleares/metabolismo , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Trasplante Homólogo/métodos , Adulto Joven
6.
J Immunol ; 189(5): 2274-82, 2012 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-22821960

RESUMEN

There is an increasing body of evidence suggesting that the transfer of preformed MHC class I:peptide complexes between a virus-infected cell and an uninfected APC, termed cross-dressing, represents an important mechanism of Ag presentation to CD8+ T cells in host defense. However, although it has been shown that memory CD8+ T cells can be activated by uninfected dendritic cells (DCs) cross-dressed by Ag from virus-infected parenchymal cells, it is unknown whether conditions exist during virus infection in which naive CD8+ T cells are primed and differentiate to cytolytic effectors through cross-dressing, and indeed which DC subset would be responsible. In this study, we determine whether the transfer of MHC class I:peptide complexes between infected and uninfected murine DC plays a role in CD8+ T cell priming to viral Ags in vivo. We show that MHC class I:peptide complexes from peptide-pulsed or virus-infected DCs are indeed acquired by splenic CD8α⁻ DCs in vivo. Furthermore, the acquired MHC class I:peptide complexes are functional in that they induced Ag-specific CD8+ T cell effectors with cytolytic function. As CD8α⁻ DCs are poor cross-presenters, this may represent the main mechanism by which CD8α⁻ DCs present exogenously encountered Ag to CD8+ T cells. The sharing of Ag as preformed MHC class I:peptide complexes between infected and uninfected DCs without the restraints of Ag processing may have evolved to accurately amplify the response and also engage multiple DC subsets critical in the generation of strong antiviral immunity.


Asunto(s)
Antivirales/metabolismo , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Inmunidad Celular , Complejo Mayor de Histocompatibilidad/inmunología , Fragmentos de Péptidos/metabolismo , Adenoviridae/inmunología , Adenoviridae/metabolismo , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/administración & dosificación , Ovalbúmina/metabolismo , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/biosíntesis
7.
Sci Adv ; 9(49): eadj6174, 2023 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-38055824

RESUMEN

Clonotypic αß T cell responses to cargoes presented by major histocompatibility complex (MHC), MR1, or CD1 proteins underpin adaptive immunity. Those responses are mostly mediated by complementarity-determining region 3 motifs created by quasi-random T cell receptor (TCR) gene rearrangements, with diversity being highest for TCRγδ. Nonetheless, TCRγδ also displays nonclonotypic innate responsiveness following engagement of germline-encoded Vγ-specific residues by butyrophilin (BTN) or BTN-like (BTNL) proteins that uniquely mediate γδ T cell subset selection. We now report that nonclonotypic TCR engagement likewise induces distinct phenotypes in TCRαß+ cells. Specifically, antibodies to germline-encoded human TCRVß motifs consistently activated naïve or memory T cells toward core states distinct from those induced by anti-CD3 or superantigens and from others commonly reported. Those states combined selective proliferation and effector function with activation-induced inhibitory receptors and memory differentiation. Thus, nonclonotypic TCRVß targeting broadens our perspectives on human T cell response modes and might offer ways to induce clinically beneficial phenotypes in defined T cell subsets.


Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta , Receptores de Antígenos de Linfocitos T gamma-delta , Humanos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Subgrupos de Linfocitos T , Butirofilinas/genética , Butirofilinas/metabolismo , Fenotipo , Inmunoterapia
8.
J Invest Dermatol ; 141(3): 523-532.e2, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-32890627

RESUMEN

Slac2-b, also known as exophilin-5, is a Rab27b effector protein with a role in exosome transport and is encoded by the EXPH5 gene. We previously described biallelic loss-of-function mutations in EXPH5 in an autosomal recessive form of epidermolysis bullosa simplex. However, how the loss of Slac2-b expression leads to skin fragility and erosions is unknown. In this study, we demonstrate that keratinocytes (KCs) isolated from two different individuals with mutations in EXPH5 have significant defects in cell‒matrix adhesion. EXPH5-mutant KCs also showed increased perinuclear accumulation and significantly reduced trafficking of CD63+ vesicles. These phenotypes were also seen in Slac2-b‒deficient KCs. This was coincident with a reduction in Rab27a protein expression in Slac2-b‒mutant KCs as well as reduced secretion of extracellular vesicles containing extracellular matrix proteins. Live imaging analysis revealed a strong correlation between CD63+ vesicle trafficking to the plasma membrane and focal adhesion dynamics. These findings support a role for Slac2-b in regulating local focal adhesion dynamics to support effective KC adhesion and provide insight into the underlying pathophysiology of inherited skin blistering.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Epidermis/patología , Epidermólisis Ampollosa Simple/patología , Vesículas Extracelulares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Biopsia , Adhesión Celular/genética , Movimiento Celular/genética , Niño , Epidermólisis Ampollosa Simple/genética , Humanos , Microscopía Intravital , Queratinocitos/patología , Masculino , Mutación , Tetraspanina 30/metabolismo , Imagen de Lapso de Tiempo , Proteínas rab27 de Unión a GTP/metabolismo
9.
Cancer Cell ; 39(2): 257-275.e6, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33476581

RESUMEN

Given the immune system's importance for cancer surveillance and treatment, we have investigated how it may be affected by SARS-CoV-2 infection of cancer patients. Across some heterogeneity in tumor type, stage, and treatment, virus-exposed solid cancer patients display a dominant impact of SARS-CoV-2, apparent from the resemblance of their immune signatures to those for COVID-19+ non-cancer patients. This is not the case for hematological malignancies, with virus-exposed patients collectively displaying heterogeneous humoral responses, an exhausted T cell phenotype and a high prevalence of prolonged virus shedding. Furthermore, while recovered solid cancer patients' immunophenotypes resemble those of non-virus-exposed cancer patients, recovered hematological cancer patients display distinct, lingering immunological legacies. Thus, while solid cancer patients, including those with advanced disease, seem no more at risk of SARS-CoV-2-associated immune dysregulation than the general population, hematological cancer patients show complex immunological consequences of SARS-CoV-2 exposure that might usefully inform their care.


Asunto(s)
COVID-19/inmunología , Neoplasias/inmunología , Neoplasias/virología , Síndrome Respiratorio Agudo Grave/inmunología , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/etiología , COVID-19/mortalidad , Femenino , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/mortalidad , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/virología , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Nasofaringe/virología , Neoplasias/mortalidad , Neoplasias/terapia , Síndrome Respiratorio Agudo Grave/etiología , Síndrome Respiratorio Agudo Grave/mortalidad , Síndrome Respiratorio Agudo Grave/virología , Linfocitos T/virología , Esparcimiento de Virus , Adulto Joven
10.
Nat Commun ; 11(1): 3412, 2020 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-32641742

RESUMEN

Regulatory B cells restrict immune and inflammatory responses across a number of contexts. This capacity is mediated primarily through the production of IL-10. Here we demonstrate that the induction of a regulatory program in human B cells is dependent on a metabolic priming event driven by cholesterol metabolism. Synthesis of the metabolic intermediate geranylgeranyl pyrophosphate (GGPP) is required to specifically drive IL-10 production, and to attenuate Th1 responses. Furthermore, GGPP-dependent protein modifications control signaling through PI3Kδ-AKT-GSK3, which in turn promote BLIMP1-dependent IL-10 production. Inherited gene mutations in cholesterol metabolism result in a severe autoinflammatory syndrome termed mevalonate kinase deficiency (MKD). Consistent with our findings, B cells from MKD patients induce poor IL-10 responses and are functionally impaired. Moreover, metabolic supplementation with GGPP is able to reverse this defect. Collectively, our data define cholesterol metabolism as an integral metabolic pathway for the optimal functioning of human IL-10 producing regulatory B cells.


Asunto(s)
Linfocitos B Reguladores/metabolismo , Colesterol/metabolismo , Interleucina-10/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Animales , Antígenos CD19/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Técnicas de Cocultivo , Enfermedades Autoinflamatorias Hereditarias/metabolismo , Humanos , Macrófagos/metabolismo , Síndrome Metabólico/metabolismo , Deficiencia de Mevalonato Quinasa/metabolismo , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Análisis de Componente Principal , Transducción de Señal , Células TH1/metabolismo , Receptor Toll-Like 9/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
12.
Nat Med ; 26(10): 1623-1635, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32807934

RESUMEN

Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.


Asunto(s)
Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Infecciones por Coronavirus/inmunología , Citocinas/inmunología , Células Dendríticas/inmunología , Neumonía Viral/inmunología , Linfocitos T/inmunología , Anciano , Subgrupos de Linfocitos B/inmunología , Basófilos/inmunología , Betacoronavirus , COVID-19 , Estudios de Casos y Controles , Ciclo Celular , Quimiocina CXCL10/inmunología , Quimiocinas/inmunología , Estudios de Cohortes , Infecciones por Coronavirus/sangre , Progresión de la Enfermedad , Femenino , Citometría de Flujo , Hospitalización , Humanos , Memoria Inmunológica , Inmunofenotipificación , Interleucina-10/inmunología , Interleucina-6/inmunología , Recuento de Leucocitos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/sangre , Pronóstico , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Subgrupos de Linfocitos T/inmunología , Regulación hacia Arriba
14.
Methods Mol Biol ; 2020: 165-174, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31177499

RESUMEN

The important role of the local mucosal environment in both the initiation and progression of allergic disease is well established. Analysis of tissue-resident lymphocyte subsets by flow cytometry requires isolation of viable cells from mucosal samples.Here we describe an advanced method to dissociate lymphocytes from human mucosal (e.g., nasal, bronchial) biopsies. Single-cell suspensions are obtained through a combination of gentle mechanical disruption and incubation of tissue with proteolytic enzymes. This method fully utilizes limited clinical samples and is amenable to a variety of downstream applications for phenotypic, single-cell analysis of tissue lymphocytes or pooled lymphocyte subsets.


Asunto(s)
Separación Celular/métodos , Linfocitos/citología , Pólipos Nasales/patología , Tonsila Palatina/patología , Piel/patología , Biopsia , Técnicas de Cultivo de Célula , Células Cultivadas , Citometría de Flujo , Humanos , Fenómenos Mecánicos , Pólipos Nasales/inmunología , Tonsila Palatina/inmunología , Análisis de la Célula Individual , Piel/inmunología
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