RESUMEN
BACKGROUND: Flow cytometric immunophenotyping is essential for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Most cases have easy to interpret flow cytometry profiles with red cells, neutrophils and monocytes showing complete deficiency of glycophosphatidylinositol (GPI) linked antigen expression. Some cases are more challenging to interpret due to the presence of multiple populations of PNH cells and variable levels of GPI antigen expression. METHODS: We studied 46 known PNH patients, many with complex immunophenotypic profiles using a novel, single tube, multi-parameter 7-color immunophenotyping assay that allowed simultaneous detection and assessment of PNH clones within multiple lineages of peripheral blood leucocytes. Red cell PNH clones were also assessed in total and immature (CD71+) components by CD59 expression. RESULTS: For individual patients, total PNH clones in each cell lineage were highly correlated. Monocytes, eosinophils and basophils showed the highest proportions of PNH cells. Red cell PNH clones were typically smaller than monocyte and neutrophil PNH clones. In most cases, PNH clones were detectable in minor leucocyte populations where multiple populations of PNH cells were present, variability in the proportions of type II and type III cells was seen across different cell lineages, even though total PNH clones remained similar. CONCLUSIONS: This study shows that PNH patients with multiple PNH clones do not always display the same abnormality across all cell lineages routinely tested. There is no simple explanation for this but is likely due to a combination of complex molecular, genetic and biochemical dysfunction in different blood cell types.
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Hemoglobinuria Paroxística , Humanos , Inmunofenotipificación , Linaje de la Célula , Citometría de Flujo , Antígenos CD59/metabolismo , Células ClonalesRESUMEN
Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec-associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNα secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.
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Neoplasias Hepáticas , Viroterapia Oncolítica , Virus Oncolíticos , Humanos , Leucocitos Mononucleares , Neoplasias Hepáticas/terapia , Terapia Neoadyuvante , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Virus Vaccinia/genéticaRESUMEN
OBJECTIVES: A retrospective population-based study to determine the incidence and prevalence of patients with the rare blood disease paroxysmal nocturnal haemoglobinuria (PNH). METHODS: All patients were identified by flow cytometric detection of blood cells deficient in glycosylphosphatidylinositol (GPI) linked proteins at a single diagnostic reference laboratory that serves the Yorkshire based, Haematological Malignancy Research Network (HMRN) with a population of 3.8 million. RESULTS: One hundred and ninety-seven patients with detectable PNH clones at a level of >0.01% in at least two lineages of cells (neutrophils, monocytes and/or red cells) were identified over a 15-year period (2004-2018). Of these, 88% had aplastic anaemia (AA), 8% classical PNH and 3% myelodysplastic syndrome. The overall incidence rate was estimated at 0.35 cases per 100 000 people per year. This equates to 220 cases newly diagnosed in the United Kingdom each year. The overall prevalence rate was 3.81 per 100 000, this equates to an estimated 2400 prevalent cases in the UK. The overall and relative 5-year survival rates were 72% and 82.7%, respectively. CONCLUSIONS: This study showed that classical haemolytic PNH is a rare disease and represents only a small proportion overall of patients with detectable PNH cells, the majority of which have aplastic anaemia.
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Anemia Aplásica/complicaciones , Anemia Aplásica/epidemiología , Hemoglobinuria Paroxística/complicaciones , Hemoglobinuria Paroxística/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anemia Aplásica/diagnóstico , Anemia Aplásica/historia , Biomarcadores , Niño , Preescolar , Femenino , Hemoglobinuria Paroxística/diagnóstico , Hemoglobinuria Paroxística/historia , Historia del Siglo XXI , Humanos , Inmunofenotipificación , Incidencia , Masculino , Persona de Mediana Edad , Vigilancia de la Población , Prevalencia , Estudios Retrospectivos , Síndrome , Reino Unido/epidemiología , Adulto JovenRESUMEN
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder, characterized by complement-mediated intravascular hemolysis and thrombosis. The increased incidence of PNH-driven thrombosis is still poorly understood, but unlike other thrombotic disorders, is thought to largely occur through complement-mediated mechanisms. Treatment with a C5 inhibitor, eculizumab, has been shown to significantly reduce the number of thromboembolic events in these patients. Based on previously described links between changes in fibrin clot structure and thrombosis in other disorders, our aim was to investigate clot structure as a possible mechanism of thrombosis in patients with PNH and the anti-thrombotic effects of eculizumab treatment on clot structure. Clot structure, fibrinogen levels and thrombin generation were examined in plasma samples from 82 patients from the National PNH Service in Leeds, UK. Untreated PNH patients were found to have increased levels of fibrinogen and thrombin generation, with subsequent prothrombotic changes in clot structure. No link was found between increasing disease severity and fibrinogen levels, thrombin generation, clot formation or structure. However, eculizumab treated patients showed decreased fibrinogen levels, thrombin generation and clot density, with increasing time spent on treatment augmenting these antithrombotic effects. These data suggest that PNH patients have a prothrombotic clot phenotype due to increased fibrinogen levels and thrombin generation, and that the antithrombotic effects of eculizumab are, in-part, due to reductions in fibrinogen and thrombin generation with downstream effects on clot structure.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Inactivadores del Complemento/uso terapéutico , Hemoglobinuria Paroxística/líquido cefalorraquídeo , Hemoglobinuria Paroxística/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/farmacología , Inactivadores del Complemento/farmacología , Femenino , Voluntarios Sanos , Hemoglobinuria Paroxística/complicaciones , Humanos , Masculino , FenotipoRESUMEN
A retrospective analysis of presentation clinical, laboratory and immunophenotypic features of 1 081 patients with paroxysmal nocturnal haemoglobinuria (PNH) clones [glycosylphosphatidylinositol (GPI)-deficient blood cells] identified at our hospital by flow cytometry over the past 25 years was undertaken. Three distinct clusters of patients were identified and significant correlations between presentation disease type and PNH clone sizes were evident. Smaller PNH clones predominate in cytopenic and myelodysplastic subtypes; large PNH clones were associated with haemolytic, thrombotic and haemolytic/thrombotic subtypes. Rare cases with an associated chronic myeloproliferative disorder had either large or small PNH clones. Cytopenia was a frequent finding, highlighting bone marrow failure as the major underlying feature associated with the detection of PNH clones in the peripheral blood. Red cell PNH clones showed significant correlations between the presence of type II (partial GPI deficiency) red cells and thrombotic disease. Haemolytic PNH was associated with type III (complete GPI deficiency) red cell populations of >20%. Those with both haemolytic and thrombotic features had major type II and type III red cell populations. Distinct patterns of presentation age decade were evident for clinical subtypes with a peak incidence of haemolytic PNH in the 30-49 year age group and a biphasic age distribution for the cytopenia group.
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Glicosilfosfatidilinositoles/deficiencia , Hemoglobinuria Paroxística/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anemia Aplásica/etiología , Anemia Hemolítica/etiología , Antígenos CD55/deficiencia , Antígenos CD59/deficiencia , Niño , Preescolar , Evolución Clonal , Células Clonales/patología , Progresión de la Enfermedad , Femenino , Citometría de Flujo , Hemoglobinuria Paroxística/complicaciones , Hemoglobinuria Paroxística/genética , Hemoglobinuria Paroxística/patología , Humanos , Inmunofenotipificación , Lactante , Linfocitos/patología , Masculino , Persona de Mediana Edad , Trastornos Mieloproliferativos/etiología , Neutrófilos/patología , Receptores de Transferrina/sangre , Estudios Retrospectivos , Trombosis/etiología , Adulto JovenRESUMEN
OBJECTIVE: To assess the prevalence of the MYD88 L265P mutation and variants within NLRP3 and evaluate the status of oligoclonal hematopoiesis in 30 patients with Schnitzler syndrome (SchS). METHODS: Thirty patients with SchS were recruited from 3 clinical centers. Six patients with known acquired cryopyrin-associated periodic syndromes (aCAPS) were included as controls. Allele-specific oligonucleotide-polymerase chain reaction was used for the detection of the MYD88 L265P variant, next-generation sequencing was applied to analyze NLRP3 and 28 genes associated with myelodysplastic syndrome, and gene scanning was performed for the detection of X chromosome inactivation. RESULTS: Activating NLRP3 mutations were not present in 11 SchS patients who had not been sequenced for this gene previously. The MYD88 L265P variant was present in 9 of 30 SchS patients, and somatic mutations associated with clonal hematopoiesis were identified in 1 of 30 patients with SchS and 1 of 6 patients with aCAPS. Evidence of nonrandom X chromosome inactivation was detected in 1 female patient with SchS and 1 female patient with aCAPS. CONCLUSION: A shared molecular mechanism accounting for the pathogenesis of inflammation in SchS remains elusive. Clonal hematopoiesis is not associated with other somatic mutations found in individuals with SchS or aCAPS.
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Hematopoyesis/genética , Mutación/genética , Factor 88 de Diferenciación Mieloide/análisis , Proteína con Dominio Pirina 3 de la Familia NLR/análisis , Síndrome de Schnitzler/genética , Síndromes Periódicos Asociados a Criopirina/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Factor 88 de Diferenciación Mieloide/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Reacción en Cadena de la Polimerasa , PrevalenciaRESUMEN
Paroxysmal nocturnal hemoglobinuria is a rare acquired hematologic disorder, the most serious complication of which is thrombosis. The increased incidence of thrombosis in paroxysmal nocturnal hemoglobinuria is still poorly understood, but unlike many other thrombotic disorders, predominantly involves complement-mediated mechanisms. This review article discusses the different factors that contribute to the increased risk of thrombosis in paroxysmal nocturnal hemoglobinuria. Paroxysmal nocturnal hemoglobinuria leads to a complex and multifaceted prothrombotic state due to the pathological effects of platelet activation, intravascular hemolysis and neutrophil/monocyte activation. Platelet and endothelial microparticles as well as oxidative stress may play a role. Impaired fibrinolysis has also been observed and may be caused by several mechanisms involving interactions between complement activation, coagulation and fibrinolysis. While many factors may affect thrombosis in paroxysmal nocturnal hemoglobinuria, the relative contribution of each mechanism that has been implicated is difficult to quantify. Further studies, including novel in vivo and in vitro thrombosis models, are required in order to define the role of the individual mechanisms contributing to thrombosis, impaired fibrinolysis and clarify other complement-driven prothrombotic mechanisms in paroxysmal nocturnal hemoglobinuria.
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Coagulación Sanguínea , Hemoglobinuria Paroxística/sangre , Hemoglobinuria Paroxística/etiología , Animales , Biomarcadores , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Hemoglobinas/metabolismo , Hemoglobinuria Paroxística/complicaciones , Hemoglobinuria Paroxística/diagnóstico , Humanos , Fenotipo , Activación Plaquetaria , Especies Reactivas de Oxígeno , Trombosis/sangre , Trombosis/etiologíaRESUMEN
Long-lived human plasma cells (PCs) play central roles in immunity and autoimmunity and are enriched among the subpopulation of CD19neg human PCs. However, whether human CD19neg PCs are necessarily aged cells that have gradually lost CD19 expression is not known. Assessing peripheral blood samples at steady-state and during the acute response to influenza vaccination in healthy donors, we identify the presence of phenotypic CD19neg plasmablasts, the proliferative precursor state to mature PCs, and demonstrate by ELISPOT that these are Ab-secreting cells (ASCs). During the acute response to influenza vaccination, CD19pos, CD19low, and CD19neg ASCs secrete vaccine-specific Abs and show linked IGHV repertoires. To address precursor/product relationships, we use in vitro models that mimic T-dependent and T-independent differentiation, finding that the CD19neg state can be established at the plasmablast to PC transition, that CD19neg PCs increase as a percentage of surviving PCs in vitro, and that CD19neg and CD19pos PCs can be maintained independently. These data provide proof-of-principle for the view that newly generated ASCs can acquire a mature PC phenotype that is accompanied by loss of CD19 expression at an early stage of differentiation and that aging is not an obligate requirement for a CD19neg state to be established.
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Células Productoras de Anticuerpos/inmunología , Antígenos CD19/inmunología , Diferenciación Celular , Células Plasmáticas/inmunología , Células Productoras de Anticuerpos/fisiología , Antígenos CD19/biosíntesis , Antígenos CD19/genética , Células de la Médula Ósea/inmunología , Senescencia Celular/inmunología , Citometría de Flujo , Humanos , Inmunofenotipificación , Activación de Linfocitos/inmunología , Células Plasmáticas/fisiologíaRESUMEN
Minimal residual disease (MRD) negativity, defined as <1 chronic lymphocytic leukemia (CLL) cell detectable per 10 000 leukocytes, has been shown to independently predict for clinical outcome in patients receiving combination chemoimmunotherapy in the frontline setting. However, the long-term prognostic value of MRD status in other therapeutic settings remains unclear. Here, we retrospectively analyzed, with up to 18 years follow-up, all patients at our institution who achieved at least a partial response (PR) with various therapies between 1996 and 2007, and received a bone marrow MRD assessment at the end of treatment according to the international harmonized approach. MRD negativity correlated with both progression-free survival (PFS) and overall survival (OS) independent of the type and line of treatment, as well as known prognostic factors including adverse cytogenetics. The greatest impact of achieving MRD negativity was seen in patients receiving frontline treatment, with 10-year PFS of 65% vs 10% and 10-year OS of 70% vs 30% for MRD-negative vs MRD-positive patients, respectively. Our results demonstrate the long-term benefit of achieving MRD negativity, regardless of the therapeutic setting and treatment modality, and support its use as a prognostic marker for long-term PFS and as a potential therapeutic goal in CLL.
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Supervivencia sin Enfermedad , Leucemia Linfocítica Crónica de Células B/patología , Neoplasia Residual/patología , Análisis Citogenético , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Análisis Multivariante , Resultado del TratamientoRESUMEN
Antimicrobial proteins influence intestinal microbial ecology and limit proliferation of pathogens, yet the regulation of their expression has only been partially elucidated. Here, we have identified a putative pathway involving epithelial cells and intestinal intraepithelial lymphocytes (iIELs) that leads to antimicrobial protein (AMP) production by Paneth cells. Mice lacking γδ iIELs (TCRδ(-/-)) express significantly reduced levels of the AMP angiogenin 4 (Ang4). These mice were also unable to up-regulate Ang4 production following oral challenge by Salmonella, leading to higher levels of mucosal invasion compared to their wild type counterparts during the first 2 hours post-challenge. The transfer of γδ iIELs from wild type (WT) mice to TCRδ(-/-) mice restored Ang4 production and Salmonella invasion levels were reduced to those obtained in WT mice. The ability to restore Ang4 production in TCRδ(-/-) mice was shown to be restricted to γδ iIELs expressing Vγ7-encoded TCRs. Using a novel intestinal crypt co-culture system we identified a putative pathway of Ang4 production initiated by exposure to Salmonella, intestinal commensals or microbial antigens that induced intestinal epithelial cells to produce cytokines including IL23 in a TLR-mediated manner. Exposure of TCR-Vγ7(+) γδ iIELs to IL-23 promoted IL22 production, which triggered Paneth cells to secrete Ang4. These findings identify a novel role for γδ iIELs in mucosal defence through sensing immediate epithelial cell cytokine responses and influencing AMP production. This in turn can contribute to the maintenance of intestinal microbial homeostasis and epithelial barrier function, and limit pathogen invasion.
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Comunicación Celular , Enterocitos/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Linfocitos/metabolismo , Células de Paneth/metabolismo , Ribonucleasa Pancreática/biosíntesis , Animales , Línea Celular , Interleucina-23/biosíntesis , Interleucinas/farmacología , Mucosa Intestinal/microbiología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Salmonella/inmunología , Estrés Fisiológico , Interleucina-22RESUMEN
Although gammadelta T cells play a role in protecting tissues from pathogen-elicited damage to bacterial, viral and parasitic pathogens, the mechanisms involved in the damage and in the protection have not been clearly elucidated. This has been addressed using a murine model of listeriosis, which in mice lacking gammadelta T cells (TCRdelta(-/-)) is characterised by severe and extensive immune-mediated hepatic necrosis. We show that these hepatic lesions are caused by Listeria-elicited CD8(+) T cells secreting high levels of TNF-alpha that accumulate in the liver of Listeria-infected TCRdelta(-/-) mice. Using isolated populations of gammadelta T cells from wild-type and cytokine-deficient strains of mice to reconstitute TCRdelta(-/-) mice, the TCR variable gene 4 (Vgamma4)(+) subset of gammadelta T cells was shown to protect against liver injury. Hepatoprotection was dependent upon their ability to produce IL-10 after TCR-mediated interactions with Listeria-elicited macrophages and CD8(+) T cells. IL-10-producing Vgamma4(+) T cells also contribute to controlling CD8(+) T cell expansion and to regulating and reducing TNF-alpha secretion by activated CD8(+) T cells. This effect on TNF-alpha production was directly attributed to IL-10. These findings identify a novel mechanism by which pathogen-elicited CD8(+) T cells are regulated via interactions with, and activation of, IL-10-producing hepatoprotective gammadelta T cells.
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Linfocitos T CD8-positivos/inmunología , Interleucina-10/biosíntesis , Listeriosis/inmunología , Hígado/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Subgrupos de Linfocitos T/inmunología , Animales , Listeriosis/patología , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Although gammadelta T cells are involved in the response to many pathogens, the dynamics and heterogeneity of the local gammadelta T cell response remains poorly defined. We recently identified gammadelta T cells as regulators of macrophages and dendritic cells during the resolution of Streptococcus pneumoniae-mediated lung inflammation. Here, using PCR, spectratype analysis and flow cytometry, we show that multiple gammadelta T cell subsets, including those bearing Vgamma1, Vgamma4 and Vgamma6 TCR, increase in number in the lungs of infected mice, but not in associated lymphoid tissue. These gammadelta T cells displayed signs of activation, as defined by CD69 and CD25 expression. In vivo BrdU incorporation suggested that local expansion, rather than recruitment, was the principal mechanism underlying this increase in gammadelta T cells. This conclusion was supported by the finding that pulmonary gammadelta T cells, but not alphabeta T cells, isolated from mice that had resolved infection exhibited lung-homing capacity in both naive and infected recipients. Together, these data provide novel insights into the origins of the heterogeneous gammadelta T cell response that accompanies lung infection, and the first evidence that inflammation-associated gammadelta T cells may exhibit distinct tissue-homing potential.
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Pulmón/inmunología , Neumonía Neumocócica/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Streptococcus pneumoniae/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Quimiotaxis de Leucocito , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T , Subunidad alfa del Receptor de Interleucina-2/análisis , Activación de Linfocitos , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Neumonía Neumocócica/patología , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Receptores de Antígenos de Linfocitos T gamma-delta/genéticaRESUMEN
BACKGROUND & AIMS: Intestinal epithelial integrity and permeability is dependent on intercellular tight junction (TJ) complexes. How TJ integrity is regulated remains unclear, although phosphorylation and dephosphorylation of the integral membrane protein occludin is an important determinant of TJ formation and epithelial permeability. We have investigated the role intestinal intraepithelial lymphocytes (iIELs) play in regulating epithelial permeability in response to infection. METHODS: Recombinant strains of Toxoplasma gondii were used to assess intestinal epithelial barrier function and TJ integrity in mice with intact or depleted populations of iIELs. Alterations in epithelial permeability were correlated with TJ structure and the state of phosphorylation of occludin. iIEL in vivo reconstitution experiments were used to identify the iIELs required to maintain epithelial permeability and TJ integrity. RESULTS: In the absence of gammadelta+ iIELs, intestinal epithelial barrier function and the ability to restrict epithelial transmigration of Toxoplasma and the unrelated intracellular bacterial pathogen Salmonella typhimurium was severely compromised. Leaky epithelium in gammadelta+ iIEL-deficient mice was associated with the absence of phosphorylation of serine residues of occludin and lack of claudin 3 and zona occludens-1 proteins in TJ complexes. These deficiencies were attributable to the absence of a single subset of gammadelta T-cell receptor (TCR-Vgamma7+) iIELs that, after reconstituting gammadelta iIEL-deficient mice, restored epithelial barrier function and TJ complexes, resulting in increased resistance to infection. CONCLUSIONS: These findings identify a novel role for gammadelta+ iIELs in maintaining TJ integrity and epithelial barrier function that have implications for understanding the pathogenesis of intestinal inflammatory diseases associated with disruption of TJ complexes.
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Permeabilidad de la Membrana Celular/inmunología , Uniones Intercelulares/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología , Animales , Western Blotting , Modelos Animales de Enfermedad , Inmunohistoquímica , Inmunoprecipitación , Mucosa Intestinal/inmunología , Mucosa Intestinal/parasitología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ocludina , Fosforilación , ARN/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismo , Toxoplasma/aislamiento & purificación , Toxoplasma/patogenicidad , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/metabolismo , Toxoplasmosis Animal/patologíaRESUMEN
To ensure invading pathogens are eliminated with minimal damage to host tissues it is essential that macrophage activation be tightly regulated. Previously we demonstrated that a subset of gammadelta T cells (Vgamma1(+)) contributes to resolving pathogen-induced immune responses by killing activated macrophages. However, the exaggerated macrophage response seen in infected Vgamma1(+) T cell-deficient mice suggests that gammadelta T cells play a broader role in macrophage homeostasis and other subsets might promote macrophage activation. Using a macrophage:gammadelta T cell co-culture system we have shown that gammadelta T cells increase the activity of macrophages activated in vivo by Listeria monocytogenes infection. In a dose-dependent manner, gammadelta T cells up-regulated production of cytokines (TNF-alpha, IL-6, IL-10) and chemokines (MIP-1alpha, MIP-1beta) by Listeria-elicited macrophages. The ability to increase macrophage cytokine production was prominent among Vgamma4(+) gammadelta T cells. Reciprocally, Vgamma4(+) gammadelta T cells were activated by Listeria-elicited macrophages, resulting in production of the anti-inflammatory cytokine, IL-10. gammadelta T cell adoptive transfer experiments showed that Vgamma4(+) T cells protected TCRdelta(-/-) mice against Listeria-induced liver injury and necrosis. These findings identify distinct and non-overlapping roles for gammadelta T cell subsets in regulating macrophage function during pathogen-induced immune responses.
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Homeostasis/inmunología , Macrófagos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Células Cultivadas , Quimiocinas/metabolismo , Técnicas de Cocultivo , Relación Dosis-Respuesta Inmunológica , Listeria monocytogenes/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subgrupos de Linfocitos T/microbiologíaRESUMEN
Although gammadelta T cells are a common feature of many pathogen-induced immune responses, the factors that influence, promote, or regulate the response of individual gammadelta T-cell subsets to infection is unknown. Here we show that in the absence of Vgamma1+ T cells, novel subsets of gammadelta T cells, expressing T-cell receptor (TCR)-Vgamma chains that normally define TCRgammadelta+ dendritic epidermal T cells (DETCs) (Vgamma5+), intestinal intraepithelial lymphocytes (iIELs) (Vgamma7+), and lymphocytes associated with the vaginal epithelia (Vgamma6+), are recruited to the spleen in response to bacterial infection in TCR-Vgamma1-/- mice. By comparison of phenotype and structure of TCR-Vgamma chains and/or -Vdelta chains expressed by these novel subsets with those of their epithelium-associated counterparts, the Vgamma6+ T cells elicited in infected Vgamma1-/- mice were shown to be identical to those found in the reproductive tract, from where they are presumably recruited in the absence of Vgamma1+ T cells. By contrast, Vgamma5+ and Vgamma7+ T cells found in infected Vgamma1-/- mice were distinct from Vgamma5+ DETCs and Vgamma7+ iIELs. Functional analyses of the novel gammadelta T-cell subsets identified for infected Vgamma1-/- mice showed that whereas the Vgamma5+ and Vgamma7+ subsets may compensate for the absence of Vgamma1+ T cells by producing similar cytokines, they do not possess cytocidal activity and they cannot replace the macrophage homeostasis function of Vgamma1+ T cells. Collectively, these findings identify novel subsets of gammadelta T cells, the recruitment and activity of which is under the control of Vgamma1+ T cells.
Asunto(s)
Listeria monocytogenes/patogenicidad , Listeriosis/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T , Genes Codificadores de la Cadena gamma de los Receptores de Linfocito T , Homeostasis , Humanos , Listeria monocytogenes/inmunología , Listeriosis/microbiología , Activación de Linfocitos , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T gamma-delta/deficiencia , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Análisis de Secuencia de ADN , Bazo/citología , Bazo/inmunologíaRESUMEN
Gammadelta T cells play important but poorly defined roles in pathogen-induced immune responses and in preventing chronic inflammation and pathology. A major obstacle to defining their function is establishing the degree of functional redundancy and heterogeneity among gammadelta T cells. Using mice deficient in Vgamma1+ T cells which are a major component of the gammadelta T cell response to microbial infection, a specific immunoregulatory role for Vgamma1+ T cells in macrophage and gammadelta T cell homeostasis during infection has been established. By contrast, Vgamma1+ T cells play no significant role in pathogen containment or eradication and cannot protect mice from immune-mediated pathology. Pathogen-elicited Vgamma1+ T cells also display different functional characteristics at different stages of the host response to infection that involves unique and different populations of Vgamma1+ T cells. These findings, therefore, identify distinct and nonoverlapping roles for gammadelta T cell subsets in infection and establish the complexity and adaptability of a single population of gammadelta T cells in the host response to infection that is not predetermined, but is, instead, shaped by environmental factors.
Asunto(s)
Listeriosis/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/microbiología , Animales , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Citotoxicidad Inmunológica/genética , Femenino , Inmunofenotipificación , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/inmunología , Listeria monocytogenes/patogenicidad , Listeriosis/genética , Listeriosis/patología , Cirrosis Hepática/genética , Cirrosis Hepática/inmunología , Cirrosis Hepática/microbiología , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Receptores de Antígenos de Linfocitos T gamma-delta/deficiencia , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Subgrupos de Linfocitos T/metabolismo , Factores de TiempoRESUMEN
PROBLEM: Detailed analysis of the expression of natural killer (NK) cell activatory and inhibitory receptors by human decidual leukocyte subpopulations has not been undertaken. METHOD OF STUDY: Expression of the natural cytotoxicity receptors NKp30, NKp44 and NKp46 by decidual leukocytes were studied by reverse transcriptase polymerase chain reaction. Expression of the killer cell Ig-like receptors CD158a and CD158b on decidual T cells were studied by flow cytometry. RESULTS: First trimester decidual leukocytes expressed mRNA for the NKp30 and NKp46 receptors but expression of NKp44, a marker of activated NK cells, was not detected. A mean of 11.8 and 15.8% of decidual T cells expressed CD158a or 158b, respectively, while only around 1% of peripheral blood T cells were CD158a+ or CD158b+. CONCLUSIONS: Like peripheral blood NK cells, decidual NK cells express the natural cytotoxicity receptors NKp30 and NKp46 but the significance of this will not become apparent until ligands for these molecules have been identified. Only a minority of decidual T cells express CD158, indicating that this is not a mechanism for inhibition of cytotoxicity mediated by all decidual T cells.
Asunto(s)
Decidua/inmunología , Células Asesinas Naturales/inmunología , Primer Trimestre del Embarazo/inmunología , Receptores Inmunológicos/biosíntesis , Linfocitos T/metabolismo , Citotoxicidad Inmunológica , Decidua/citología , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Receptor 3 Gatillante de la Citotoxidad Natural , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunologíaRESUMEN
Inflammatory mediators including chemokines play a critical role in acute pancreatitis. The precise nature of early inflammatory signals within the pancreas remains, however, unclear. We examined the ability of isolated pancreatic acini to synthesize CC chemokine monocyte chemotactic protein-1 (MCP-1) and CXC chemokine cytokine-induced neutrophil chemoattractant (CINC) and the response to the secretagogue cerulein at physiological and supraphysiological concentrations. Isolated rat pancreatic acini maintained in short-term (< or =48 h) primary culture constitutively synthesized MCP-1 and CINC. Cerulein (10(-7) M; supramaximal dose) increased production of MCP-1 but not CINC. Cerulein-induced increase in MCP-1 synthesis was accompanied by increase in nuclear factor (NF)-kappaB activation shown by EMSA. Pretreatment with NF-kappaB inhibitors N-acetylcysteine (NAC) and N-tosylphenyalanine chloromethyl ketone (TPCK) blocked cerulein-induced NF-kappaB activation and abolished cerulein's effect on MCP-1 synthesis. Pretreatment with calcium antagonist BAPTA-AM also blocked cerulein's effect on MCP-1 synthesis. These results indicate that isolated acini synthesize MCP-1 and CINC and support the idea of acinar-derived chemokines as early mediators of inflammatory response in acute pancreatitis. Although cerulein hyperstimulation increased MCP-1 synthesis by a calcium-dependent mechanism involving NF-kappaB activation, CINC synthesis was not affected. This suggests that regulation of CC and CXC chemokines within acinar cells may be quite different.