RESUMEN
Immunological memory, the hallmark of adaptive immunity, is orchestrated by T and B lymphocytes. In circulation and different organs, there are billions of unique T and B cell clones, and each one can bind a specific antigen, leading to proliferation, differentiation and/or cytokine secretion. The vast heterogeneity in T and B cells is generated by random recombination of different genetic segments. Next-generation sequencing (NGS) technologies, developed in the last decade, enable an unprecedented in-depth view of the T and B cell receptor immune repertoire. Studies in various inflammatory conditions, immunodeficiencies, infections and malignancies demonstrated marked changes in clonality, gene usage, and biophysical properties of immune repertoire, providing important insights about the role of adaptive immune responses in different disorders. Here, we provide a detailed protocol for NGS of immune repertoire of T and B cells from blood and tissue. We present a pipeline starting from DNA isolation through library preparation, sequencing on NGS sequencer and ending with basic analyses. This method enables exploration of specific T and B cells at the nucleotide or amino-acid level, and thus can identify dynamic changes in lymphocyte populations and diversity parameters in different diseases. This technique is slowly entering clinical practice and has the potential for identification of novel biomarkers, risk stratification and precision medicine.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Inmunidad Adaptativa , Linfocitos B/inmunología , ADN/genética , ADN/aislamiento & purificación , Biblioteca de Genes , Humanos , Memoria Inmunológica , Linfocitos T/inmunologíaRESUMEN
OBJECTIVES: To determine the current role of bisantrene, an anthracene with anthracycline-like activity which was shown in earlier studies to be effective therapy in relapsed/refractory acute myeloid leukemia with no discernible cardiotoxicity, in the treatment of patients with R/R AML. METHODS: This phase 2, single-center study (NCT03820908) enrolled adult R/R AML to receive bisantrene (250 mg/m2 daily for 7 days) which was administered via an intravenous infusion over 2 hours on days 1-7. Disease assessment included routine blood work and bone marrow studies. RESULTS: In all, 10 patients were enrolled with a median of 3 lines of prior therapy including seven patients who had relapsed following allogeneic stem cell transplantation. The most frequently reported grade ≥3 treatment-attributed hematologic AE was thrombocytopenia, whereas the most frequently reported grade ≥3 treatment-attributed non-hematologic AE was mucositis. Of the 10 patients, one (10%) achieved a complete remission and three patients achieved a partial remission resulting in an overall response rate of 40%. Next-generation sequencing of patient samples identified a wide array of mutations associated with activated signaling, splicing, and epigenetic modification. CONCLUSIONS: In view of the observed low toxicity, a follow-up study combining bisantrene with complementary anti-leukemic therapy is planned.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Antracenos/administración & dosificación , Antracenos/efectos adversos , Antracenos/uso terapéutico , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/efectos adversos , Biopsia , Médula Ósea , Susceptibilidad a Enfermedades , Resistencia a Antineoplásicos , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Tomografía de Emisión de Positrones , Recurrencia , Retratamiento , Resultado del Tratamiento , Adulto JovenRESUMEN
A substantial segment of patients with acute myeloid leukemia (AML) will relapse following an initial response to induction therapy or will prove to be primary refractory. High-dose cytarabine and mitoxantrone (HiDAC/MITO) is an established salvage therapy for these patients. We studied all adult patients with relapsed/refractory (R/R) AML who were treated with HiDAC/MITO in our center between the years 2008-2017. To determine whether responding patients harbored a unique molecular signature, we performed targeted next-generation sequencing (NGS) on a subset of patients. The study cohort consisted of 172 patients with a median age of 54 years (range 18-77). The composite complete remission rate was 58%; 11 patients (6%) died during salvage therapy. Median survival was 11.4 months with a 1-year survival rate of 48%. In multivariate analysis favorable risk cytogenetics [Odds ratio (OR)=0.34, confidence interval (CI) 95%, 0.17-0.68; P = 0.002], and de-novo AML (OR = 0.4, CI 95%, 0.16-0.98; P = 0.047) were independently associated with a favorable response. Patients who attained a complete remission had a median survival of 43.7 months compared with 5.2 months for refractory patients (p < 0.0001). Neither the FLT3-ITD and NPM1 mutational status nor the indication for salvage therapy significantly impacted on the response to HiDAC/MITO salvage. NGS analysis identified 20 different mutations across the myeloid gene spectrum with a distinct TP53 signature detected in non-responding patients. HiDAC/MITO is an effective salvage regimen in R/R AML, however patients with adverse cytogenetics or secondary disease may not benefit as much from this approach.
RESUMEN
Both JAK2V617F and calreticulin (CALR) mutated essential thrombocythemia (ET) patients have different clinical characteristics, with lower thrombosis risk in patients with CALR mutations. To elucidate the mechanism for this lower risk we studied platelet function in ET patients with either JAK2V617F or a CALR mutation. Platelet activation state was similar in ET and healthy controls at baseline using P-selectin and PAC1 flow cytometry analysis. However, CALR mutated platelets were significantly less activated following ADP stimulation, compared to control or JAK2 mutated platelets (P < .001). In live-cell imaging of platelet attachment to immobilized fibrinogen by Interference Reflection Microscopy (IRM), the number of attached CALR mutated platelets was lower compared to control and JAK2 mutated platelets, with lower fractions of platelets achieving the fully spread state (90%, 78% and 54% of adherent cells for control, JAK2 and CALR mutated subjects, respectively). Compared to controls, ET patients, regardless of the mutation type, had increased numbers of immature platelets (IP) and leukocyte platelet aggregates (LPA), as well as plasma sP-selectin. These were all correlated with the platelet count and not to the state of platelet activation. We also found that intracellular free Ca2+ was increased in resting ET compared to control platelets. Note, CALR had a more dispersed localization in activated ET platelets compared to healthy controls, and mutated CALR interact physically with TpoR in CALR mutated platelets. We hypothesize that defects in platelet activation and spreading in CALR mutated patients can explain, at least in part, the lower thrombotic tendency in CALR mutated ET patients.
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Plaquetas/efectos de los fármacos , Calreticulina/genética , Janus Quinasa 2/genética , Activación Plaquetaria/efectos de los fármacos , Trombocitemia Esencial/sangre , Trombofilia/etiología , Adenosina Difosfato/farmacología , Adulto , Calcio/sangre , Forma de la Célula , Femenino , Humanos , Leucocitos/patología , Masculino , Microscopía de Interferencia , Persona de Mediana Edad , Mutación Missense , Selectina-P/sangre , Receptores de Trombopoyetina/metabolismo , Trombocitemia Esencial/complicaciones , Trombocitemia Esencial/genética , Trombomodulina/sangre , Trombofilia/genéticaRESUMEN
Calreticulin mutation represents the second most frequent mutation after JAK2 V617F in myeloproliferative disorder and is considered to be a driving mutation. Herein the mutation burden was evaluated in patients with essential thrombocythemia or myelofibrosis and found to increase by 5.7% over time unrelated to the time elapsed from the initial to the final positive test. The longer the course of the disease when first tested (range 0-30 years, mean 7.9 years) the lower mutation burden was observed. The mutated clone was larger in type II in comparison with type I mutation when first tested but the difference in mutation burden from the final to the first positive test was significantly higher in those with type I. Similarly, the difference in mutation burden was higher in patients with essential thrombocythemia reaching almost 8% in comparison to 1.3% in post-essential thrombocythemia myelofibrosis. Thus a repeat calreticulin quantitative test is not warranted.
Asunto(s)
Calreticulina/genética , Mutación , Mielofibrosis Primaria/genética , Trombocitemia Esencial/genética , Evolución Clonal/genética , Femenino , Proteínas de Fusión bcr-abl/genética , Humanos , Janus Quinasa 2/genética , Masculino , FenotipoRESUMEN
Liver stiffness and spleen stiffness in patients with myelofibrosis have traditionally been assessed through manual palpation and thus influenced by interobserver variability. In this article, for the first time, liver stiffness and spleen stiffness of patients with myelofibrosis were evaluated through FibroScan and shear wave elastography (SWE). Nine patients with myelofibrosis comprised the study group. They were compared with 11 patients with liver cirrhosis and 8 healthy volunteers. Before the FibroScan study, all patients underwent ultrasound study to delineate the left intercostal space for validated measurements. In patients with myelofibrosis, the mean stiffness of the spleen was 41.3 and 32.9 kilopascals (kPa) through FibroScan and SWE, respectively. The mean stiffness of the liver was 7.8 kPa through FibroScan and 10.4 kPa through SWE. The stiffness of the spleen in patients with cirrhosis was even higher, reaching a mean of 58.5 kPa through FibroScan and 40.5 kPa through SWE. The means were considerably lower among the healthy controls (13.5 and 18.1 kPa, respectively). The correlation between spleen stiffness among the patients with cirrhosis is negative and opposite in direction (r = -0.35) in comparison with the patients with myelofibrosis (r = 0.78). Among the patients with liver cirrhosis and myelofibrosis, spleen size was weakly related to spleen stiffness as assessed through SWE (r = 0.49) but had almost no relation to the FibroScan measure (r = 0.13). The FibroScan and SWE of the spleen have little ability to distinguish between the patients with myelofibrosis and cirrhosis, but they do differentiate both patient groups from the healthy controls. The stiffness of spleen and liver as measured through FibroScan and SWE was not correlated to the longevity of myelofibrosis.
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Diagnóstico por Imagen de Elasticidad/métodos , Interpretación de Imagen Asistida por Computador/métodos , Hígado/diagnóstico por imagen , Hígado/fisiopatología , Mielofibrosis Primaria/diagnóstico por imagen , Bazo/diagnóstico por imagen , Bazo/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Módulo de Elasticidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Variaciones Dependientes del Observador , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: A study was designed to identify the source of fever in a patient with post-polycythemia myelofibrosis, associated with clonal Janus Kinase 2 (JAK2) mutation involving duplication of exon 12. The patient presented with 1-2 day long self-limited periodic episodes of high fever that became more frequent as the hematologic disease progressed. METHODS: After ruling out other causes for recurrent fever, analysis of the pyrin encoding Mediterranean fever gene (MEFV) was carried out by Sanger sequencing in peripheral blood DNA samples obtained 4 years apart, in buccal cells, laser dissected kidney tubular cells, and FACS-sorted CD3-positive or depleted mononucleated blood cells. Hematopoeitc cells results were validated by targeted deep sequencing. A Sanger sequence based screen for pathogenic variants of the autoinflammatory genes NLRP3, TNFRSF1A and MVK was also performed. RESULTS: A rare, c.1955G>A, p.Arg652His MEFV gene variant was identified at negligible levels in an early peripheral blood DNA sample, but affected 46 % of the MEFV alleles and was restricted to JAK2-positive, polymorphonuclear and CD3-depleted mononunuclear DNA samples obtained 4 years later, when the patient experienced fever bouts. The patient was also heterozygous for the germ line, non-pathogenic NLRP3 gene variant, p.Q705K. Upon the administration of colchicine, the gold standard treatment for familial Mediterranean fever (FMF), the fever attacks subsided. CONCLUSIONS: This is the first report of non-transmitted, acquired FMF, associated with a JAK2 driven clonal expansion of a somatic MEFV exon 10 mutation. The non-pathogenic germ line NLRP3 p.Q705K mutation possibly played a modifier role on the disease phenotype.
Asunto(s)
Proteínas del Citoesqueleto/genética , Fiebre Mediterránea Familiar/genética , Janus Quinasa 2/genética , Mutación , Policitemia Vera/genética , Mielofibrosis Primaria/genética , Biopsia , Células Clonales , Colchicina/uso terapéutico , Exones/genética , Fiebre Mediterránea Familiar/diagnóstico , Fiebre Mediterránea Familiar/tratamiento farmacológico , Femenino , Supresores de la Gota/uso terapéutico , Humanos , Persona de Mediana Edad , Policitemia Vera/diagnóstico , Policitemia Vera/tratamiento farmacológico , Polimorfismo de Longitud del Fragmento de Restricción , Mielofibrosis Primaria/diagnóstico , Mielofibrosis Primaria/tratamiento farmacológico , PirinaRESUMEN
INTRODUCTION: Patients with severe combined immunodeficiency (SCID) may present with residual circulating T cells. While all cells are functionally deficient, resulting in high susceptibility to infections, only some of these cells are causing autoimmune symptoms. METHODS: Here we compared T-cell functions including the number of circulating CD3(+) T cells, in vitro responses to mitogens, T-cell receptor (TCR) repertoire, TCR excision circles (TREC) levels, and regulatory T cells (Tregs) enumeration in several immunodeficinecy subtypes, clinically presenting with nonreactive residual cells (MHC-II deficiency) or reactive cells. The latter includes patients with autoreactive clonal expanded T cell and patients with alloreactive transplacentally maternal T cells. RESULTS: MHC-II deficient patients had slightly reduced T-cell function, normal TRECs, TCR repertoires, and normal Tregs enumeration. In contrast, patients with reactive T cells exhibited poor T-cell differentiation and activity. While the autoreactive cells displayed significantly reduced Tregs numbers, the alloreactive transplacentally acquired maternal lymphocytes had high functional Tregs. CONCLUSION: SCID patients presenting with circulating T cells show different patterns of T-cell activity and regulatory T cells enumeration that dictates the immunodeficient and autoimmune manifestations. We suggest that a high-tolerance capacity of the alloreactive transplacentally acquired maternal lymphocytes represents a toleration advantage, yet still associated with severe immunodeficiency.
Asunto(s)
Inmunodeficiencia Combinada Grave/inmunología , Linfocitos T Reguladores/inmunología , Diferenciación Celular/inmunología , Humanos , Tolerancia Inmunológica/inmunología , Lactante , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Lymphocytes establish dynamic cell-cell interactions with the cells they scan. Previous studies show that upon cell contact, various membrane-associated proteins, such as Ras-family proteins, transfer from B to T and NK lymphocytes. Mutations in RAS genes that encode constitutively active, GTP-bound, oncoproteins are rather common in human cancers; for instance, melanoma. Cancer immunoediting has been postulated to contribute to the elimination of malignant melanoma. Thus, we asked whether Ras oncoproteins can transfer from melanoma to T cells, including tumor-infiltrating lymphocytes (TILs), and subsequently induce functional effects in the adopting T cells. To explore this issue, we genetically engineered an HLA-A2(+) melanoma cell line, MEL526, to express GFP or GFP-tagged H-Ras mutants stably. In this study, we show by an in vitro coculture system that GFP-tagged H-Ras, but not GFP, transfers from MEL526 to T cells and localizes to the inner aspect of their plasma membrane. This cell-contact-dependent process was increased by TCR stimulation and did not require strict Ag specificity. Importantly, we found a positive correlation between the levels of the acquired constitutively active H-RasG12V and ERK1/2 phosphorylation within the adopting TILs. We also show a significant increase in IFN-γ production and cytotoxic activity in TILs that acquired H-RasG12V compared to TILs that acquired a different H-Ras mutant. In conclusion, our findings demonstrate a hitherto unknown phenomenon of intercellular transfer of Ras oncoproteins from melanoma to TILs that consequently augments their effector functions.
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Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Melanoma/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Línea Celular , Técnicas de Cocultivo , Humanos , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Melanoma/patología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Linfocitos T/patología , Transfección , Células Tumorales CultivadasRESUMEN
The pathogenesis of juvenile idiopathic arthritis (JIA) is thought to involve multiple components of the cellular immune system, including subsets of γδ T cells. In this study, we conducted experiments to define the functional roles of one of the major synovial fluid (SF) T cell subsets, Vγ9(+)Vδ2(+) (Vγ9(+)) T cells, in JIA. We found that as opposed to CD4(+) T cells, equally high percentages (â¼35%) of Vγ9(+) T cells in SF and peripheral blood (PB) produced TNF-α and IFN-γ. Furthermore, stimulation with isopentenyl pyrophosphate (IPP), a metabolite in the mevalonate pathway, which is a specific potent Ag for Vγ9Jγ1.2(+) T cells, similarly amplified cytokine secretion by SF and PB Vγ9(+) T cells. Significantly, the SF subset expressed higher levels of CD69 in situ, suggesting their recent activation. Furthermore, 24-h coculturing with SF-derived fibroblasts enhanced CD69 on the SF > PB Vγ9(+) T cells, a phenomenon strongly augmented by zoledronate, a farnesyl pyrophosphate synthase inhibitor that increases endogenous intracellular IPP. Importantly, although Vγ9(+) T cell proliferation in response to IPP was significantly lower in SF than PBMC cultures, it could be enhanced by depleting SF CD4(+)CD25(+)FOXP3(+) cells (regulatory T cells). Furthermore, coculture with the Vγ9(+) T cells in medium containing zoledronate or IPP strongly increased SF-derived fibroblasts' apoptosis. The findings that IPP-responsive proinflammatory synovial Vγ9(+) T cells for which proliferation is partly controlled by regulatory T cells can recognize and become activated by SF fibroblasts and then induce their apoptosis suggest their crucial role in the pathogenesis and control of synovial inflammation.
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Artritis Juvenil/inmunología , Comunicación Celular/inmunología , Fibroblastos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Líquido Sinovial/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Artritis Juvenil/patología , Conservadores de la Densidad Ósea/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Niño , Preescolar , Técnicas de Cocultivo , Difosfonatos/inmunología , Difosfonatos/farmacología , Femenino , Fibroblastos/patología , Geraniltranstransferasa/antagonistas & inhibidores , Geraniltranstransferasa/inmunología , Hemiterpenos/inmunología , Humanos , Imidazoles/inmunología , Imidazoles/farmacología , Interferón gamma/inmunología , Lectinas Tipo C/inmunología , Masculino , Compuestos Organofosforados/inmunología , Linfocitos T Reguladores/patología , Factor de Necrosis Tumoral alfa/inmunología , Ácido ZoledrónicoRESUMEN
Emerging data suggest that regulatory T cell (Treg) dysfunction and consequent breakdown of immunological self-tolerance in autoimmunity can be mediated by factors that are not Treg-intrinsic (e.g., cytokines). Indeed, recent studies show that in rheumatoid arthritis the proinflammatory cytokine TNF reduces the suppressive function of Tregs, whereas in vivo TNF blockade restores this function and accordingly self-tolerance. However, until now a coherent mechanism by which TNF regulates the Treg has not been described. In this paper, we show that TNF induces preferential and significant activation of the canonical NF-kappaB pathway in human Tregs as compared with CD25(-) conventional T cells. Furthermore, TNF induced primarily in CD45RA(-) Tregs a transcription program highly enriched for typical NF-kappaB target genes, such as the cytokines lymphotoxin-alpha and TNF, the TNFR superfamily members FAS, 4-1BB, and OX-40, various antiapoptotic genes, and other important immune-response genes. FACS analysis revealed that TNF also induced upregulation of cell surface expression of 4-1BB and OX40 specifically in CD45RA(-)FOXP3(+) Tregs. In contrast, TNF had only a minimal effect on the Treg's core transcriptional signature or on the intracellular levels of the FOXP3 protein in Tregs. Importantly, TNF treatment modulated the capacity of Tregs to suppress the proliferation and IFN-gamma secretion by conventional T cells, an effect that was fully reversed by cotreatment with anti-TNFR2 mAbs. Our findings thus provide new mechanistic insight into the role of TNF and TNFR2 in the pathogenesis of autoimmunity.
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Regulación de la Expresión Génica/inmunología , Tolerancia Inmunológica/inmunología , FN-kappa B/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Autoinmunidad/inmunología , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/inmunología , Expresión Génica , Humanos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Antígenos Comunes de Leucocito/biosíntesis , Antígenos Comunes de Leucocito/inmunología , Activación de Linfocitos/inmunología , FN-kappa B/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/biosíntesis , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Omenn syndrome (OS) is characterized by a peculiar severe T-cell immune deficiency associated with autoimmunelike manifestations. Dysregulations of the central and peripheral immune tolerance, mediated by the protein autoimmune regulator (AIRE) and regulatory T cells, respectively, were proposed as possible mechanisms of this aberrant inflammatory process. OBJECTIVE: We studied mechanisms of central and peripheral tolerance in patients with OS and also examined the gene expression profile associated with OS features. METHODS: T-cell receptor diversity, DNA rearrangement, and the expression of AIRE and forkhead box P3 mRNA as well as the expression of regulatory T cells in cells obtained from patients with OS were studied. Characterization of gene expression in these cells was carried out by using the TaqMan Low-Density Array. RESULTS: Transcript expression of peripheral blood AIRE but not forkhead box P3 was reduced in patients with OS. The expression of natural killer T and regulatory T cells was normal, although the latter showed an abnormal CD4-negative population. Patients with OS have oligoclonal T cells with limited DNA recombination activity, including the presence of early but not late T-cell maturation events, regardless of the genetic defect underlying the syndrome. The transcriptional profile associated with OS features reveals significant changes in 25.5% of the tested genes compared with normal control. CONCLUSION: Our findings suggest that T-cell oligoclonal expansion in OS emanates from an incomplete block before the maturation stage of negative selection, which may explain escape of autoreactive T cells from the thymus. Dysregulated genes in patients with OS are closely involved with self-tolerance and autoimmunity.
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Autoinmunidad , Linfocitos T CD4-Positivos/inmunología , Tolerancia Inmunológica , Receptores de Antígenos de Linfocitos T/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Linfocitos T Reguladores/inmunología , Autoinmunidad/genética , Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Clonales/inmunología , Células Clonales/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Femenino , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Expresión Génica , Reordenamiento Génico de Linfocito T , Humanos , Masculino , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/metabolismo , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología , Proteína AIRERESUMEN
The transcription factor forkhead box P3 (FOXP3 in humans; Foxp3 in mice) controls the development and function of regulatory T cells (Treg). In mice, CD4(+)CD25(-) T cells do not express Foxp3 following TCR activation. Whether FOXP3 is a common activation-induced molecule in human T cells--hence not Treg restricted--is currently a controversial issue. As FOXP3 can significantly modulate the function of T cells, understanding the mode (and regulation) of FOXP3 expression in human T cells is vital. Here we show that in conventional CD4(+)CD25(-) T cells, the induction of FOXP3 expression following TCR activation is both restricted to a fraction of the progeny and transient. Moreover, FOXP3 expression in vivo is particularly infrequent in activated effector CD4(+) T cells that accumulate within inflamed joints. We next demonstrate that the repression of FOXP3 transcription in resting conventional human CD25(-) T cells is linked to complete methylation of an evolutionarily conserved intronic CpG island. The dense methylation pattern is furthermore inherited after activation by progeny. This intronic CpG island, on the other hand, is frequently unmethylated in CD4(+)CD25(+) T cells. Importantly, blocking maintenance DNA methylation, by pharmacological inhibition of DNA methyltransferase-1, induced significant and stable activation-dependent FOXP3 expression in cycling conventional T cells, which was further amplified by co-treatment with transforming growth factor beta. In contrast to natural Treg, such induced CD4(+)FOXP3(+) T cells could produce pro-inflammatory cytokines upon activation. These results indicate that DNA methylation normally restricts FOXP3 transcription in conventional human T cells.