RESUMEN
The presence of leukocytes in the cerebral spinal fluid (CSF) of patients with acute lymphoblastic leukemia may indicate a relapse in the central nervous system. CD19-directed immunotherapy may increase the blood-brain barrier permeability, leading to neurologic toxicity and infiltrate the CNS. We studied the CSF cell and protein content in 71 consecutive patients who received either CD19 chimeric antigen receptor T cells or blinatumomab. Responding patients had an incidence of 66% and 61% of pleocytosis following blinatumomab or chimeric antigen receptor T cells, respectively. CSF parameters did not correlate with toxicity or prior CNS disease. Routine CSF flow cytometry following immunotherapy to distinguish T-cell infiltration from CNS relapse should be considered.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores Quiméricos de Antígenos , Niño , Humanos , Adulto Joven , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Inmunoterapia , RecurrenciaRESUMEN
Defects of the glycosylphosphatidylinositol (GPI) biosynthesis pathway constitute an emerging subgroup of congenital disorders of glycosylation with heterogeneous phenotypes. A mutation in the promoter of PIGM, resulting in a syndrome with portal vein thrombosis and persistent absence seizures, was previously described in three patients. We now report four additional patients in two unrelated families, with further clinical, biochemical and molecular delineation of this unique entity. We also describe the first prenatal diagnosis of PIGM deficiency, allowing characterization of the natural history of the disease from birth. The patients described herein expand the phenotypic spectrum of PIGM deficiency to include macrocephaly and infantile-onset cerebrovascular thrombotic events. Finally, we offer insights regarding targeted treatment of this rare disorder with sodium phenylbutyrate.
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Glicosilfosfatidilinositoles/deficiencia , Manosiltransferasas/genética , Megalencefalia/etiología , Vena Porta/patología , Convulsiones/etiología , Trombosis/etiología , Adolescente , Niño , Preescolar , Femenino , Glicosilfosfatidilinositoles/genética , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Megalencefalia/diagnóstico por imagen , Mutación , Regiones Promotoras Genéticas , Convulsiones/complicaciones , Convulsiones/genéticaRESUMEN
BACKGROUND: Graft manipulation using selective depletion of αß-T cells provides a source of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) enriched in effector cells. We report our experience implementing this haplo-HSCT for high-risk malignancies in pediatric patients focusing on the conditioning regimen. PROCEDURE: We performed a retrospective study of patients who underwent T-cell receptor αß-depleted haplo-HSCT for high-risk pediatric malignancies. RESULTS: Eighteen patients underwent haplo-HSCT using this method. The initial reduced-toxicity chemotherapy-based conditioning regimen was given to eight patients, and resulted in a high rate of graft rejections (six of eight patients). Thus, total body irradiation (TBI) based regimen was introduced in the following 10 patients and resulted in engraftment in all patients. Neutrophil and platelet engraftment were rapid (median time to engraft, 10 days and 12 days, respectively). Significant treatment-related complications for both cohorts were all due to graft failure in patients receiving chemotherapy-based conditioning, with a treatment-related mortality rate of 17%. None of the patients developed hepatic sinusoidal-obstruction syndrome, and no grade III-IV acute graft versus host disease (GVHD) was observed. The majority of patients were free of immunosuppression in the first 100 days post-HSCT, and only two patients developed chronic GVHD. The cumulative incidence of relapse was 39%. Compared to patients conditioned with chemotherapy, patients conditioned with TBI had superior actuarial overall survival (66% vs. 37%, P = 0.05) and event-free survival (61% vs. 33%, P = 0.04). CONCLUSIONS: A TBI-based conditioning for haplo-HSCT using αß-T-cell depletion for malignant diseases ensured engraftment and resulted in acceptable outcomes.
Asunto(s)
Supervivencia de Injerto , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Depleción Linfocítica , Neoplasias , Receptores de Antígenos de Linfocitos T alfa-beta , Acondicionamiento Pretrasplante , Adolescente , Niño , Preescolar , Femenino , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/terapia , Humanos , Masculino , Neoplasias/mortalidad , Neoplasias/terapia , Estudios Retrospectivos , Factores de TiempoRESUMEN
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.
Asunto(s)
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
Non-cell-autonomous proteins are incorporated into cells that form tight contacts or are invaded by bacteria, but identifying the full repertoire of transferred proteins has been a challenge. Here we introduce a quantitative proteomics approach to sort out non-cell-autonomous proteins synthesized by other cells or intracellular pathogens. Our approach combines stable-isotope labeling of amino acids in cell culture (SILAC), high-purity cell sorting and bioinformatics analysis to identify the repertoire of relevant non-cell-autonomous proteins. This 'trans-SILAC' method allowed us to discover many proteins transferred from human B to natural killer cells and to measure biosynthesis rates of Salmonella enterica proteins in infected human cells. Trans-SILAC should be a useful method to examine protein exchange between different cells of multicellular organisms or pathogen and host.
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Marcaje Isotópico/métodos , Transporte de Proteínas , Proteoma , Antígeno B7-2/metabolismo , Proteínas Bacterianas/biosíntesis , Comunicación Celular , Células Cultivadas , Humanos , Biosíntesis de ProteínasRESUMEN
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
The T cell receptor (TCR) signaling pathway is an ensemble of numerous proteins that are crucial for an adequate immune response. Disruption of any protein involved in this pathway leads to severe immunodeficiency and unfavorable clinical outcomes. Here, we describe an infant with severe immunodeficiency who was found to have novel biallelic mutations in SLP76. SLP76 is a key protein involved in TCR signaling and in other hematopoietic pathways. Previous studies of this protein were performed using Jurkat-derived human leukemic T cell lines and SLP76-deficient mice. Our current study links this gene, for the first time, to a human immunodeficiency characterized by early-onset life-threatening infections, combined T and B cell immunodeficiency, severe neutrophil defects, and impaired platelet aggregation. Hereby, we characterized aspects of the patient's immune phenotype, modeled them with an SLP76-deficient Jurkat-derived T cell line, and rescued some consequences using ectopic expression of wild-type SLP76. Understanding human diseases due to SLP76 deficiency is helpful in explaining the mixed T cell and neutrophil defects, providing a guide for exploring human SLP76 biology.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Plaquetas/patología , Neutrófilos/patología , Fosfoproteínas/deficiencia , Inmunodeficiencia Combinada Grave/metabolismo , Inmunodeficiencia Combinada Grave/patología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Plaquetas/metabolismo , Resultado Fatal , Humanos , Lactante , Recién Nacido , Células Jurkat , Mutación/genética , Neutrófilos/metabolismo , Fenotipo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Inmunodeficiencia Combinada Grave/inmunología , Transducción de SeñalRESUMEN
BACKGROUND: CD19 chimeric antigen receptor T (CAR-T) cells demonstrate remarkable remission rates in pediatric and adult patients with refractory or relapsed (r/r) acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). In 2016, we initiated a clinical trial with in-house produced CD19 CAR-T cells with a CD28 co-stimulatory domain. We analyzed, for the first time, differences in production features and phenotype between ALL and NHL patients. METHODS: Non-cryopreserved CAR-T cells were produced from patients' peripheral blood mononuclear cells within 9 to 10 days. 93 patients with r/r ALL and NHL were enrolled under the same study. CAR-T cells of ALL and NHL patients were produced simultaneously, allowing the head-to-head comparison. RESULTS: All patients were heavily pretreated. Three patients dropped out from the study due to clinical deterioration (n=2) or production failure (n=1). Cells of ALL patients (n=37) expanded significantly better and contained more CAR-T cells than of NHL patients (n=53). Young age had a positive impact on the proliferation capacity. The infusion products from ALL patients contained significantly more naïve CAR-T cells and a significantly higher expression of the chemokine receptor CXCR3. PD-1, LAG-3, TIM-3, and CD28 were equally expressed. 100% of ALL patients and 94% of NHL patients received the target dose of 1×10e6 CAR-T/kg. The overall response rate was 84% (30/36) in ALL and 62% (32/52) in NHL. We further compared CAR-T cell infusion products to tumor infiltrating lymphocytes (TIL), another common type of T cell therapy, mainly clinically effective in solid tumors. CAR-T cells contained significantly more naïve T cells and central memory T cells and significantly less CCR5 compared to TIL infusion products. CONCLUSIONS: The in-house production of CAR-T cells is highly efficient and fast. Clinical response rate is high. CAR-T cells can be successfully produced for 99% of patients in just 9 to 10 days. Cells derived from ALL patients demonstrate a higher proliferation rate and contain higher frequencies of CAR-T cells and naïve T cells than of NHL patients. In addition, understanding the differences between CAR-T and TIL infusion products, may provide an angle to develop CAR-T cells for the treatment of solid tumors in the future. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov; CAR-T: NCT02772198, First posted: May 13, 2016; TIL: NCT00287131, First posted: February 6, 2006.
Asunto(s)
Antígenos CD19/inmunología , Inmunoterapia Adoptiva/métodos , Leucocitos Mononucleares/inmunología , Linfoma no Hodgkin/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores Quiméricos de Antígenos/inmunología , Adolescente , Adulto , Factores de Edad , Antígenos CD19/genética , Antígenos CD19/metabolismo , Resistencia a Antineoplásicos , Femenino , Humanos , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores Quiméricos de Antígenos/genética , Resultado del Tratamiento , Adulto JovenRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Metilación de ADN , Factores de Transcripción Forkhead , Linfocitos T Reguladores/metabolismo , Artritis Juvenil/genética , Artritis Juvenil/inmunología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Citocinas/metabolismo , Metilasas de Modificación del ADN/antagonistas & inhibidores , Decitabina , Femenino , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Subunidad alfa del Receptor de Interleucina-2 , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Líquido Sinovial/citología , Líquido Sinovial/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Haploidntical hematopoietic stem cell transplantation has been increasingly used in recent years for patients without a matched donor. The αßTCR+/CD19+ depletion technique provide a graft that is enriched with CD34 cells, γδ-T-cells and natural killer. The current experience with αßTCR+/CD19+ depleted grafts in pediatric patients with malignant and non-malignant disorders, demonstrated rapid engraftment, improved immune reconstitution and low risk of GVHD. Future studies will need to define the optimal conditioning regimen in order to improve transplant outcome.
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Antígenos CD19/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Depleción Linfocítica/métodos , Acondicionamiento Pretrasplante/métodos , Trasplante Haploidéntico/métodos , Adolescente , Niño , Preescolar , Femenino , Humanos , MasculinoRESUMEN
Leukocyte adhesion deficiency type 1 (LAD-1) is an autosomal recessive primary immunodeficiency, hallmarked by defective polymorphonuclear transmigration. It is caused by mutations in the gene encoding CD18, which interfere with the CD18/CD11 heterodimerization and expression on leukocyte cell surface. LAD-1 diagnosis rests primarily on the measurement of CD18 expression. However, CD18 measurement entails its pitfalls. Here we present a cohort of ten LAD patients and a review of the relevant literature illustrating the difficulties in sole reliance on CD18 measurement for initial diagnosis. These include normal range expression in some mutations, great variability between patients with the same mutation and subjective interpretation of results. We think there is a need for additional markers as part of the initial LAD diagnostic algorithm. We suggest CD11a expression, which was near absent in all patients in our cohort. The dual use of CD18 and CD11a can increase testing sensitivity and prevent delayed diagnosis of LAD-1.
Asunto(s)
Antígenos CD18/metabolismo , Síndrome de Deficiencia de Adhesión del Leucocito/diagnóstico , Síndrome de Deficiencia de Adhesión del Leucocito/metabolismo , Biomarcadores , Antígenos CD18/genética , Estudios de Casos y Controles , Femenino , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunofenotipificación , Lactante , Recién Nacido , Recuento de Leucocitos , Síndrome de Deficiencia de Adhesión del Leucocito/genética , Síndrome de Deficiencia de Adhesión del Leucocito/terapia , Leucocitos/inmunología , Leucocitos/metabolismo , Masculino , Mutación , Fenotipo , Reproducibilidad de los Resultados , Trasplante HomólogoRESUMEN
Monosomy 21 is an extremely rare genetic disorder presenting with a wide array of symptoms. Recurrent infections, some life threatening, have been reported in several monosomy 21 patients and attributed to an, as of yet, undefined immunodeficiency. Here we report on a 3-year-old boy with mosaic monosomy 21 who presented with clinical and laboratory evidence of immunodeficiency. Despite suffering from infections highly suggestive of a cell-mediated immune defect, the patient's T cells displayed normal counts, subsets and proliferation capability. T cell receptor repertoire was diverse, and de novo T cell production was intact. Consistent with earlier case reports, our patient displayed mildly low B cell counts with hypogammaglobulinemia. B cell subsets demonstrated mainly naïve and marginal zone B cells that have not undergone class switch. Subsequently, IgG, IgA and IgE levels were near absent, whereas IgM level was normal. De novo B cell production and B cell receptor diversity were normal. Together, these results are indicative of a defect in immunoglobulin class switching as the principal cause of immunodeficiency in monosomy 21. A better understanding of the immunodeficiency in this syndrome will enable targeted treatment and prevention of infections in order to prevent morbidity and mortality in these patients.
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Agammaglobulinemia/genética , Linfocitos B/inmunología , Cromosomas Humanos Par 21 , Síndromes de Inmunodeficiencia/genética , Infecciones/genética , Monosomía/genética , Linfocitos T/inmunología , Preescolar , Cromosomas Humanos Par 21/genética , Humanos , Cambio de Clase de Inmunoglobulina/genética , Síndromes de Inmunodeficiencia/diagnóstico , Memoria Inmunológica , Infecciones/diagnóstico , MasculinoAsunto(s)
Antígenos CD19/inmunología , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , Inmunoterapia Adoptiva/métodos , Leucemia Mieloide Aguda/terapia , Linfocitos T/trasplante , Translocación Genética , Adulto , Quimerismo , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Masculino , Pronóstico , Inducción de Remisión , Linfocitos T/inmunología , Linfocitos T/metabolismo , Donantes de Tejidos/provisión & distribuciónRESUMEN
Immune cells establish dynamic adhesive cell-cell interactions at a specific contact region, termed the immunological synapse (IS). Intriguing features of the IS are the formation of regions of plasma membrane fusion and the intercellular exchange of membrane fragments between the conjugated cells. It is not known whether upon IS formation, intact intracellular proteins can transfer from target cells to lymphocytes to allow the transmission of signals across cell boundaries. Here we show by both FACS and confocal microscopy that human lymphocytes acquire from the cells they scan the inner-membrane protein H-Ras, a G-protein vital for common lymphocyte functions and a prominent participant in human cancer. The transfer was cell contact-dependent and occurred in the context of cell-conjugate formation. Moreover, the acquisition of oncogenic H-RasG12V by natural killer (NK) and T lymphocytes had important biological functions in the adopting lymphocytes: the transferred H-RasG12V induced ERK phosphorylation, increased interferon-gamma and tumor necrosis factor-alpha secretion, enhanced lymphocyte proliferation, and augmented NK-mediated target cell killing. Our findings reveal a novel mode of cell-to-cell communication-allowing lymphocytes to extend the confines of their own proteome-which may moreover play an important role in natural tumor immunity.