RESUMEN
Systemic sclerosis (scleroderma, SSc) is an incurable autoimmune disease with high morbidity and mortality rates. Here, we conducted a population-scale single-cell genomic analysis of skin and blood samples of 56 healthy controls and 97 SSc patients at different stages of the disease. We found immune compartment dysfunction only in a specific subtype of diffuse SSc patients but global dysregulation of the stromal compartment, particularly in a previously undefined subset of LGR5+-scleroderma-associated fibroblasts (ScAFs). ScAFs are perturbed morphologically and molecularly in SSc patients. Single-cell multiome profiling of stromal cells revealed ScAF-specific markers, pathways, regulatory elements, and transcription factors underlining disease development. Systematic analysis of these molecular features with clinical metadata associates specific ScAF targets with disease pathogenesis and SSc clinical traits. Our high-resolution atlas of the sclerodermatous skin spectrum will enable a paradigm shift in the understanding of SSc disease and facilitate the development of biomarkers and therapeutic strategies.
Asunto(s)
Esclerodermia Sistémica , Células Cultivadas , Fibroblastos/metabolismo , Fibrosis , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/genética , Piel/metabolismoRESUMEN
Mutations affecting T-cell receptor (TCR) signaling typically cause combined immunodeficiency (CID) due to varying degrees of disturbed T-cell homeostasis and differentiation. Here, we describe two cousins with CID due to a novel nonsense mutation in LCK and investigate the effect of this novel nonsense mutation on TCR signaling, T-cell function, and differentiation. Patients underwent clinical, genetic, and immunological investigations. The effect was addressed in primary cells and LCK-deficient T-cell lines after expression of mutated LCK. RESULTS: Both patients primarily presented with infections in early infancy. The LCK mutation led to reduced expression of a truncated LCK protein lacking a substantial part of the kinase domain and two critical regulatory tyrosine residues. T cells were oligoclonal, and especially naïve CD4 and CD8 T-cell counts were reduced, but regulatory and memory including circulating follicular helper T cells were less severely affected. A diagnostic hallmark of this immunodeficiency is the reduced surface expression of CD4. Despite severely impaired TCR signaling mTOR activation was partially preserved in patients' T cells. LCK-deficient T-cell lines reconstituted with mutant LCK corroborated partially preserved signaling. Despite detectable differentiation of memory and effector T cells, their function was severely disturbed. NK cell cytotoxicity was unaffected. Residual TCR signaling in LCK deficiency allows for reduced, but detectable T-cell differentiation, while T-cell function is severely disturbed. Our findings expand the previous report on one single patient on the central role of LCK in human T-cell development and function.
Asunto(s)
Síndromes de Inmunodeficiencia , Enfermedades de Inmunodeficiencia Primaria , Humanos , Codón sin Sentido , Síndromes de Inmunodeficiencia/diagnóstico , Síndromes de Inmunodeficiencia/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Fosforilación , Enfermedades de Inmunodeficiencia Primaria/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T-cell (CAR T) therapy shows remarkable efficacy in patients with relapsed and/or refractory (R/R) multiple myeloma (MM). HBI0101, a novel second generation optimized anti- BCMA CAR T-cell therapy, was developed in an academic setting. We conducted a phase I dose-escalation study of HBI0101 (cohort 1: 150x106 CAR T cells, n=6; cohort 2: 450x106 CAR T cells, n=7; cohort 3: 800x106 CAR T cells, n=7) in 20 heavily pre-treated R/R MM patients. Grade 1-2 cytokine release syndrome (CRS) was reported in 18 patients (90%). Neither grade 3-4 CRS nor neurotoxicity of any grade were observed. No dose-limiting toxicities were observed in any cohort. The overall response rate (ORR), (stringent) complete response (CR/sCR), and very good partial response rates were 75%, 50%, and 25%, respectively. Response rates were dose-dependent with 85% ORR, 71% CR, and 57% minimal residual disease negativity in the high-dose cohort 3. Across all cohorts, the median overall survival (OS) was 308 days (range 25-466+), with an estimated OS of 55% as of June 27th (data cut-off). The median progression-free survival was 160 days, with 6 subjects remaining progression free at the time of data cut-off. Our findings demonstrate the manageable safety profile and efficacy of HBI0101. These encouraging data support the decentralization of CAR T production in an academic setting, ensuring sufficient CAR T supply to satisfy the increasing local demand. Clinicaltrials.gov NCT04720313.
Asunto(s)
Mieloma Múltiple , Receptores Quiméricos de Antígenos , Humanos , Mieloma Múltiple/tratamiento farmacológico , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Linfocitos T , AnticuerposRESUMEN
Chimeric antigen receptor (CAR) T-cell based immunotherapy has become a promising treatment mainly for hematological malignancies. Following the major success of CD19-targeted CAR, new potential targets for other malignancies are required. As such, B-cell maturation antigen (BCMA) is an attractive tumor-associated antigen to be targeted in multiple myeloma (MM). Herein, we aimed at assessing the function and optimal configuration of different BCMA-specific CAR, based on the same targeting moiety but with a different hinge and co-stimulatory domain. We compared their function to that of a previously characterized BCMA-CAR used in clinical trials. All constructs were expressed at high levels by primary human T cells and could trigger cytokine production and cytotoxicity upon co-culture with multiple myeloma targets. Nonetheless, critical differences were observed in off-target activation, exhaustion, and activation marker expression and in vivo antitumoral activity mediated by these different constructs. Interestingly, we noted that CD8-based hinge, combined with a 4-1BB intracellular domain, proved superior compared to IgG4-connecting regions, and/or a CD28-signaling moiety respectively. Overall, this study emphasizes the influence of CAR primary structure on its function and led to the identification of a highly efficient BCMA-specific CAR, namely H8BB, which displayed superior anti-tumoral activity both in vitro and long-term in vivo efficacy.
Asunto(s)
Mieloma Múltiple , Receptores Quiméricos de Antígenos , Antígeno de Maduración de Linfocitos B/metabolismo , Antígenos CD28 , Citocinas , Humanos , Inmunoglobulina G , Inmunoterapia Adoptiva , Mieloma Múltiple/patologíaRESUMEN
Mutations in Sp110 are the underlying cause of veno-occlusive disease with immunodeficiency (VODI), a combined immunodeficiency that is difficult to treat and often fatal. Because early treatment is critically important for patients with VODI, broadly usable diagnostic tools are needed to detect Sp110 protein deficiency. Several factors make establishing the diagnosis of VODI challenging: (1) Current screening strategies to identify severe combined immunodeficiency are based on measuring T cell receptor excision circles (TREC). This approach will fail to identify VODI patients because the disease is not associated with severe T cell lymphopenia at birth; (2) the SP110 gene contains 17 exons, making it a challenge for Sanger sequencing. The recently developed next-generation sequencing (NGS) platforms that can rapidly determine the sequence of all 17 exons are available in only a few laboratories; (3) there is no standard functional assay to test for the effects of novel mutations in Sp110; and (4) it has been difficult to use flow cytometry to identify patients who lack Sp110 because of the low level of Sp110 protein in peripheral blood lymphocytes. We report here a novel flow cytometric assay that is easily performed in diagnostic laboratories and might thus become a standard assay for the evaluation of patients who may have VODI. In addition, the assay will facilitate investigations directed at understanding the function of Sp110.
Asunto(s)
Citometría de Flujo/métodos , Enfermedad Veno-Oclusiva Hepática/diagnóstico , Síndromes de Inmunodeficiencia/diagnóstico , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Nucleares/metabolismo , Linfocitos T/metabolismo , Adenoviridae/genética , Línea Celular Tumoral , Niño , Preescolar , Femenino , Enfermedad Veno-Oclusiva Hepática/metabolismo , Humanos , Síndromes de Inmunodeficiencia/metabolismo , Leucocitos Mononucleares/citología , Masculino , Antígenos de Histocompatibilidad Menor/genética , Proteínas Nucleares/genéticaRESUMEN
Adenosine deaminase 2 deficiency is an autoinflammatory disease, characterized by various forms of vasculitis. We describe 5 patients with adenosine deaminase 2 deficiency with various hematologic manifestations, including pure red cell aplasia, with no evidence for vasculitis.
Asunto(s)
Adenosina Desaminasa/deficiencia , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Adenosina Desaminasa/genética , Niño , Preescolar , Femenino , Humanos , Lactante , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/genética , FenotipoRESUMEN
Glucocorticoid (GC) hormones have been introduced as therapeutic agents in blood cancers six decades ago. The effectiveness of GC treatment stems from its ability to induce apoptotic death of hemopoietic cells. A major impediment in GC therapy is the acquisition of resistance to the drug upon repeated treatment. In addition, some blood cancers are a priori resistant to GC therapy. Usually, resistance to GC correlates with poor prognosis. Albeit the wide use of GC in clinical practice, their mode of action is not fully understood. The cellular response to GC is initiated by its binding to the cytosolic GC receptor (GR) that translocates to the nucleus and modulates gene expression. However, nuclear activities of GR occur in both apoptosis-sensitive and apoptosis-resistant cells. These apparent controversies can be resolved by deciphering non-genomic effects of GCs and the mode by which they modulate the apoptotic response. We suggest that non-genomic consequences of GC stimulation determine the cell fate toward survival or death. Understanding the cellular mechanisms of GC apoptotic sensitivity contributes to the development of new modalities for overcoming GC resistance.
Asunto(s)
Apoptosis , Resistencia a Antineoplásicos , Glucocorticoides/química , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/terapia , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Citosol/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Linfocitos/citología , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Neoplasias/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Glucocorticoides/metabolismoRESUMEN
ABSTRACT: HBI0101 is an academic chimeric antigen receptor T-cell (CART)-targeted to B-cell maturation antigen (BCMA) for the treatment of relapsed and refractory multiple myeloma (R/RMM) and light chain amyloidosis. Herein, we present the phase 1b/2 results of 50 heavily pretreated patients with R/RMM dosed with 800 × 106 CART cells. Inclusion criteria were relatively permissive (i.e., performance status and baseline organ function) and consequently, approximately half of the enrolled patients would have been ineligible for pivotal clinical trials. The median time elapsed from patient enrollment until CART delivery was 25 days (range, 14-65). HBI0101-related toxicities included grade 1 to 3 cytokine release syndrome, grade 3 to 4 hematologic toxicities, and grade 1 to 2 immune effector cell-associated neurotoxicity syndrome. Responses were achieved in 90% of the patients, 56% achieved stringent and complete response, and 70% reached a minimal residual disease negativity. Within a median follow-up of 12.3 months, the median progression-free survival (PFS) was 11.0 months (95% confidence interval [CI], 6.2-14.6), and the overall survival was not reached (95% CI, 13.3 to not reached). Multivariable analysis on patient/disease and CART-related characteristics revealed that high-risk cytogenetic, extramedullary disease, and increased number of effector-memory T cells in CART products were independently associated with inferior PFS. In conclusion, comprehensive analyses of the parameters affecting the response to CART therapy are essential for improving patients' outcome. This trial was registered at www.ClinicalTrials.gov as #NCT04720313.
Asunto(s)
Antígeno de Maduración de Linfocitos B , Mieloma Múltiple , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/mortalidad , Mieloma Múltiple/terapia , Antígeno de Maduración de Linfocitos B/antagonistas & inhibidores , Masculino , Persona de Mediana Edad , Anciano , Femenino , Adulto , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Resultado del Tratamiento , Receptores Quiméricos de Antígenos/uso terapéutico , Recurrencia , Anciano de 80 o más Años , Anticuerpos Monoclonales HumanizadosRESUMEN
PURPOSE: Although chimeric antigen receptor T therapy (CAR-T) cells are an established therapy for relapsed/refractory multiple myeloma (RRMM), there are no established models predicting outcome to identify patients who may benefit the most from CAR-T. PATIENTS AND METHODS: This is an international retrospective observational study including patients with RRMM infused with currently available commercial or academically produced anti-B-cell maturation antigen (BCMA) CAR-T. We describe characteristics and outcomes in Europe (n = 136) and the United States (n = 133). Independent predictors of relapse/progression built a simple prediction model (Myeloma CAR-T Relapse [MyCARe] model) in the training cohort (Europe), which was externally validated (US cohort) and tested within patient- and treatment-specific subgroups. RESULTS: The overall response rate was 87% and comparable between both cohorts, and complete responses were seen in 48% (Europe) and 49% (the United States). The median time to relapse was 5 months, and early relapse <5 months from infusion showed poor survival across cohorts, with the 12-month overall survival of 30% (Europe) and 14% (the United States). The presence of extramedullary disease or plasma cell leukemia, lenalidomide-refractoriness, high-risk cytogenetics, and increased ferritin at the time of lymphodepletion were independent predictors of early relapse or progression. Each factor received one point, forming the three-tiered MyCARe model: scores 0-1 (low risk), scores 2-3 (intermediate risk), and a score of 4 (high risk). The MyCARe model was significantly associated with distinct 5-month incidence of relapse/progression (P < .001): 7% for low-risk, 27% for intermediate-risk, and 53% for high-risk groups. The model was validated in the US cohort and maintained prognostic utility for response, survival, and outcomes across subgroups. CONCLUSION: Outcomes of patients with RRMM after CAR-T are comparable between Europe and the United States. The MyCARe model may facilitate optimal timing of CAR-T cells in patient-specific subgroups.
Asunto(s)
Antígeno de Maduración de Linfocitos B , Inmunoterapia Adoptiva , Mieloma Múltiple , Humanos , Mieloma Múltiple/terapia , Mieloma Múltiple/mortalidad , Mieloma Múltiple/inmunología , Persona de Mediana Edad , Masculino , Estudios Retrospectivos , Femenino , Anciano , Inmunoterapia Adoptiva/métodos , Antígeno de Maduración de Linfocitos B/inmunología , Estados Unidos , Adulto , Receptores Quiméricos de Antígenos/inmunología , Europa (Continente) , Resultado del Tratamiento , Recurrencia Local de Neoplasia/terapiaRESUMEN
Thymic epithelial cells (TECs) play a central role in T-cell development by presenting self-antigens on MHC proteins. Double-positive (DP) thymocytes that fail to interact with TEC via their TCR die by 'Death by Neglect'. We demonstrated a role for TEC-derived glucocorticoids (GCs) in this process. In a previous study, we used an in vitro system recapitulating Death by Neglect, to demonstrate the involvement of nitric oxide (NO) and inducible NO synthase (iNOS) in this process. In this study, we show that NO synergizes with GCs to induce apoptosis of DP thymocytes in a fetal thymic organ culture. Also, DP thymocytes from iNOSâ»/â» mice are less sensitive to GC-induced apoptosis. Furthermore, the number of DP thymocytes in iNOSâ»/â» mice is higher than in wild-type mice, suggesting a role for NO in Death by Neglect. This phenomenon effects T-cell function profoundly: iNOSâ»/â» T cells do not respond to TCR-mediated activation signals, measured by up-regulation of CD69, IL-2R and IFNγ secretion. This failure to activate is a result of TCR incompetence because iNOâ»/â» T cells respond to TCR-independent stimuli (phorbol myristate acetate and calcium ionophore). This study suggests that NO and GCs synergize to execute TEC-induced death of DP thymocytes.
Asunto(s)
Apoptosis , Células Epiteliales/efectos de los fármacos , Glucocorticoides/farmacología , Óxido Nítrico/metabolismo , Células Precursoras de Linfocitos T/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Timo/inmunología , Animales , Presentación de Antígeno/efectos de los fármacos , Autoantígenos/inmunología , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Células Cultivadas , Selección Clonal Mediada por Antígenos/efectos de los fármacos , Células Epiteliales/inmunología , Humanos , Interferón gamma/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética , Células Precursoras de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Liver fibrosis, which involves activation of hepatic stellate cells (HSC), is a major health problem and is the end outcome of all chronic liver diseases. The liver is populated with lymphocytes, among which are natural killer (NK) cells, whose activity is controlled by inhibitory and activating receptors. NKp46, one of the major NK activating receptors expressed by NK cells, is also a specific NK marker that discriminates NK cells from all other lymphocyte subsets. It recognises viral haemagglutinins and unknown cellular ligands. METHODS: The anti-fibrotic activity of the NKp46 receptor was assessed in vivo and in vitro using NKp46-deficient mice (NCR1(gfp/gfp)), the carbon tetrachloride model and in vitro NK killing assays. Primary murine and human HSC were stained for the expression of the NKp46 ligand using fusion proteins composed of the extracellular portions of the murine and human NKp46 receptors fused to human IgG1. RESULTS: It was shown that murine HSC express a ligand for the murine orthologue of the NKp46 receptor, NCR1. NCR1 inhibited liver fibrosis in vivo; in vitro, murine HSC were killed in an NCR1-dependent manner. In humans it was shown that human HSC also express a ligand for the human NKp46 receptor and that the killing of human HSC is NKp46 dependent. CONCLUSIONS: In addition to NKG2D, NKp46/NCR1 play an important role in inhibition of liver fibrosis. This suggests that fibrosis can be better controlled through the manipulation of NKp46 activity.
Asunto(s)
Células Estrelladas Hepáticas/fisiología , Cirrosis Hepática/fisiopatología , Receptor 1 Gatillante de la Citotoxidad Natural/fisiología , Animales , Antígenos Ly/fisiología , Humanos , Inmunoglobulinas/fisiología , Células Asesinas Naturales/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
BACKGROUNDChronic pain is a debilitating illness with currently limited therapy, in part due to difficulties in translating treatments derived from animal models to patients. The transient receptor potential vanilloid 1 (TRPV1) channel is associated with noxious heat detection and inflammatory pain, and reports of adverse effects in human trials have hindered extensive efforts in the clinical development of TRPV1 antagonists as novel pain relievers.METHODSWe examined 2 affected individuals (A1 and A2) carrying a homozygous missense mutation in TRPV1, rendering the channel nonfunctional. Biochemical and functional assays were used to analyze the mutant channel. To identify possible phenotypes of the affected individuals, we performed psychophysical and medical examinations.RESULTSWe demonstrated that diverse TRPV1 activators, acting at different sites of the channel protein, were unable to open the cloned mutant channel. This finding was not a consequence of impairment in the expression, cellular trafficking, or assembly of protein subunits. The affected individuals were insensitive to application of capsaicin to the mouth and skin and did not demonstrate aversive behavior toward capsaicin. Furthermore, quantitative sensory testing of A1 revealed an elevated heat-pain threshold but also, surprisingly, an elevated cold-pain threshold and extensive neurogenic inflammatory, flare, and pain responses following application of the TRPA1 channel activator mustard oil.CONCLUSIONOur study provides direct evidence in humans for pain-related functional changes linked to TRPV1, which is a prime target in the development of pain relievers.FUNDINGSupported by the Israel Science Foundation (368/19); Teva's National Network of Excellence in Neuroscience grant (no. 0394886) and Teva's National Network of Excellence in Neuroscience postdoctoral fellowship.
Asunto(s)
Canales de Potencial de Receptor Transitorio , Animales , Humanos , Capsaicina/farmacología , Nocicepción , Canales Catiónicos TRPV/metabolismo , Dolor/genéticaRESUMEN
Multiple myeloma (MM) is characterized by recurrent relapses. Consequently, patients receive multiple therapy lines, including alkylating agents and immune modulators, which have been associated with secondary malignancies such as myelodysplastic syndrome (MDS). Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T cell (CART) therapy is efficacious in patients with relapsed/refractory (R/R) MM. However, the long-term complications, particularly MDS, are not well understood. Whether CART therapy causes or promotes MDS has not been thoroughly investigated. In this study, we explored the causal relationship between MDS and CART therapy. We retrospectively examined the prevalence of MDS-related morphological and mutational changes before and after administration of CART therapy in five patients. Among them, four developed MDS after CART therapy, while one had pre-existing MDS prior to CART. None of the four patients who developed post-CART MDS showed morphological MDS changes prior to CART therapy. However, all four patients exhibited molecular alterations associated with MDS in their pre-CART as well as post-CART therapy bone marrow. No new mutations were observed. Our findings provide initial evidence suggesting that anti-BCMA CART therapy in MM may promote expansion of pre-existing MDS clones rather than causing development of new clones.
RESUMEN
PURPOSE: AL amyloidosis (AL) treatments are generally based on those employed for multiple myeloma. Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T (CART)-cell therapy, already approved for multiple myeloma, might be too toxic for patients with AL. EXPERIMENTAL DESIGN: Here we describe the ex vivo applicability of a novel in-house, academic anti-BCMA CAR construct on AL primary cells, as well as the safety and efficacy in 4 patients with relapsed/refractory (RR) primary AL, treated in a phase I clinical trial (NCT04720313). RESULTS: Three had MAYO stage IIIa cardiac involvement at enrollment. The treatment proved relatively safe, with a short and manageable grade 3 cytokine release syndrome evident in 2 patients and no neurotoxicity in any. Cardiac decompensations, observed in 2 patients, were also short and manageable. The overall hematologic response and complete response rates were observed in all patients with an organ response evident in all four. Within a median follow-up period of 5.2 (2.5-9.5) months, all 4 patients maintained their responses. CONCLUSIONS: BCMA-CART cells provide a first proof-of-concept that this therapy is safe enough and highly efficacious for the treatment of patients with advanced, RR AL.
Asunto(s)
Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Mieloma Múltiple , Receptores Quiméricos de Antígenos , Humanos , Estudios de Factibilidad , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/tratamiento farmacológico , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/etiología , Inmunoterapia Adoptiva/efectos adversos , Mieloma Múltiple/tratamiento farmacológicoAsunto(s)
Síndromes de Inmunodeficiencia/diagnóstico , Síndromes de Inmunodeficiencia/genética , Mutación con Pérdida de Función/genética , Subunidad p50 de NF-kappa B/genética , Adulto , Femenino , Humanos , Síndromes de Inmunodeficiencia/inmunología , Mediadores de Inflamación/inmunología , Mutación con Pérdida de Función/inmunología , Subunidad p50 de NF-kappa B/inmunología , Linaje , Adulto JovenRESUMEN
The GTPase of immunity-associated proteins (GIMAPs) are a family of genes believed to contribute to lymphocyte development, signaling, and apoptosis, thus playing an important role in immune system homeostasis. While models of gene derangement have been described in both mice and immortalized cell lines, human examples of these diseases remain exceptionally rare. In this manuscript we describe the first documented human cases of a homozygous deleterious GIMAP6 variant in the GIMAP6 gene and their subsequent clinical and immunological phenotype. In order to interrogate the patients' immune defect, we performed whole-exome sequencing, western blot, flow cytometry analysis, lymphocyte activation and proliferation studies, cytokine release assays, and apoptosis studies. We found two siblings with a predicted deleterious homozygous variant in the GIMAP6 gene with no expression of GIMAP6 protein on western blot. Patients demonstrated accelerated apoptosis, but largely normal lymphocyte subpopulations, activation and proliferation and cytokine release. There appears to be a spectrum of clinical features associated with deficiency of GIMAP6 protein, with one patient suffering lymphopenia and recurrent sinopulmonary infections, and the other clinically asymptomatic. Biallelic variants in the GIMAP6 gene have now been shown to demonstrate disease in humans. The absence of GIMAP6 protein is associated with a spectrum of clinical manifestations and much remains to be learnt about the pathogenic mechanisms underlying this disease. We suggest that biallelic variants in the gene for GIMAP6 should be considered in children with lymphopenia and recurrent sinopulmonary infections.
Asunto(s)
GTP Fosfohidrolasas/genética , Enfermedades de Inmunodeficiencia Primaria/genética , Niño , Citocinas/metabolismo , Femenino , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/metabolismo , Homocigoto , Humanos , Células Jurkat , Subgrupos Linfocitarios/inmunología , Masculino , Mutación , Enfermedades de Inmunodeficiencia Primaria/patologíaRESUMEN
T cell development in the thymus is controlled by thymic epithelial cells (TE). While it is accepted that TE interact with maturing T cells, the mechanisms by which they trigger 'death by neglect' of double-positive (DP) thymocytes are poorly understood. We and others have demonstrated a role for TE-derived glucocorticoids (GCs) in this process. We have studied TE-induced apoptosis using an in vitro system based on co-culturing a thymic epithelial cell line (TEC) with DP thymic lymphoma cells or thymocytes (DP thymic cells). Here, we demonstrate that nitric oxide (NO*) is also involved in this death process. The inducible nitric oxide synthase (iNOS) inhibitors N(G)-methyl-L-arginine and 1,4-PBIT attenuated TEC-induced apoptosis of DP thymic cells. Co-cultivation of TEC with DP thymic cells increased the expression of iNOS in TEC. A concomitant increase in NO* was detected by staining with DAF-FM diacetate. Moreover, the iNOS-regulating cytokines IL-1alpha, IL-1beta and IFNgamma were up-regulated upon interaction of TEC with DP thymic cells. Neutralizing IL-1R or IFNgamma reduced TEC-induced apoptosis of DP thymic cells. Cardinally, NO* synergizes with GCs in eliciting apoptosis of DP thymic cells. Our data indicate that a cross-talk between DP thymic cells and TEC is required for proper induction of iNOS-up-regulating cytokines with a subsequent increase in iNOS expression and NO* production in TEC. NO*, in turn, cooperates with GCs in promoting death by neglect. We suggest that NO* together with GCs fine-tune the T cell selection process.
Asunto(s)
Apoptosis/inmunología , Células Epiteliales/inmunología , Glucocorticoides/inmunología , Óxido Nítrico Sintasa de Tipo II/inmunología , Óxido Nítrico/inmunología , Timo/inmunología , Animales , Apoptosis/efectos de los fármacos , Técnicas de Cocultivo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glucocorticoides/metabolismo , Antagonistas de Hormonas/farmacología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-1alfa/inmunología , Interleucina-1alfa/metabolismo , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mifepristona/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/inmunología , Receptores de Glucocorticoides/metabolismo , Tiourea/análogos & derivados , Tiourea/farmacología , Timo/efectos de los fármacos , Timo/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunología , omega-N-Metilarginina/farmacologíaRESUMEN
Glucocorticoids (GCs) are commonly used in the treatment of hematopoietic malignancies owing to their ability to induce apoptosis of these cancerous cells. Whereas some types of lymphoma and leukemia respond well to this drug, others are resistant. Also, GC-resistance gradually develops upon repeated treatments ultimately leading to refractory relapsed disease. Understanding the mechanisms regulating GC-induced apoptosis is therefore uttermost important for designing novel treatment strategies that overcome GC-resistance. This review discusses updated data describing the complex regulation of the cell's susceptibility to apoptosis triggered by GCs. We address both the genomic and nongenomic effects involved in promoting the apoptotic signals as well as the resistance mechanisms opposing these signals. Eventually we address potential strategies of clinical relevance that sensitize GC-resistant lymphoma and leukemia cells to this drug. The major target is the nongenomic signal transduction machinery where the interplay between protein kinases determines the cell fate. Shifting the balance of the kinome towards a state where Glycogen synthase kinase 3alpha (GSK3alpha) is kept active, favors an apoptotic response. Accumulating data show that it is possible to therapeutically modulate GC-resistance in patients, thereby improving the response to GC therapy.
Asunto(s)
Apoptosis , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Glucocorticoides/metabolismo , Neoplasias Hematológicas/metabolismo , Leucemia/metabolismo , Linfoma/metabolismo , Animales , Linaje de la Célula , Resistencia a Antineoplásicos , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Inmunosupresores/metabolismo , Ratones , Receptores de Glucocorticoides/metabolismoRESUMEN
Glucocorticoid (GC) hormones are an important ingredient of leukemia therapy since they are potent inducers of lymphoid cell apoptosis. However, the development of GC resistance remains an obstacle in GC-based treatment. In the present investigation we found that miR-103 is upregulated in GC-sensitive leukemia cells treated by the hormone. Transfection of GC resistant cells with miR-103 sensitized them to GC induced apoptosis (GCIA), while miR-103 sponging of GC sensitive cells rendered them partially resistant. miR-103 reduced the expression of cyclin dependent kinase (CDK2) and its cyclin E1 target, thereby leading to inhibition of cellular proliferation. miR-103 is encoded within the fifth intron of PANK3 gene. We demonstrate that the GC receptor (GR) upregulates miR-103 by direct interaction with GC response element (GRE) in the PANK3 enhancer. Consequently, miR-103 targets the c-Myc activators c-Myb and DVL1, thereby reducing c-Myc expression. Since c-Myc is a transcription factor of the miR-17~92a poly-cistron, all six miRNAs of the latter are also downregulated. Of these, miR-18a and miR-20a are involved in GCIA, as they target GR and BIM, respectively. Consequently, GR and BIM expression are elevated, thus advancing GCIA. Altogether, this study highlights miR-103 as a useful prognostic biomarker and drug for leukemia management in the future.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/genética , Regulación Leucémica de la Expresión Génica , Glucocorticoides/farmacología , Leucemia/tratamiento farmacológico , Leucemia/genética , MicroARNs/metabolismo , Apoptosis/genética , Proteína 11 Similar a Bcl2/metabolismo , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteínas Dishevelled/genética , Proteínas Dishevelled/metabolismo , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Regulación de la Expresión Génica , Glucocorticoides/uso terapéutico , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Intrones/genética , Leucemia/patología , MicroARNs/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Glucocorticoides/metabolismo , Análisis de Secuencia de ARN , Activación Transcripcional/genética , Transfección , Regulación hacia ArribaRESUMEN
Notch1 is a transcription factor important for T-cell development. Notch1 is active in double negative (DN) thymocytes, while being depressed in double positive (DP) thymocytes. Synchronously, the expression of Bcl-2 becomes downregulated during the transition from DN to DP thymocytes. We previously observed that overexpression of an intracellular active Notch1 (ICN) in Bcl-2-positive 2B4 T cells leads to the transcription of Notch1-regulated genes. However, these genes were not induced in Bcl-2-negative DP PD1.6 thymic lymphoma cells overexpressing ICN. Here we show that, when Bcl-2 is simultaneously introduced into these cells, Notch-regulated genes are transcribed. Only in the presence of both Bcl-2 and ICN, PD1.6 thymic lymphoma cells become resistant to glucocorticoid (GC)-induced apoptosis. Our data suggest that Bcl-2 plays a role in modulating Notch1 function in T cells.