RESUMEN
Rheumatoid arthritis (RA) is one of the most common autoimmune diseases worldwide. Due to high heterogeneity in disease manifestation, accurate and fast diagnosis of RA is difficult. This study analyzed the potential relationship between the infrared (IR) spectra obtained by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and the presence of autoantibodies and antibodies against urease in sera. Additionally, the wave number of the IR spectrum that enabled the best differentiation between patients and healthy blood donors was investigated. Using a mathematical model involving principal component analysis and discriminant analysis, it was shown that the presence of anti-citrullinated protein antibody, rheumatoid factor, anti-neutrophil cytoplasmic antibodies, and anti-nuclear antibodies correlated significantly with the wave numbers in the IR spectra of the tested sera. The most interesting findings derived from determination of the best predictors for distinguishing RA. Characteristic features included an increased reaction with urease mimicking peptides and a correspondence with particular nucleic acid bands. Taken together, the results demonstrated the potential application of ATR-FTIR in the study of RA and identified potential novel markers of the disease.
Asunto(s)
Artritis Reumatoide/inmunología , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Autoanticuerpos/inmunología , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Péptidos/inmunología , Péptidos Cíclicos/inmunología , Factor Reumatoide/sangre , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Background: Only a proportion of patients with bladder cancer may benefit from durable response to immune checkpoint inhibitor (ICI) therapy. More precise indicators of response to immunotherapy are warranted. Our study aimed to construct a more precise classifier for predicting the benefit of immune checkpoint inhibitor therapy. Methods: This multi-cohort study examined the top 20 frequently mutated genes in five cohorts of patients with bladder cancer and developed the TP53/PIK3CA/ATM mutation classifier based on the MSKCC ICI cohort. The classifier was then validated in a validation set consisting of IMvigor210 cohort and Broad/Dana-Farber cohort. The molecular profile and immune infiltration characteristics in each subgroup as defined by this classifier were explored. Results: Among all 881 patients with bladder cancer, the mutation frequency of TP53, PIK3CA, and ATM ranked in the top 20 mutated genes. The TP53/PIK3CA/ATM mutation classifier was constructed based on the Memorial Sloan Kettering Cancer Center (MSKCC) ICI cohort and only showed predictive value for patients with bladder cancer who received ICI therapy (median overall survival: low-risk group, not reached; moderate-risk group, 13.0 months; high-risk group, 8.0 months; P<0.0001). Similar results were found in subgroups of MSKCC ICI cohort defined by tumor mutation burden. Multivariate Cox analysis revealed that the risk group defined by the classifier served as an independent prognostic factor for overall survival in patients with bladder cancer. Efficacy of the classifier was verified in a validation set consisting of IMvigor210 cohort and Broad/Dana-Farber cohort. Lower expression of PD-1/PD-L1 and less tumor immune infiltration were observed in the high-risk group than the other two groups of the TCGA cohort and the IMvigor210 cohort. Conclusion: Our study constructed a TP53/PIK3CA/ATM mutation classifier to predict the benefit of immune checkpoint inhibitor therapy for patients with bladder cancer. This classifier can potentially complement the tumor mutation burden and guide clinical ICI treatment decisions according to distinct risk levels.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Fosfatidilinositol 3-Quinasa Clase I , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Mutación , Proteína p53 Supresora de Tumor , Neoplasias de la Vejiga Urinaria , Adulto , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/inmunología , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Tasa de Supervivencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/mortalidadRESUMEN
Inflammatory bowel disease (IBD) is a chronic inflammatory condition driven by diverse genetic and nongenetic programs that converge to disrupt immune homeostasis in the intestine. We have reported that, in murine intestinal epithelium with telomere dysfunction, DNA damage-induced activation of ataxia-telangiectasia mutated (ATM) results in ATM-mediated phosphorylation and activation of the YAP1 transcriptional coactivator, which in turn up-regulates pro-IL-18, a pivotal immune regulator in IBD pathogenesis. Moreover, individuals with germline defects in telomere maintenance genes experience increased occurrence of intestinal inflammation and show activation of the ATM/YAP1/pro-IL-18 pathway in the intestinal epithelium. Here, we sought to determine the relevance of the ATM/YAP1/pro-IL-18 pathway as a potential driver of IBD, particularly older-onset IBD. Analysis of intestinal biopsy specimens and organoids from older-onset IBD patients documented the presence of telomere dysfunction and activation of the ATM/YAP1/precursor of interleukin 18 (pro-IL-18) pathway in the intestinal epithelium. Employing intestinal organoids from healthy individuals, we demonstrated that experimental induction of telomere dysfunction activates this inflammatory pathway. In organoid models from ulcerative colitis and Crohn's disease patients, pharmacological interventions of telomerase reactivation, suppression of DNA damage signaling, or YAP1 inhibition reduced pro-IL-18 production. Together, these findings support a model wherein telomere dysfunction in the intestinal epithelium can initiate the inflammatory process in IBD, pointing to therapeutic interventions for this disease.
Asunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Telómero/inmunología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Humanos , Enfermedades Inflamatorias del Intestino/genética , Interleucina-18/genética , Interleucina-18/inmunología , Mucosa Intestinal/inmunología , Ratones , Telomerasa/genética , Telomerasa/inmunología , Telómero/genética , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/inmunologíaRESUMEN
Class switch recombination (CSR) changes the effector functions of antibodies and is carried out by classical and alternative nonhomologous end joining (c-NHEJ and A-EJ) of repetitive switch (S) region double-strand breaks (DSBs). The master DNA damage response (DDR) kinase ataxia-telangiectasia mutated (ATM) is critical for CSR in part by suppressing S region DSB resection. However, whether another related DDR kinase ATM- and Rad3-related (ATR) plays similar role in CSR remains elusive. In this study, we investigated the requirement for ATR kinase activity on CSR in both c-NHEJ competent and deficient B cell lines with high-throughput sequencing of S-S junctions. We found that ATR kinase inhibition efficiently blocked both c-NHEJ- and A-EJ-mediated CSR without affecting germline transcription and activation-induced cytosine deaminase expression. In contrast to ATM, ATR does not suppress S region DSB resection and microhomology usage. In addition, ATR kinase inhibition did not affect Cas9-generated DSB end joining by either c-NHEJ and A-EJ. ATR kinase-inhibited stimulated B cells proliferate much slower than controls and exhibited altered cell cycle profile with increased G1 and G2/M phase cells. In summary, our data revealed a role for ATR in promoting both c-NHEJ- and A-EJ-mediated CSR through regulating cell proliferation upon damage without negatively influencing DSB end-joining features.
Asunto(s)
Linfocitos B/inmunología , Ciclo Celular , Roturas del ADN de Doble Cadena , Cambio de Clase de Inmunoglobulina/inmunología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Linfocitos B/metabolismo , Ciclo Celular/inmunología , Línea Celular , Reparación del ADN por Unión de Extremidades/inmunología , RatonesAsunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Daño del ADN , ADN de Neoplasias , Proteínas de Neoplasias , Proteínas Nucleares , Neoplasias de la Próstata/inmunología , Proteínas Represoras , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Daño del ADN/genética , Daño del ADN/inmunología , ADN de Neoplasias/genética , ADN de Neoplasias/inmunología , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Neoplasias de la Próstata/genética , Proteínas Represoras/genética , Proteínas Represoras/inmunologíaRESUMEN
Novel approaches are needed to boost the efficacy of immune checkpoint blockade (ICB) therapy. Ataxia telangiectasia mutated (ATM) protein plays a central role in sensing DNA double-stranded breaks (DSBs) and coordinating their repair. Recent data indicated that ATM might be a promising target to enhance ICB therapy. However, the molecular mechanism involved has not been clearly elucidated. Here, we show that ATM inhibition could potentiate ICB therapy by promoting cytoplasmic leakage of mitochondrial DNA (mtDNA) and activation of the cGAS/STING pathway. We show that genetic depletion of ATM in murine cancer cells delayed tumor growth in syngeneic mouse hosts in a T cell-dependent manner. Furthermore, chemical inhibition of ATM potentiated anti-PD-1 therapy of mouse tumors. ATM inhibition potently activated the cGAS/STING pathway and enhanced lymphocyte infiltration into the tumor microenvironment by downregulating mitochondrial transcription factor A (TFAM), which led to mtDNA leakage into the cytoplasm. Moreover, our analysis of data from a large patient cohort indicated that ATM mutations, especially nonsense mutations, predicted for clinical benefits of ICB therapy. Our study therefore provides strong evidence that ATM may serve as both a therapeutic target and a biomarker to enable ICB therapy.
Asunto(s)
ADN Mitocondrial , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunoterapia , Proteínas de la Membrana , Proteínas de Neoplasias , Neoplasias Experimentales , Nucleotidiltransferasas , Transducción de Señal , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Línea Celular Tumoral , Codón sin Sentido , ADN Mitocondrial/genética , ADN Mitocondrial/inmunología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Mechanistic understanding of how ionizing radiation induces type I interferon signaling and how to amplify this signaling module should help to maximize the efficacy of radiotherapy. In the current study, we report that inhibitors of the DNA damage response kinase ATR can significantly potentiate ionizing radiation-induced innate immune responses. Using a series of mammalian knockout cell lines, we demonstrate that, surprisingly, both the cGAS/STING-dependent DNA-sensing pathway and the MAVS-dependent RNA-sensing pathway are responsible for type I interferon signaling induced by ionizing radiation in the presence or absence of ATR inhibitors. The relative contributions of these two pathways in type I interferon signaling depend on cell type and/or genetic background. We propose that DNA damage-elicited double-strand DNA breaks releases DNA fragments, which may either activate the cGAS/STING-dependent pathway or-especially in the case of AT-rich DNA sequences-be transcribed and initiate MAVS-dependent RNA sensing and signaling. Together, our results suggest the involvement of two distinct pathways in type I interferon signaling upon DNA damage. Moreover, radiation plus ATR inhibition may be a promising new combination therapy against cancer.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Roturas del ADN de Doble Cadena/efectos de la radiación , Interferón Tipo I/inmunología , Radiación Ionizante , Transducción de Señal/efectos de la radiación , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular Tumoral , Humanos , Interferón Tipo I/genética , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
HIV infection leads to a phenomenon of inflammaging, in which chronic inflammation induces an immune aged phenotype, even in individuals on combined antiretroviral therapy (cART) with undetectable viremia. In this study, we investigated T cell homeostasis and telomeric DNA damage and repair machineries in cART-controlled HIV patients at risk for inflammaging. We found a significant depletion of CD4 T cells, which was inversely correlated with the cell apoptosis in virus-suppressed HIV subjects compared to age-matched healthy subjects (HS). In addition, HIV CD4 T cells were prone to DNA damage that extended to chromosome ends-telomeres, leading to accelerated telomere erosion-a hallmark of cell senescence. Mechanistically, the DNA double-strand break (DSB) sensors MRE11, RAD50, and NBS1 (MRN complex) remained intact, but both expression and activity of the DNA damage checkpoint kinase ataxia-telangiectasia mutated (ATM) and its downstream checkpoint kinase 2 (CHK2) were significantly suppressed in HIV CD4 T cells. Consistently, ATM/CHK2 activation, DNA repair, and cellular functions were also impaired in healthy CD4 T cells following ATM knockdown or exposure to the ATM inhibitor KU60019 in vitro, recapitulating the biological effects observed in HIV-derived CD4 T cells in vivo. Importantly, ectopic expression of ATM was essential and sufficient to reduce the DNA damage, apoptosis, and cellular dysfunction in HIV-derived CD4 T cells. These results demonstrate that failure of DSB repair due to ATM deficiency leads to increased DNA damage and renders CD4 T cells prone to senescence and apoptotic death, contributing to CD4 T cell depletion or dysfunction in cART-controlled, latent HIV infection.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Daño del ADN , Infecciones por VIH/inmunología , Linfocitos T/inmunología , Antirretrovirales/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Senescencia Celular , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Humanos , TelómeroRESUMEN
Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15âCD122/É£c signaling in ODN-primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients.
Asunto(s)
Ciclina D2/inmunología , Daño del ADN/inmunología , Interleucina-15/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Factor de Transcripción STAT5/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Proteínas de Ciclo Celular/inmunología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , Proteína 1 de Unión al Supresor Tumoral P53/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Ataxia telangiectasia (AT) is a genetic condition caused by mutations involving ATM (Ataxia Telangiectasia Mutated). This gene is responsible for the expression of a DNA double stranded break repair kinase, the ATM protein kinase. The syndrome encompasses combined immunodeficiency and various degrees of neurological abnormalities and increased risk of malignancy. Typically, patients present early in life with delay in neurological milestones, but very infrequently, with life threatening infections typical of a profound T cell deficiency. It would therefore be unexpected to identify this condition immediately after birth using T cell receptor excision circle (TREC)-based newborn screening (NBS) for SCID. We sought to evaluate the frequency of AT detected by NBS, and to assess immunity as well as the genetic aberrations associated with this early presentation. Here, we describe the clinical, laboratory, and genetic features of patients diagnosed with AT through the Ontario NBS program for SCID, and followed in our center since its inception in 2013. Four patients were diagnosed with AT as a result of low TRECs on NBS. In each case, whole exome sequencing was diagnostic. All of our patients had compound heterozygous mutations involving the FRAP-ATM-TRRAP (FAT) domain of the ATM gene, which appears critical for kinase activity and is highly sensitive to mutagenesis. Our patients presented with profound lymphopenia involving both B and T cells. The ratio of naïve/memory CD45+RA/RO T cells population was variable. T cell repertoire showed decreased T cell diversity. Two out of four patients had decreased specific antibody response to vaccination and hypogammaglobulinemia requiring IVIG replacement. In two patients, profound decreased responses to phytohemagglutinin stimulation was observed. In the other two patients, the initial robust response declined with time. In summary, the rate of detection of AT through NBS had been surprisingly high at our center. One case was identified per year, while the total rate for SCID has been five new cases per year. This early detection may allow for better prospective evaluation of AT shortly after birth, and may assist in formulating early and more effective interventions both for the neurological as well as the immune abnormalities in this syndrome.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Ataxia Telangiectasia , Mutación , Tamizaje Neonatal , Inmunodeficiencia Combinada Grave , Ataxia Telangiectasia/diagnóstico , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/inmunología , Ataxia Telangiectasia/terapia , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Estudios de Casos y Controles , Humanos , Inmunoglobulinas Intravenosas/administración & dosificación , Lactante , Recién Nacido , Masculino , Ontario , Inmunodeficiencia Combinada Grave/diagnóstico , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/terapia , Linfocitos T/inmunología , Linfocitos T/patología , Vacunación , Secuenciación Completa del GenomaRESUMEN
Activation of T cells, a major fraction of peripheral blood lymphocytes (PBLCS), is essential for the immune response. Genotoxic stress resulting from ionizing radiation (IR) and chemical agents, including anticancer drugs, has serious impact on T cells and, therefore, on the immune status. Here we compared the sensitivity of non-stimulated (non-proliferating) vs. CD3/CD28-stimulated (proliferating) PBLC to IR. PBLCs were highly sensitive to IR and, surprisingly, stimulation to proliferation resulted in resistance to IR. Radioprotection following CD3/CD28 activation was observed in different T-cell subsets, whereas stimulated CD34+ progenitor cells did not become resistant to IR. Following stimulation, PBLCs showed no significant differences in the repair of IR-induced DNA damage compared with unstimulated cells. Interestingly, ATM is expressed at high level in resting PBLCs and CD3/CD28 stimulation leads to transcriptional downregulation and reduced ATM phosphorylation following IR, indicating ATM to be key regulator of the high radiosensitivity of resting PBLCs. In line with this, pharmacological inhibition of ATM caused radioresistance of unstimulated, but not stimulated, PBLCs. Radioprotection was also achieved by inhibition of MRE11 and CHK1/CHK2, supporting the notion that downregulation of the MRN-ATM-CHK pathway following CD3/CD28 activation results in radioprotection of proliferating PBLCs. Interestingly, the crosslinking anticancer drug mafosfamide induced, like IR, more death in unstimulated than in stimulated PBLCs. In contrast, the bacterial toxin CDT, damaging DNA through inherent DNase activity, and the DNA methylating anticancer drug temozolomide induced more death in CD3/CD28-stimulated than in unstimulated PBLCs. Thus, the sensitivity of stimulated vs. non-stimulated lymphocytes to genotoxins strongly depends on the kind of DNA damage induced. This is the first study in which the killing response of non-proliferating vs. proliferating T cells was comparatively determined. The data provide insights on how immunotherapeutic strategies resting on T-cell activation can be impacted by differential cytotoxic effects resulting from radiation and chemotherapy.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Antígenos CD28/inmunología , Complejo CD3/inmunología , Rayos gamma , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Clorometilcetonas de Aminoácidos/farmacología , Anticuerpos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Antígenos CD28/antagonistas & inhibidores , Antígenos CD28/genética , Complejo CD3/antagonistas & inhibidores , Complejo CD3/genética , Caspasas/genética , Caspasas/inmunología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Cromonas/farmacología , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/inmunología , Resistencia a Medicamentos/genética , Resistencia a Medicamentos/inmunología , Regulación de la Expresión Génica , Humanos , Isoxazoles/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/efectos de la radiación , Proteína Homóloga de MRE11/antagonistas & inhibidores , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/inmunología , Morfolinas/farmacología , Cultivo Primario de Células , Pirazinas/farmacología , Pironas/farmacología , Tolerancia a Radiación/genética , Tolerancia a Radiación/inmunología , Transducción de Señal , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/efectos de la radiación , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/efectos de la radiación , Tiofenos/farmacología , Tioxantenos/farmacologíaRESUMEN
CD40L/interleukin-4 (IL-4) stimulation occurs in vivo in the tumor microenvironment and induces global translation to varying degrees in individuals with chronic lymphocytic leukemia (CLL) in vitro. However, the implications of CD40L/IL-4 for the translation of specific genes is not known. To determine the most highly translationally regulated genes in response to CD40L/IL-4, we carried out ribosome profiling, a next-generation sequencing method. Significant differences in the translational efficiency of DNA damage response genes, specifically ataxia-telangiectasia-mutated kinase (ATM) and the MRE11/RAD50/NBN (MRN) complex, were observed between patients, suggesting different patterns of translational regulation. We confirmed associations between CD40L/IL-4 response and baseline ATM levels, induction of ATM, and phosphorylation of the ATM targets, p53 and H2AX. X-irradiation was used to demonstrate that CD40L/IL-4 stimulation tended to improve DNA damage repair. Baseline ATM levels, independent of the presence of 11q deletion, correlated with overall survival (OS). Overall, we suggest that there are individual differences in translation of specific genes, including ATM, in response to CD40L/IL-4 and that these interpatient differences might be clinically important.
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Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Ligando de CD40/inmunología , Daño del ADN , Interleucina-4/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Biosíntesis de Proteínas/inmunología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Ligando de CD40/genética , Femenino , Rayos gamma , Histonas/genética , Histonas/inmunología , Humanos , Interleucina-4/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/efectos de la radiación , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Microambiente Tumoral/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
Epstein-Barr virus (EBV) is an ubiquitous herpesvirus with a tropism for epithelial cells (where lytic replication occurs) and B-cells (where latency is maintained). EBV persists throughout life and chronic infection is asymptomatic in most individuals. However, immunocompromised patients may be unable to control EBV infection and are at increased risk of EBV-related malignancies, such as diffuse large B-cell lymphomas or Hodgkin's lymphomas. Ataxia telangiectasia (AT) is a primary immunodeficiency caused by mutations in the ATM gene and associated with an increased incidence of cancers, particularly EBV-associated lymphomas. However, the immune deficiency present in AT patients is often too modest to explain the increased incidence of EBV-related malignancies. The ATM defect in these patients could therefore impair the normal regulation of EBV latency in B-cells, thus promoting lymphomagenesis. This suggests that ATM plays a role in the normal regulation of EBV latency. ATM is a serine/threonine kinase involved in multiple cell functions such as DNA damage repair, cell cycle regulation, oxidative stress, and gene expression. ATM is implicated in the lytic cycle of EBV, where EBV uses the activation of DNA damage repair pathway to promote its own replication. ATM regulates the latent cycle of the EBV-related herpesvirus KSHV and MHV68. However, the contribution of ATM in the control of the latent cycle of EBV is not yet known. A better understanding of the regulation of EBV latency could be harnessed in the conception of novel therapeutic strategies in AT and more generally in all ATM deficient EBV-related malignancies.
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Proteínas de la Ataxia Telangiectasia Mutada/genética , Ataxia Telangiectasia/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Trastornos Linfoproliferativos/inmunología , Animales , Ataxia Telangiectasia/complicaciones , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Huésped Inmunocomprometido/genética , Huésped Inmunocomprometido/inmunología , Incidencia , Trastornos Linfoproliferativos/epidemiología , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/virología , Ratones , Ratones Transgénicos , Activación Viral/inmunología , Latencia del Virus/inmunología , Replicación Viral/inmunologíaRESUMEN
T-cell immunity requires extremely rapid clonal proliferation of rare, antigen-specific T lymphocytes to form effector cells. Here we identify a critical role for ETAA1 in this process by surveying random germ line mutations in mice using exome sequencing and bioinformatic annotation to prioritize mutations in genes of unknown function with potential effects on the immune system, followed by breeding to homozygosity and testing for immune system phenotypes. Effector CD8+ and CD4+ T-cell formation following immunization, lymphocytic choriomeningitis virus (LCMV) infection, or herpes simplex virus 1 (HSV1) infection was profoundly decreased despite normal immune cell development in adult mice homozygous for two different Etaa1 mutations: an exon 2 skipping allele that deletes Gly78-Leu119, and a Cys166Stop truncating allele that eliminates most of the 877-aa protein. ETAA1 deficiency decreased clonal expansion cell autonomously within the responding T cells, causing no decrease in their division rate but increasing TP53-induced mRNAs and phosphorylation of H2AX, a marker of DNA replication stress induced by the ATM and ATR kinases. Homozygous ETAA1-deficient adult mice were otherwise normal, healthy, and fertile, although slightly smaller, and homozygotes were born at lower frequency than expected, consistent with partial lethality after embryonic day 12. Taken together with recently reported evidence in human cancer cell lines that ETAA1 activates ATR kinase through an exon 2-encoded domain, these findings reveal a surprisingly specific requirement for this ATR activator in adult mice restricted to rapidly dividing effector T cells. This specific requirement may provide new ways to suppress pathological T-cell responses in transplantation or autoimmunity.
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Antígenos de Superficie/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , División Celular/inmunología , Inmunidad Celular , Mutación , Animales , Antígenos de Superficie/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , División Celular/genética , Herpes Simple/genética , Herpes Simple/inmunología , Herpes Simple/patología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/inmunología , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/patología , Virus de la Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Mutantes , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
Human papillomavirus (HPV) is the most common sexually transmitted viral infection. Infection with certain types of HPV pose a major public health risk as these types are associated with multiple human cancers, including cervical cancer, other anogenital malignancies and an increasing number of head and neck cancers. The HPV life cycle is closely tied to host cell differentiation with late viral events such as structural gene expression and viral genome amplification taking place in the upper layers of the stratified epithelium. The DNA damage response (DDR) is an elaborate signaling network of proteins that regulate the fidelity of replication by detecting, signaling and repairing DNA lesions. ATM and ATR are two kinases that are major regulators of DNA damage detection and repair. A multitude of studies indicate that activation of the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia and Rad3-related) pathways are critical for HPV to productively replicate. This review outlines how HPV interfaces with the ATM- and ATR-dependent DNA damage responses throughout the viral life cycle to create an environment supportive of viral replication and how activation of these pathways could impact genomic stability.
Asunto(s)
Proteínas de Ciclo Celular/genética , Genoma Viral , Interacciones Huésped-Patógeno , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Daño del ADN , Reparación del ADN , Regulación de la Expresión Génica , Humanos , Queratinocitos/metabolismo , Queratinocitos/virología , Proteínas Oncogénicas Virales/inmunología , Papillomaviridae/crecimiento & desarrollo , Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , Transducción de Señal , Replicación ViralRESUMEN
The innate immune response constitutes the first line of defense against infections by pathogens. Successful pathogens such as human papillomaviruses (HPVs) have evolved mechanisms that target several points in these pathways including sensing of viral genomes, blocking the synthesis of interferons and inhibiting the action of JAK/STAT transcription factors. Disruption of these inhibitory mechanisms contributes to the ability of HPVs to establish persistent infections, which is the major etiological factor in the development of anogenital cancers. Interestingly, HPVs also positively activate several members of these pathways such as STAT-5 that are important for their differentiation-dependent life cycle. STAT-5 activation induces the ATM and ATR DNA damage response pathways that play critical roles in HPV genome amplification. Targeting of these pathways by pharmaceuticals can provide novel opportunities to inhibit infections by these important human pathogens.
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Genoma Viral , Evasión Inmune , Queratinocitos/inmunología , FN-kappa B/genética , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Interferones/genética , Interferones/inmunología , Quinasas Janus/genética , Quinasas Janus/inmunología , Queratinocitos/virología , FN-kappa B/inmunología , Proteínas Oncogénicas Virales/inmunología , Papillomaviridae/crecimiento & desarrollo , Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/patología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transducción de Señal , Replicación ViralRESUMEN
DNA double-strand breaks (DSBs) arise during physiological transcription, DNA replication, and antigen receptor diversification. Mistargeting or misprocessing of DSBs can result in pathological structural variation and mutation. Here we describe a sensitive method (END-seq) to monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We utilized END-seq to determine the frequency and spectrum of restriction-enzyme-, zinc-finger-nuclease-, and RAG-induced DSBs. Beyond sequence preference, chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least one DSB per cell among 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class-switch recombination. Thus, END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice.
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Cromatina/química , Roturas del ADN de Doble Cadena , ADN/genética , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Cromatina/inmunología , ADN/inmunología , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Regulación de la Expresión Génica , Histonas/genética , Histonas/inmunología , Cambio de Clase de Inmunoglobulina/genética , Ratones , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Recombinación Genética , Timocitos/citología , Timocitos/inmunologíaRESUMEN
The airway mucosa is responsible for mounting a robust innate immune response (IIR) upon encountering pathogen-associated molecular patterns. The IIR produces protective gene networks that stimulate neighboring epithelia and components of the immune system to trigger adaptive immunity. Little is currently known about how cellular reactive oxygen species (ROS) signaling is produced and cooperates in the IIR. We discuss recent discoveries about 2 nuclear ROS signaling pathways controlling innate immunity. Nuclear ROS oxidize guanine bases to produce mutagenic 8-oxoguanine, a lesion excised by 8-oxoguanine DNA glycosylase1/AP-lyase (OGG1). OGG1 forms a complex with the excised base, inducing its nuclear export. The cytoplasmic OGG1:8-oxoG complex functions as a guanine nucleotide exchange factor, triggering small GTPase signaling and activating phosphorylation of the nuclear factor (NF)x03BA;B/RelA transcription factor to induce immediate early gene expression. In parallel, nuclear ROS are detected by ataxia telangiectasia mutated (ATM), a PI3 kinase activated by ROS, triggering its nuclear export. ATM forms a scaffold with ribosomal S6 kinases, inducing RelA phosphorylation and resulting in transcription-coupled synthesis of type I and type III interferons and CC and CXC chemokines. We propose that ATM and OGG1 are endogenous nuclear ROS sensors that transmit nuclear signals that coordinate with outside-in pattern recognition receptor signaling, regulating the IIR.
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Núcleo Celular/inmunología , Inmunidad Innata , Pulmón/inmunología , Especies Reactivas de Oxígeno/inmunología , Transducción de Señal/inmunología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Núcleo Celular/patología , ADN Glicosilasas/inmunología , Guanina/análogos & derivados , Guanina/inmunología , Humanos , Pulmón/patología , Oxidación-Reducción , Fosfatidilinositol 3-Quinasas/inmunología , Proteínas Quinasas S6 Ribosómicas/inmunología , Factor de Transcripción ReIA/inmunologíaRESUMEN
The serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M(+) B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell-like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors.
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Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Vigilancia Inmunológica , Linfoma de Células B Grandes Difuso/inmunología , Proteínas Supresoras de Tumor/deficiencia , Animales , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Caspasas/genética , Caspasas/inmunología , Línea Celular Tumoral , Inestabilidad Cromosómica/inmunología , Sitios Genéticos/inmunología , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones Noqueados , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/genética , FN-kappa B/inmunología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Linfocitos T/inmunología , Linfocitos T/patología , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
Polymorphisms of genes involved in innate and adaptive immunity have become an object of major interest in regard to hematopoietic stem cell transplantation (HSCT) complications. Regimen-related gastrointestinal toxicity (RR-GIT) is the dominant complication during the pre-engraftment period and has been linked to increased risk of graft-versus-host disease (GVHD) development. According to our hypothesis, functional variants of genes participating in DNA damage response (DDR) may have an impact on the extent of tissue damage caused by the conditioning regimen. In our single-center study, we analyzed 62 patients who underwent HSCT from HLA-identical donors after reduced conditioning. The patients were genotyped for 5 single nucleotide polymorphisms (SNPs, rs4585 T/G, rs189037 A/G, rs227092 T/G, rs228590 C/T, and rs664677 T/C) of the ATM gene-the essential member of the DDR pathways, using allele-specific matrix-assisted laser desorption/ionization, time-of-flight (MALDI-TOF) mass spectrometry assay. Because of almost absolute linkage disequilibrium observed among all 5 SNPs, association of 2 major ATM haplotypes (ATM1/ATM2) with RR-GIT and acute GVHD (aGVHD) was analyzed. Importantly, the univariate and multivariate analysis showed that patients homozygous for ATM2 haplotype (rs4585*T, rs189037*A, rs227092*T, rs228590*C, and rs664677*T) are more likely to suffer from high-grade RR-GIT than ATM1 homozygous patients. The association with aGVHD was not significant. To our knowledge, this is the first report showing the ATM gene variability in relation to RR-GIT in the allogeneic HSCT setting.