RESUMEN
Chimeric antigen receptor T (CAR-T) cell therapy initiates new methods and turns the scale of clinical treatment on relapsed/refractory acute T lymphoblastic leukemia (T-ALL). In this study, we generated the second-generation CD7-targeting CAR-T cells with a new antigen-binding single-chain variable fragment sequence and made it universal via CRISPR-based knockout of TRAC and CD7 genes (termed UCAR-T). The CD7 UCAR-T cells can efficiently proliferate and lyse T-ALL tumor cell in vitro, along with prominent proinflammatory cytokines secretion. A Jurkat-based xenograft mouse model further verified the superior cytotoxicity of the UCAR-T cells in vivo. During the UCAR-T construction, we observed a CD4/CD8 ratio shift among CD7-/- T/CAR-T cells, which motivated us to further analyze the effects of CD7 antigen on T/CAR-T cells. We sorted out CD7+/- T or anti-CD19 CAR-T cells after partially CD7 knockout and performed functional, phenotypic detection, as well as translational analysis. CD7-/- CAR-T cells tended to be CD8 negative and showed slightly better cytotoxicity at long-term assay. RNA-seq further confirmed an elevation of activated CD4 memory cell subpopulation. However, limited distinction on crucial regulatory genes and pathways was revealed, suggesting the safety and feasibility of UCAR-T application as well as the potential translational rather than transcriptional regulation of CD7 antigen.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Animales , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Antígenos CD7/genética , Inmunoterapia Adoptiva/métodos , Linfocitos T CD4-Positivos , Expresión Génica , Antígenos CD19RESUMEN
Targeting T cell malignancies with universal CD7-targeting chimeric antigen receptor T cells (UCART7) can lead to profound immune deficiency due to loss of normal T and NK cells. While a small population of endogenous CD7- T cells exists, these cells are unlikely to be able to repopulate the entire immune repertoire after UCART7 treatment, as they are limited in number and proliferative capacity. To rescue T and NK cells after UCART7, we created hematopoietic stem cells genetically deleted for CD7 (CD7-KO HSCs). CD7-KO HSCs were able to engraft immunodeficient mice and differentiate into T and NK cells lacking CD7 expression. CD7-KO T and NK cells could perform effector functions as robustly as control T and NK cells. Furthermore, CD7-KO T cells were phenotypically and functionally distinct from endogenous CD7- T cells, indicating that CD7-KO T cells can supplement immune functions lacking in CD7- T cells. Mice engrafted with CD7-KO HSCs maintained T and NK cell numbers after UCART7 treatment, while these were significantly decreased in control mice. These studies support the development of CD7-KO HSCs to augment host immunity in patients with T cell malignancies after UCART7 treatment.
Asunto(s)
Antígenos CD7/genética , Citotoxicidad Inmunológica , Trasplante de Células Madre Hematopoyéticas/métodos , Inmunoterapia Adoptiva/efectos adversos , Animales , Ingeniería Celular/métodos , Edición Génica , Técnicas de Inactivación de Genes , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Leucemia de Células B/inmunología , Leucemia de Células B/terapia , Ratones , RNA-Seq , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Análisis de la Célula Individual , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplante , Quimera por TrasplanteRESUMEN
Although defined as a lymphoid surface marker, CD7 is aberrantly expressed on a subtype of acute myeloid leukemia cells and appears to be associated with an inferior response to chemotherapy. Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative modality but no data has been reported in CD7-positive AML patients. We performed a retrospective analysis involving 141 AML patients who underwent allo-HCT in first morphological complete remission (CR1). The results showed that CD7-positive AML patients had a poor 2-year overall survival (64.5% vs 82.0%, P = 0.040), relapse-free survival (RFS) (56.5% vs 79.4%, P = 0.005), and higher cumulative incidence of relapse (27.0% vs 9.7%, P = 0.003) post-HCT. In addition, expression of CD7 was related to RAS and RUNX1 mutation, and high residual disease level pre-HCT. Multivariate analyses showed CD7 expression at diagnosis was an independent risk factor for RFS (P = 0.016, HR = 0.418) and relapse (P = 0.014, HR = 0.307). We concluded that for AML patients in CR1, CD7 is a negative predictor for allo-transplant outcomes.
Asunto(s)
Antígenos CD7/metabolismo , Biomarcadores de Tumor , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Antígenos CD7/genética , Terapia Combinada , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/terapia , Masculino , Mutación , Cuidados Posoperatorios , Pronóstico , Inducción de Remisión , Estudios Retrospectivos , Factores de Riesgo , Trasplante Homólogo , Resultado del TratamientoRESUMEN
Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by 'T v T' fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in 'self-enrichment' yielding populations 99.6% TCR-/CD3-/CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic 'off-target' activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.
Asunto(s)
Antígenos CD7/genética , Complejo CD3/genética , Inmunoterapia Adoptiva/métodos , Leucemia de Células T/terapia , Linfocitos T/inmunología , Animales , Antígenos CD7/química , Antígenos CD7/metabolismo , Complejo CD3/antagonistas & inhibidores , Complejo CD3/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Femenino , Edición Génica , Humanos , Leucemia de Células T/inmunología , Leucemia de Células T/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The CD7 antigen is a member of the immunoglobulin superfamily that expresses on the surface of all thymocytes, a majority of mature T cells, and also natural killer cells. Interestingly, under physiological and different pathological conditions, the loss of CD7 antigen occurred in the subset of CD4+ memory T cells. Various functions have been proposed for CD7, including its role in the activation and intercellular adhesiveness of T cells. Several studies indicate that the number of CD4+ CD7- T cells increases in diseases such as chronic inflammation and T-cell malignancies, these being skin inflammatory lesions. Therefore, this can be useful for the diagnosis of cancer cells, especially with reference to blood origin, treatment monitoring, and establishment of new therapies. Therefore, a comprehensive review could be useful to increase our knowledge about the clinical importance of these cells in human disease.
Asunto(s)
Antígenos CD7/inmunología , Linfocitos T CD4-Positivos/inmunología , Inflamación/inmunología , Activación de Linfocitos , Neoplasias/inmunología , Animales , Antígenos CD7/genética , Antígenos CD7/metabolismo , Apoptosis , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Neoplasias/metabolismo , Neoplasias/patología , Fenotipo , Transducción de Señal , Escape del TumorRESUMEN
OBJECTIVE: To improve monitoring of myeloid neoplasms by flow cytometry-based minimal residual disease (MRD) analysis, we analyzed the significance of leukemia-associated immunophenotype (LAIP) markers in 44 patients. METHODS: In a pilot study cohort, peripheral blood or bone marrow samples from 13 patients with myeloid neoplasms and one case of B lymphoblastic leukemia in complete hematologic remission after allogeneic bone marrow or stem cell transplantation were subjected to selection for leukemia-specific phenotypes by fluorescence-activated cell sorting using individual marker combinations, followed by PCR-based chimerism analysis. RESULTS: The feasibility of this method could be demonstrated, with selection being successful in 12 cases, including two cases where mixed chimerism was found exclusively in sorted cells. Interestingly, four specimens displayed full donor chimerism in cells expressing the presumably aberrant combination CD34+ /CD7+ . Further analyses, including assessment of an independent cohort of 25 patients not affected by neoplastic bone marrow infiltration, revealed that normal myeloid precursors usually include a population coexpressing CD34, CD13, CD33, and CD7. CONCLUSION: We conclude that the combination CD34+ /CD7+ might not be suitable as an LAIP for MRD diagnostics and that a subset of normal myeloid precursors in the bone marrow expresses CD7.
Asunto(s)
Antígenos CD34/metabolismo , Antígenos CD7/metabolismo , Células Progenitoras Mieloides/metabolismo , Antígenos CD7/genética , Biomarcadores , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Expresión Génica , Humanos , Inmunofenotipificación , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Células Progenitoras Mieloides/patología , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Neoplasia Residual/metabolismoRESUMEN
Regulatory T cells in rejected allograft patients display an inability to control responder T cells. Galectin-1 (Gal1) inhibits responder T cells through binding CD7. We investigated whether the dysfunctional immunoregulation in liver allograft rejection patients results from reduced regulatory T-cell Gal1 expression and/or responder T-cell CD7 expression. Circulating regulatory T cells and responder T cells were profiled from 31 acute rejection transplant patients, 85 transplant patients in remission, and 40 healthy controls. CD7+ and CD7- responder T cells were co-cultured with regulatory T cells to assess regulatory T-cell suppressor function. Gal1-small interfering RNA was used to silence regulatory T-cell Gal1. The CD7+ cell percentage was inversely correlated with AST, ALT, and GGT levels. The proportions of CD7+ responder T cells and Gal1+ regulatory T cells were higher in healthy controls than in transplant patients in remission and lowest in acute rejection transplant patients. Notably, CD7+ responder T-cell susceptibility to Gal1+ regulatory T-cell control was ranked in the same manner. Silencing Gal1 expression in regulatory T cells reduced their ability to suppress CD7+ (but not CD7-) responder T cells. Additionally, the proportions of CD43+ and CD45+ responder T cells were higher in healthy controls than in acute rejection transplant patients. CD43 co-expression (but not CD45 co-expression) on CD7+ responder T cells promoted their apoptosis in a Gal1-dependent manner. In sum, dysfunctional immunoregulation in liver allograft rejection patients can be partly attributed to reduced regulatory T-cell Gal1 expression and reduced responder T-cell CD7 expression. Responder T-cell CD43 downregulation in acute rejection patients may further contribute to reduced responder T-cell responsiveness to regulatory T-cell control.
Asunto(s)
Aloinjertos/inmunología , Antígenos CD7/inmunología , Galectina 1/inmunología , Rechazo de Injerto/inmunología , Hígado/cirugía , Adulto , Anciano , Antígenos CD7/genética , Apoptosis , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/fisiopatología , Carcinoma Hepatocelular/cirugía , Femenino , Galectina 1/genética , Rechazo de Injerto/genética , Humanos , Leucosialina/genética , Leucosialina/inmunología , Hígado/inmunología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/fisiopatología , Neoplasias Hepáticas/cirugía , Trasplante de Hígado , Masculino , Persona de Mediana Edad , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Adulto JovenRESUMEN
T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here, we describe a fratricide-resistant "off-the-shelf" CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide-resistant, allo-tolerant "off-the-shelf" CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphoma without a requirement for autologous T cells.
Asunto(s)
Inmunoterapia Adoptiva , Leucemia de Células T/inmunología , Leucemia de Células T/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Animales , Antígenos CD7/genética , Antígenos CD7/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Sistemas CRISPR-Cas , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Edición Génica , Orden Génico , Vectores Genéticos/genética , Humanos , Inmunoterapia Adoptiva/métodos , Leucemia de Células T/genética , Leucemia de Células T/terapia , Masculino , Ratones , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Extending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies is problematic because most target antigens are shared between normal and malignant cells, leading to CAR T-cell fratricide. CD7 is a transmembrane protein highly expressed in acute T-cell leukemia (T-ALL) and in a subset of peripheral T-cell lymphomas. Normal expression of CD7 is largely confined to T cells and natural killer (NK) cells, reducing the risk of off-target-organ toxicity. Here, we show that the expression of a CD7-specific CAR impaired expansion of transduced T cells because of residual CD7 expression and the ensuing fratricide. We demonstrate that targeted genomic disruption of the CD7 gene prevented this fratricide and enabled expansion of CD7 CAR T cells without compromising their cytotoxic function. CD7 CAR T cells produced robust cytotoxicity against malignant T-cell lines and primary tumors and were protective in a mouse xenograft model of T-ALL. Although CD7 CAR T cells were also toxic against unedited (CD7+) T and NK lymphocytes, we show that the CD7-edited T cells themselves can respond to viral peptides and therefore could be protective against pathogens. Hence, genomic disruption of a target antigen overcomes fratricide of CAR T cells and establishes the feasibility of using CD7 CAR T cells for the targeted therapy of T-cell malignancies.
Asunto(s)
Antígenos CD7/inmunología , Citotoxicidad Inmunológica , Inmunoterapia Adoptiva/métodos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/trasplante , Animales , Antígenos CD7/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Expresión Génica , Silenciador del Gen , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Masculino , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/genética , Linfocitos T/citología , Linfocitos T/inmunología , Transducción Genética , Trasplante HeterólogoRESUMEN
Although numerous recent publications have demonstrated interest in multiparameter flow cytometry in the investigation of myelodysplastic disorders, it is perceived by many laboratory hematologists as difficult and expensive, requiring a high level of expertise. We report a multicentric open real-life study aimed at evaluating the added value of the technically simple flow cytometry score described by the Ogata group for the diagnosis of myelodysplastic syndromes. A total of 652 patients were recruited prospectively in four different centers: 346 myelodysplastic syndromes, 53 myelodysplastic/myeloproliferative neoplasms, and 253 controls. The Ogata score was assessed using CD45 and CD34 staining, with the addition of CD10 and CD19. Moreover, labeling of CD5, CD7 and CD56 for the evaluation of myeloid progenitors and monocytes was tested on a subset of 294 patients. On the whole series, the specificity of Ogata score reached 89%. Respective sensitivities were 54% for low-risk myelodysplastic syndromes, 68% and 84% for type 1 and type 2 refractory anemia with excess of blasts, and 72% for myelodysplastic/myeloproliferative neoplasms. CD5 expression was poorly informative. When adding CD56 or CD7 labeling to the Ogata score, sensitivity rose to 66% for low-risk myelodysplastic syndromes, to 89% for myelodysplastic/myeloproliferative neoplasms and to 97% for refractory anemia with excess of blasts. This large multicenter study confirms the feasibility of Ogata scoring in routine flow cytometry diagnosis but highlights its poor sensitivity in low-risk myelodysplastic syndromes. The addition of CD7 and CD56 in flow cytometry panels improves the sensitivity but more sophisticated panels would be more informative.
Asunto(s)
Antígenos CD7/metabolismo , Antígenos CD5/metabolismo , Antígeno CD56/metabolismo , Inmunofenotipificación , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/metabolismo , Enfermedades Mielodisplásicas-Mieloproliferativas/diagnóstico , Enfermedades Mielodisplásicas-Mieloproliferativas/metabolismo , Anciano , Anciano de 80 o más Años , Antígenos CD7/genética , Antígenos CD5/genética , Antígeno CD56/genética , Diagnóstico Diferencial , Citometría de Flujo , Expresión Génica , Humanos , Inmunofenotipificación/métodos , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Enfermedades Mielodisplásicas-Mieloproliferativas/genética , Sensibilidad y EspecificidadRESUMEN
CD7 is an integral membrane protein which mediates an important signal to mediate the differentiation, activation, and regulation of some T cells and NK cells. However, only human and mouse CD7 have been identified and studied among mammalian species. In this study, we cloned pig CD7 cDNA and determined its complete cDNA sequence. Pig CD7 cDNA contained an open reading frame (627 bp) encoding 208 amino acids with well conserved motifs involved in signal transduction within cytoplasmic tail among mammalian species. Pig CD7 mRNA was detected by RT-PCR in mainly lymphoid tissues, indicating the conserved functions of CD7 in pigs. Moreover, we generated soluble pig CD7 fusion immunoglobulin (pig CD7Ig) containing extracellular domain of pig CD7 to test whether pig CD7 binds to pig galectin-3. Flow cytometry and immunohistochemistry analyses indicated that soluble pig CD7Ig can bind to galectin-3 expressed in macrophages and epithelial cells of small intestine. These results help to analyze the structural relationship between CD7 and its ligand transferring signal transduction among mammalian species.
Asunto(s)
Antígenos CD7/genética , Antígenos CD7/inmunología , Regulación de la Expresión Génica , Porcinos/genética , Porcinos/inmunología , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/química , Galectina 3/metabolismo , Perfilación de la Expresión Génica , Tejido Linfoide/inmunología , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Porcinos/clasificaciónRESUMEN
The purpose of the current study was to find novel rheumatoid arthritis (RA)-specific gene expression by simultaneously comparing the expression profiles of the synovial tissues from patients with RA, osteoarthritis (OA) and ankylosing spondylitis (AS). The Illumina Human HT-12 v4 Expression BeadChip was used to investigate the global gene expression profiles in synovial tissues from RA (n = 12), OA (n = 14) and AS (n = 7) patients. By comparing the profiles in synovial tissues from RA, OA and AS, we identified the CD38, ankyrin repeat domain 38 (ANKRD38), E2F transcription factor 2 (E2F2), craniofacial development protein 1 (CFDP1), cluster of differentiation (CD)7, interferon-stimulated exonuclease gene 20 kDa (ISG20) and interleukin-2 receptor gamma (IL)-2RG genes as differentially expressed gene expression in RA synovial tissues. The increased expression of CD38, E2F2 and IL-2RG, as revealed using real-time polymerase chain reaction (PCR) with synovial tissues from RA (n = 30), OA (n = 26) and AS patients (n = 20), was in agreement with the microarray data. Immunohistochemistry revealed significant CD38 expression and E2F2 in synovial membranes from RA patients (n = 5). The CD38(+) cells had high a percentage in the RA patients' blood (n = 103) and in the CD3(+) and CD56(+) subsets. The CD38(+) cell percentage was correlated significantly with RF level (P = 0·026) in RA patients. The IL-1α and IL-ß levels were depressed significantly in the culture medium of RA synovial fibroblast cells (n = 5) following treatment with siRNAs targeting the E2F2 or CD38 genes. This study suggests that the uniquely increased expression of CD38 and E2F2 in RA synovial tissues contribute to the immunoactivation of the disease.
Asunto(s)
ADP-Ribosil Ciclasa 1/genética , Artritis Reumatoide/genética , Factor de Transcripción E2F2/genética , Membrana Sinovial/metabolismo , Transcriptoma , ADP-Ribosil Ciclasa 1/sangre , ADP-Ribosil Ciclasa 1/metabolismo , Adolescente , Adulto , Anciano , Antígenos CD7/genética , Antígenos CD7/metabolismo , Artritis Reumatoide/metabolismo , Western Blotting , Factor de Transcripción E2F2/metabolismo , Exonucleasas/genética , Exonucleasas/metabolismo , Exorribonucleasas , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Interleucina-1alfa/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Nucleares , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoartritis/genética , Osteoartritis/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espondilitis Anquilosante/genética , Espondilitis Anquilosante/metabolismo , Membrana Sinovial/patología , Adulto JovenRESUMEN
OBJECTIVES: To investigate the expression and function of CD74 in normal renal tissue and clear cell-renal cell carcinoma (ccRCC), as well as related renal tubule epithelial lines. We also analyzed the association between clinicopathological characteristics of ccRCCs and the expression levels of CD74. METHODS: Immunostaining of CD74 was performed in 107 patients' renal tissue and cell lines. We evaluated the association between clinicopathological characteristics of ccRCC and CD74 levels using image analysis. CD74 expression levels were also analyzed by Western blot. Lentivirus-mediated CD74 knockdown inhibited the growth and invasion, of ccRCC cell lines 786-O in vitro and in vivo. Cell proliferation, apoptosis, and invasion as well as HIF-1α pathway-related proteins, were estimated by Western blot. All experiments were repeated at least 3 times. RESULTS: Immunostaining and image analysis showed strong immunoreactions of CD74 in all patients' ccRCC tissue and malignant cell lines, while CD74 expression levels were associated with tumor grade (P = 0.013). Western blot indicated that ccRCC tissue and malignant cell lines expressed higher levels of CD74 and hypoxia inducible factor 1α (HIF-1α) than adjacent normal renal tissue and normal cell HK-2. Vitro and vivo tests demonstrated that lentivirus-mediated CD74 knockdown inhibited the proliferation of ccRCC cell lines, induced G1/S arrest and apoptosis, and inhibited invasion. Inhibition of CD74 resulted in down-regulation of HIF-1α pathway proteins. CONCLUSIONS: CD74 was overexpressed in human ccRCCs and associated with tumor grade, and inhibition of CD74 produced ccRCC proliferation arrest, induced apoptosis, and inhibited invasion, which impinged on HIF-1α pathway-related proteins. It might represent a potential therapeutic target for ccRCC.
Asunto(s)
Antígenos CD7/metabolismo , Carcinoma de Células Renales/metabolismo , Regulación hacia Abajo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/metabolismo , Transducción de Señal , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos CD7/genética , Western Blotting , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Inmunohistoquímica , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Interferencia de ARN , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: Mutations in NPM1 and FLT3 genes represent the most frequent genetic alterations and important diagnostic and prognostic indicators in patients with acute myeloid leukemia (AML). OBJECTIVE: We investigated the prevalence and clinical characteristics of NPM1 and FLT3 mutations in 161 patients of de novo AML including adults and children. RESULTS: NPM1 mutation was found in 21% and FLT3 mutation in 25% of the AML patients. Thirteen (8%) samples were positive for both NPM1 and FLT3/ITD mutations. Adult patients had significantly higher frequency of NPM1 mutation than children (25.8% versus 8.8%; P = 0.02). Further, NPM1 mutation was found to be more frequent in patients above 45 years of age (P = 0.02). NPM1 mutation was significantly associated with higher platelet count (P = 0.05) and absence of hepatosplenomegaly (P = 0.01), while FLT3/ITD mutation was associated with higher white blood count (P = 0.01). Immunophenotypically, NPM1 mutation was associated with the lack of CD34 (P < 0.001) and HLD-DR expression (P < 0.001), while FLT3/ITD mutation was positively associated with the expression of CD7 (P = 0.04). No correlation was found between NPM1 mutation and fusion gene. Interestingly, FLT3/ITD mutation was found to be inversely associated with AML/ETO fusion gene (P = 0.04). CONCLUSIONS: The results suggest that distinct clinical and immunophenotypic characteristics of NPM1 and FLT3/ITD mutations present further insight into the molecular mechanism of leukemogenesis.
Asunto(s)
Leucemia Mieloide Aguda/genética , Mutación , Proteínas Nucleares/genética , Fenotipo , Tirosina Quinasa 3 Similar a fms/genética , Adulto , Factores de Edad , Antígenos CD34/genética , Antígenos CD34/metabolismo , Antígenos CD7/genética , Antígenos CD7/metabolismo , Niño , Femenino , Frecuencia de los Genes , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/inmunología , Masculino , Nucleofosmina , Recuento de PlaquetasRESUMEN
Most T cell responses to pathogens or self antigens are modulated through the action of regulatory T cells and tissue-specific inhibitory mechanisms. To this end, several receptor-ligand pairs have evolved which either augment or diminish T cell function. Here we describe the tissue ligand SECTM1A (Secreted and transmembrane1A) as an alternative murine CD7 ligand. We show that SECTM1A, like SECTM1B, binds strongly to CD7, and that SECTM1B was able to compete with SECTM1A for CD7 binding. SECTM1A is ubiquitously expressed and has two major alternative transcripts which differ in expression between tissues. Both immobilised soluble forms of SECTM1A and SECTM1B and cell surface anchored forms demonstrated opposing effects on CD4+ T cell activation. Whereas SECTM1A acted as a co-stimulator of T cells, enhancing IL-2 production and proliferation, SECTM1B proved inhibitory to TCR mediated T cell activation. Surprisingly, both functional outcomes proved to be CD7-independent, indicating the existence of alternative receptors for both ligands. We used a SECTM1A-Fc fusion protein to immunoprecipitate potential alternative ligands from detergent lysates of CD7(-/-) T cells and, using mass spectrometry, identified GITR as a SECTM1A binder. SECTM1A was found to bind to activated CD4+ and CD8+ T cells as well as to CHO cells expressing cell surface GITR. Binding of SECTM1A to activated primary T cells was inhibited by either GITRL-Fc or anti GITR antibodies. Thus SECTM1A and SECTM1B represent novel reciprocal alternative ligands which may function to modulate the activation of effector and regulatory T cells. The ability of SECTM1A to activate T cells may be explained by its ability to bind to GITR.
Asunto(s)
Antígenos CD7/inmunología , Activación de Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD7/genética , Antígenos CD7/metabolismo , Células CHO , Proliferación Celular , Células Cultivadas , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Proteína Relacionada con TNFR Inducida por Glucocorticoide/inmunología , Proteína Relacionada con TNFR Inducida por Glucocorticoide/metabolismo , Interleucina-2/inmunología , Interleucina-2/metabolismo , Ligandos , Espectrometría de Masas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Datos de Secuencia Molecular , Unión Proteica/inmunología , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Linfocitos T/metabolismo , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/inmunología , Factores de Necrosis Tumoral/metabolismoRESUMEN
The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti-atherosclerotic therapies. Vascular cell adhesion molecule-1 (VCAM-1) mediates both the initial tethering and the firm adhesion of leukocytes to endothelial cells. Our work evaluates the feasibility of using the cytoskeletal anchorage of VCAM-1 as a target for gene therapy. As a proof of concept, integrin alphaIIbbeta3-mediated cell adhesion with clearly defined cytoskeletal anchorage was tested. We constructed fusion proteins containing the intracellular domain of beta3 placed at various distances to the cell membrane. Using cell adhesion assays and immunofluorescence, we established fusion constructs with competitive and dominant negative inhibition of cell adhesion. With the goal being the transfer of the dominant negative mechanism towards VCAM-1 inhibition, we constructed a fusion molecule containing the cytoplasmic domain of VCAM-1. Indeed, VCAM-1 mediated leukocyte adhesion can be inhibited via transfection of DNA encoding the designed VCAM-1 fusion protein. This is demonstrated in adhesion assays under static and flow conditions using CHO cells expressing recombinant VCAM-1 as well as activated endothelial cells. Thus, we are able to describe a novel approach for dominant negative inhibition of leukocyte adhesion to endothelial cells. This approach warrants further development as a novel gene therapeutic strategy that aims for a locally restricted effect at atherosclerotic areas of the vasculature.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Técnicas de Transferencia de Gen , Proteínas Recombinantes/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Antígenos CD7/genética , Antígenos CD7/metabolismo , Células CHO , Cricetinae , Cricetulus , Selectina E/genética , Selectina E/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Monocitos/citología , Monocitos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas Recombinantes/genética , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
BACKGROUND: CD7 is a negative prognostic marker in myeloid malignancies. In acute myeloid leukemia (AML), an inverse correlation exists between expression of wild-type CEBPA and CD7. Aim of this study was to find out whether C/EBPalpha is a negative regulator of CD7 and which other regulatory mechanisms might be involved. RESULTS: As already described for primary AML cells, the majority of AML cell lines tested were either C/EBPalpha+/CD7- or C/EBPalpha-/CD7+. However, the existence of isolated CD7+ cell lines expressing wild-type C/EBPalpha challenges the notion that C/EBPalpha acts as a unique repressor of CD7. Furthermore, ectopic expression of CEBPA did not reduce CD7 in CD7+ cells and knock-down of C/EBPalpha failed to induce CD7 in CD7- cells. In contrast, the DNA demethylating agent Aza-2'deoxycytidine triggered CD7 expression in CD7- AML and in T-cell lines suggesting epigenetic regulation of CD7. Bisulfite sequencing data confirmed that CpGs in the CD7 exon1 region are methylated in CD7- cell lines, and unmethylated in CD7+ cell lines. CONCLUSION: We confirmed an inverse correlation between the expression of wild-type CEBPA and of CD7 in AML cells. Our results contradict the hypothesis that C/EBPalpha acts as repressor for CD7, and instead show that epigenetic mechanisms are responsible for CD7 regulation, in AML cells as well as in T-cells, the typical CD7 expressing cell type.
Asunto(s)
Antígenos CD7/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Epigénesis Genética , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Linfocitos T/metabolismo , Antígenos CD7/genética , Western Blotting , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína alfa Potenciadora de Unión a CCAAT/genética , Metilación de ADN , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Linfocitos T/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Expression levels of the cell surface glycoprotein, CD7, and the serine protease, elastase 2 (ELA2), in the leukemic cells of patients with chronic myeloid leukemia (CML) have been associated with clinical outcome. However, little is known about the mechanisms that underlie the variable expression of these genes in the leukemic cells. RESULTS: To address this question, we compared the level of their expression with the DNA methylation and histone acetylation status of 5' sequences of both genes in leukemic cell lines and primitive (lin-CD34+) leukemic cells from chronic phase CML patients. DNA methylation of the ELA2 gene promoter did not correlate with its expression pattern in lin-CD34+ cells from chronic phase CML patient samples even though there was clear differential DNA methylation of this locus in ELA2-expressing and non-expressing cell lines. In contrast, we found a strong relation between CD7 expression and transcription-permissive chromatin modifications, both at the level of DNA methylation and histone acetylation with evidence of hypomethylation of the CD7 promoter region in the lin-CD34+ cells from CML patients with high CD7 expression. CONCLUSION: These findings indicate a link between epigenetic modifications and CD7 expression in primitive CML cells.
Asunto(s)
Antígenos CD7/metabolismo , Biomarcadores de Tumor/metabolismo , Epigénesis Genética , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Acetilación , Adulto , Anciano , Antígenos CD34/metabolismo , Antígenos CD7/genética , Estudios de Casos y Controles , Línea Celular Tumoral , Metilación de ADN/genética , Femenino , Regulación Leucémica de la Expresión Génica , Histonas/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Persona de Mediana Edad , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transcripción GenéticaRESUMEN
In the periphery, a galectin-1 receptor, CD7, plays crucial roles in galectin-1-mediated apoptosis of activated T-cells as well as progression of T-lymphoma. Previously, we demonstrated that NF-kappaB downregulated CD7 gene expression through the p38 MAPK pathway in developing immature thymocytes. However, its regulatory pathway is not well understood in functional mature T-cells. Here, we show that CD7 expression was downregulated by Twist2 in Jurkat cells, a human acute T-cell lymphoma cell line, and in EL4 cells, a mature murine T-cell lymphoma cell line. Furthermore, ectopic expression of Twist2 in Jurkat cells reduced galectin-1-induced apoptosis. While full-length Twist2 decreased CD7 promoter activity, a C-terminal deletion form of Twist2 reversed its inhibition, suggesting an important role of the C-terminus in CD7 regulation. In addition, CD7 expression was enhanced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate, which indicates that Twist2 might be one of candidate factors involved in histone deacetylation. Based on these results, we conclude that upregulation of Twist2 increases the resistance to galectin-1-mediated-apoptosis, which may have significant implications for the progression of some T-cells into tumors such as Sezary cells.
Asunto(s)
Antígenos CD7/metabolismo , Proteínas Represoras/metabolismo , Síndrome de Sézary/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Animales , Antígenos CD7/genética , Antígenos CD7/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/genética , Progresión de la Enfermedad , Galectina 1/inmunología , Galectina 1/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Células Jurkat , Ratones , Ingeniería de Proteínas , Estructura Terciaria de Proteína/genética , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Eliminación de Secuencia , Síndrome de Sézary/genética , Síndrome de Sézary/patología , Síndrome de Sézary/fisiopatología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patología , Transfección , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/inmunologíaRESUMEN
Modulation of actin dynamics through the N-WASp/Arp2/3 pathway is important in cell locomotion, membrane trafficking, and pathogen infection. Here, we demonstrate that Nck is essential for actin remodeling stimulated by phosphatidylinositol 4,5 bisphosphate (PI(4,5)P(2)) and, conversely, that PI(4,5)P(2) is necessary for localized actin polymerization induced by Nck in vivo. Nck knockdown or knockout suppressed actin comets induced by phosphatidylinositol 5-kinase (PIP5K), and PIP5K stimulated tyrosine phosphorylation of an Nck SH2 domain binding partner, suggesting that Nck couples phosphotyrosine- and phosphoinositide-dependent signals. We show that PI(4,5)P(2) and PIP5K are both enriched at actin comets induced by Nck aggregates and that formation of actin comets was strongly inhibited by coclustering with an inositol 5-phosphatase domain to decrease local PI(4,5)P(2) levels. The extent of Nck-induced actin polymerization was also modulated by PI(4,5)P(2)-sensitive N-WASp mutants. This study uncovers a strong reciprocal interdependence between Nck and PI(4,5)P(2) in promoting localized N-WASp-mediated actin polymerization in cells.