RESUMEN
We performed a retrospective analysis on 124 patients with transfusion-dependent thalassemia who were registered in the German pediatric registry for stem cell transplantation. All patients underwent first allogeneic hematopoietic stem cell transplantation (HSCT) between 2011 and 2020 and belonged mainly to Pesaro risk class 1-2. Four-year overall (OS) and thalassemia-free survival (TFS) were 94.5% ± 2.9% and 88.0% ± 3.4% after treosulfan-fludarabine-thiotepa- and 96.9% ± 3.1% (P = 0.763) and 96.9% ± 3.1% (P = 0.155) after busulfan-fludarabine-based conditioning. Mixed chimerism below 75% occurred predominantly in treosulfan-based regimens (27.5% versus 6.2%). OS and TFS did not differ significantly between matched sibling, other matched family and matched unrelated donor (UD) HSCTs (OS: 100.0%, 100.0%, 96.3% ± 3.6%; TFS: 96.5% ± 2.4%, 90.0% ± 9.5%, 88.9% ± 6.0%). However, mismatched UD-HSCTs performed less favorable (OS: 84.7% ± 7.3% (P = 0.029); TFS: 79.9% ± 7.4% (P = 0.082)). We generated a scoring system reflecting the risk to develop mixed chimerism in our cohort. The main risk-reducing factors were a high CD3+ cell count (≥6 × 107/kg) in the graft, busulfan-conditioning, pre-conditioning therapy and low-targeted ciclosporin A trough levels. Acute GvHD grade III-IV in treosulfan-based concepts predominantly occurred in patients with UD and reduced GvHD prophylaxis but not in the context of high CD3+ cell doses. Taken together, this information might be used to develop more risk-adapted HSCT regimens for thalassemia patients.
Asunto(s)
Busulfano/análogos & derivados , Trasplante de Células Madre Hematopoyéticas , Talasemia , Humanos , Trasplante de Células Madre Hematopoyéticas/métodos , Masculino , Femenino , Niño , Talasemia/terapia , Preescolar , Estudios Retrospectivos , Adolescente , Acondicionamiento Pretrasplante/métodos , Complejo CD3 , Busulfano/uso terapéutico , Busulfano/administración & dosificación , Terapia de Inmunosupresión/métodos , LactanteRESUMEN
BACKGROUNDAdoptive transfer of EBV-specific T cells can restore specific immunity in immunocompromised patients with EBV-associated complications.METHODSWe provide results of a personalized T cell manufacturing program evaluating donor, patient, T cell product, and outcome data. Patient-tailored clinical-grade EBV-specific cytotoxic T lymphocyte (EBV-CTL) products from stem cell donors (SCDs), related third-party donors (TPDs), or unrelated TPDs from the allogeneic T cell donor registry (alloCELL) at Hannover Medical School were manufactured by immunomagnetic selection using a CliniMACS Plus or Prodigy device and the EBV PepTivators EBNA-1 and Select. Consecutive manufacturing processes were evaluated, and patient outcome and side effects were retrieved by retrospective chart analysis.RESULTSForty clinical-grade EBV-CTL products from SCDs, related TPDs, or unrelated TPDs were generated for 37 patients with refractory EBV infections or EBV-associated malignancies with and without a history of transplantation, within 5 days (median) after donor identification. Thirty-four patients received 1-14 EBV-CTL products (fresh and cryopreserved). EBV-CTL transfer led to a complete response in 20 of 29 patients who were evaluated for clinical response. No infusion-related toxicity was reported. EBV-specific T cells in patients' blood were detectable in 16 of 18 monitored patients (89%) after transfer, and their presence correlated with clinical response.CONCLUSIONPersonalized clinical-grade manufacture of EBV-CTL products via immunomagnetic selection from SCDs, related TPDs, or unrelated TPDs in a timely manner is feasible. Overall, EBV-CTLs were clinically effective and well tolerated. Our data suggest EBV-CTL transfer as a promising therapeutic approach for immunocompromised patients with refractory EBV-associated diseases beyond HSCT, as well as patients with preexisting organ dysfunction.TRIAL REGISTRATIONNot applicable.FUNDINGThis study was funded in part by the German Research Foundation (DFG, 158989968/SFB 900), the Deutsche Kinderkrebsstiftung (DKS 2013.09), Wilhelm-Sander-Stiftung (reference 2015.097.1), Ellen-Schmidt-Program of Hannover Medical School, and German Federal Ministry of Education and Research (reference 01EO0802).
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Infecciones por Virus de Epstein-Barr , Inmunoterapia Adoptiva , Humanos , Herpesvirus Humano 4 , Inmunoterapia Adoptiva/métodos , Estudios Retrospectivos , Linfocitos T Citotóxicos , Donante no EmparentadoRESUMEN
Patients with precursor B-cell acute lymphoblastic leukemia (pB-ALL) who have relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT), have relapsed more than once, or are resistant upfront have a dismal prognosis. CD19-targeted chimeric antigen receptor (CAR) T cells have evolved as potent immune therapies. Tisagenlecleucel (Tisa-cel) is a commercially available autologous CD19-directed CAR T-cell product. We performed a retrospective study inviting all CAR T-cell centers in Germany to participate. Eighty-one patients with pB-ALL were included. Twenty-eight days after CAR T-cell infusion, 71 patients (87.7%) were in complete response, and 8 (9.9%) were in nonremission. At 2 years, the probabilities of event-free survival (pEFS), relapse-free survival (pRFS), and overall survival (pOS) were 45.3%, 51.7%, and 53.2%, respectively. pEFS was not different in patients without (n = 16, 55.0%) vs with prior allo-HSCT (n = 65, 43.4%). In patients treated after allo-HSCT, the time to relapse after allo-HSCT was a strong predictor of outcome. Patients relapsing within 6 months of allo-HSCT had a disappointing pEFS of 18.4% (pOS = 16.0%); the pEFS for those relapsing later was 55.5% (pOS = 74.8%). Our study provides real-world experience in pediatric, adolescent, and young adult patients with ALL treated with Tisa-cel, where most patients were treated after having relapsed after allo-HSCT. A total of 45.3% were rescued with a single dose of Tisa-cel. Our novel finding that patients with ALL after allo-HSCT had by far a better pEFS if relapse occurred beyond 6 months might be helpful in clinical decision-making and motivates studies to uncover the reasons.
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Trasplante de Células Madre Hematopoyéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Adulto Joven , Humanos , Niño , Estudios Retrospectivos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inmunoterapia Adoptiva , Recurrencia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Linfocitos TRESUMEN
Total body irradiation (TBI)-based conditioning is associated with superior leukemia-free survival in children with ALL undergoing HSCT. However, the risk for subsequent malignant neoplasms (SMN) remains a significant concern. We analyzed 705 pediatric patients enrolled in the prospective ALL-SCT-BFM-2003 trial and its subsequent registry. Patients >2 years received conditioning with TBI 12 Gy/etoposide (n = 558) and children ≤2 years of age or with contraindications for TBI received busulfan/cyclophosphamide/etoposide (n = 110). The 5- and 10-year cumulative incidence of SMN was 0.02 ± 0.01 and 0.13 ± 0.03, respectively. In total, 39 SMN (34 solid tumors, 5 MDS/AML) were diagnosed in 33 patients at a median of 5.8 years (1.7-13.4), exclusively in the TBI group. Of 33 affected patients, 21 (64%) are alive at a median follow-up of 5.1 years (0-9.9) after diagnosis of their first SMN. In univariate analysis, neither age at HSCT, donor type, acute GVHD, chronic GVHD, nor CMV constituted a significant risk factor for SMN. The only significant risk factor was TBI versus non-TBI based conditioning. This analysis confirms and quantifies the increased risk of SMN in children with ALL after conditioning with TBI. Future strategies to avoid TBI will need careful tailoring within prospective, controlled studies to prevent unfavorable outcomes.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Neoplasias , Humanos , Niño , Irradiación Corporal Total/efectos adversos , Enfermedad Injerto contra Huésped/epidemiología , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/patología , Acondicionamiento Pretrasplante/efectos adversos , Incidencia , Estudios de Seguimiento , Trasplante Homólogo/efectos adversos , Etopósido , Estudios Prospectivos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Busulfano , Ciclofosfamida , Neoplasias/complicacionesRESUMEN
Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for patients affected by Wiskott-Aldrich syndrome (WAS). Reported HSCT outcomes have improved over time with respect to overall survival, but some studies have identified older age and HSCT from alternative donors as risk factors predicting poorer outcome. We analyzed 197 patients undergoing transplant at European Society for Blood and Marrow Transplantation centers between 2006 and 2017 who received conditioning as recommended by the Inborn Errors Working Party (IEWP): either busulfan (n = 103) or treosulfan (n = 94) combined with fludarabine ± thiotepa. After a median follow-up post-HSCT of 44.9 months, 176 patients were alive, resulting in a 3-year overall survival of 88.7% and chronic graft-versus-host disease (GVHD)-free survival (events include death, graft failure, and severe chronic GVHD) of 81.7%. Overall survival and chronic GVHD-free survival were not significantly affected by conditioning regimen (busulfan- vs treosulfan-based), donor type (matched sibling donor/matched family donor vs matched unrelated donor/mismatched unrelated donor vs mismatched family donor), or period of HSCT (2006-2013 vs 2014-2017). Patients aged <5 years at HSCT had a significantly better overall survival. The overall cumulative incidences of grade III to IV acute GVHD and extensive/moderate/severe chronic GVHD were 6.6% and 2.1%, respectively. Patients receiving treosulfan-based conditioning had a higher incidence of graft failure and mixed donor chimerism and more frequently underwent secondary procedures (second HSCT, unconditioned stem cell boost, donor lymphocyte infusion, or splenectomy). In summary, HSCT for WAS with conditioning regimens currently recommended by IEWP results in excellent survival and low rates of GVHD, regardless of donor or stem cell source, but age ≥5 years remains a risk factor for overall survival.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Síndrome de Wiskott-Aldrich , Busulfano/uso terapéutico , Preescolar , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Estudios Retrospectivos , Donantes de Tejidos , Acondicionamiento Pretrasplante/métodos , Resultado del Tratamiento , Síndrome de Wiskott-Aldrich/terapiaRESUMEN
BACKGROUND: Betibeglogene autotemcel (beti-cel) gene therapy for transfusion-dependent ß-thalassemia contains autologous CD34+ hematopoietic stem cells and progenitor cells transduced with the BB305 lentiviral vector encoding the ß-globin (ßA-T87Q) gene. METHODS: In this open-label, phase 3 study, we evaluated the efficacy and safety of beti-cel in adult and pediatric patients with transfusion-dependent ß-thalassemia and a non-ß0/ß0 genotype. Patients underwent myeloablation with busulfan (with doses adjusted on the basis of pharmacokinetic analysis) and received beti-cel intravenously. The primary end point was transfusion independence (i.e., a weighted average hemoglobin level of ≥9 g per deciliter without red-cell transfusions for ≥12 months). RESULTS: A total of 23 patients were enrolled and received treatment, with a median follow-up of 29.5 months (range, 13.0 to 48.2). Transfusion independence occurred in 20 of 22 patients who could be evaluated (91%), including 6 of 7 patients (86%) who were younger than 12 years of age. The average hemoglobin level during transfusion independence was 11.7 g per deciliter (range, 9.5 to 12.8). Twelve months after beti-cel infusion, the median level of gene therapy-derived adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q) was 8.7 g per deciliter (range, 5.2 to 10.6) in patients who had transfusion independence. The safety profile of beti-cel was consistent with that of busulfan-based myeloablation. Four patients had at least one adverse event that was considered by the investigators to be related or possibly related to beti-cel; all events were nonserious except for thrombocytopenia (in 1 patient). No cases of cancer were observed. CONCLUSIONS: Treatment with beti-cel resulted in a sustained HbAT87Q level and a total hemoglobin level that was high enough to enable transfusion independence in most patients with a non-ß0/ß0 genotype, including those younger than 12 years of age. (Funded by Bluebird Bio; HGB-207 ClinicalTrials.gov number, NCT02906202.).
Asunto(s)
Productos Biológicos/uso terapéutico , Terapia Genética/métodos , Globinas beta/genética , Talasemia beta/terapia , Adolescente , Adulto , Productos Biológicos/efectos adversos , Busulfano/uso terapéutico , Niño , Transfusión de Eritrocitos/efectos adversos , Eritropoyesis , Femenino , Vectores Genéticos , Genotipo , Hemoglobinas/análisis , Humanos , Sobrecarga de Hierro/prevención & control , Lentivirus/genética , Masculino , Persona de Mediana Edad , Agonistas Mieloablativos/uso terapéutico , Talasemia beta/sangre , Talasemia beta/genéticaRESUMEN
Germline SAMD9 and SAMD9L mutations (SAMD9/9Lmut) predispose to myelodysplastic syndromes (MDS) with propensity for somatic rescue. In this study, we investigated a clinically annotated pediatric MDS cohort (n = 669) to define the prevalence, genetic landscape, phenotype, therapy outcome and clonal architecture of SAMD9/9L syndromes. In consecutively diagnosed MDS, germline SAMD9/9Lmut accounted for 8% and were mutually exclusive with GATA2 mutations present in 7% of the cohort. Among SAMD9/9Lmut cases, refractory cytopenia was the most prevalent MDS subtype (90%); acquired monosomy 7 was present in 38%; constitutional abnormalities were noted in 57%; and immune dysfunction was present in 28%. The clinical outcome was independent of germline mutations. In total, 67 patients had 58 distinct germline SAMD9/9Lmut clustering to protein middle regions. Despite inconclusive in silico prediction, 94% of SAMD9/9Lmut suppressed HEK293 cell growth, and mutations expressed in CD34+ cells induced overt cell death. Furthermore, we found that 61% of SAMD9/9Lmut patients underwent somatic genetic rescue (SGR) resulting in clonal hematopoiesis, of which 95% was maladaptive (monosomy 7 ± cancer mutations), and 51% had adaptive nature (revertant UPD7q, somatic SAMD9/9Lmut). Finally, bone marrow single-cell DNA sequencing revealed multiple competing SGR events in individual patients. Our findings demonstrate that SGR is common in SAMD9/9Lmut MDS and exemplify the exceptional plasticity of hematopoiesis in children.
Asunto(s)
Evolución Clonal/genética , Hematopoyesis Clonal/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Síndromes Mielodisplásicos/genética , Proteínas Supresoras de Tumor/genética , Adolescente , Células de la Médula Ósea/metabolismo , Niño , Preescolar , Femenino , Factor de Transcripción GATA2/genética , Mutación de Línea Germinal/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Estimación de Kaplan-Meier , Masculino , Síndromes Mielodisplásicos/patología , Análisis de la Célula IndividualRESUMEN
GATA2 deficiency is a heterogeneous multi-system disorder characterized by a high risk of developing myelodysplastic syndrome (MDS) and myeloid leukemia. We analyzed the outcome of 65 patients reported to the registry of the European Working Group (EWOG) of MDS in childhood carrying a germline GATA2 mutation (GATA2mut) who had undergone hematopoietic stem cell transplantation (HSCT). At 5 years the probability of overall survival and disease-free survival (DFS) was 75% and 70%, respectively. Non-relapse mortality and relapse equally contributed to treatment failure. There was no evidence of increased incidence of graft-versus-host-disease or excessive rates of infections or organ toxicities. Advanced disease and monosomy 7 (-7) were associated with worse outcome. Patients with refractory cytopenia of childhood (RCC) and normal karyotype showed an excellent outcome (DFS 90%) compared to RCC and -7 (DFS 67%). Comparing outcome of GATA2mut with GATA2wt patients, there was no difference in DFS in patients with RCC and normal karyotype. The same was true for patients with -7 across morphological subtypes. We demonstrate that HSCT outcome is independent of GATA2 germline mutations in pediatric MDS suggesting the application of standard MDS algorithms and protocols. Our data support considering HSCT early in the course of GATA2 deficiency in young individuals.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Síndromes Mielodisplásicos , Adolescente , Niño , Deleción Cromosómica , Factor de Transcripción GATA2/genética , Mutación de Línea Germinal , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/terapiaRESUMEN
Outcome of 333 children with acute myeloid leukaemia relapsing after a first allogeneic haematopoietic stem cell transplantation was analyzed. Four-year probability of overall survival (4y-pOS) was 14%. 4y-pOS for 122 children receiving a second haematopoietic stem cell transplantation was 31% and 3% for those that did not (P = <0·0001). Achievement of a subsequent remission impacted survival (P = <0·0001). For patients receiving a second transplant survival with or without achieving a subsequent remission was comparable. Graft source (bone marrow vs. peripheral blood stem cells, P = 0·046) and donor choice (matched family vs. matched unrelated donor, P = 0·029) positively impacted survival after relapse. Disease recurrence and non-relapse mortality at four years reached 45% and 22%.
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Trasplante de Células Madre Hematopoyéticas/métodos , Leucemia Mieloide Aguda/terapia , Acondicionamiento Pretrasplante/métodos , Trasplante Homólogo/métodos , Niño , Femenino , Humanos , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Masculino , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
AML SCT-BFM 2007 was the first hematopoietic stem cell transplantation (HCT) trial in Germany to comply with the European Clinical Trials Directive, and aimed to standardize pediatric HCT for acute myeloid leukemia (AML) across centers in Germany, Austria, and the Czech Republic. Children with high-risk features and a good early response achieving a complete first remission (CR-1) and those in CR-2 after a first relapse were stratified to receive HCT from a matched donor after myeloablative conditioning consisting of busulfan, cyclophosphamide, and melphalan. Four-year EFS and OS were 61 and 70%. Cumulative incidence of relapse (CIR) was 22%. TRM was 15% and correlated with age reaching 9% (SE 3%) in children younger than 12 years and 31% (SE 9%) in older children and adolescents. Children with poorly responding primary disease or relapse were allocated to receive early HCT after a cytoreductive regimen with fludarabine, amsacrine, and cytarabine, followed by reduced intensity conditioning and prophylactic donor lymphocyte infusions. Four-year EFS and OS were 49 and 53%. CIR was 38% and TRM 11%. For patients with primary poor response disease, early use of RIC HCT followed by prophylactic DLI can induce long-term remissions in more than 50% (EFS 46% (SE 9%)).
RESUMEN
Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator (rtTA3). For selection of LMO2-expressing ciLPs (LMO2-ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro. LMO2-ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44+CD25- myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44+CD25- myeloid cell subpopulations expressed c-KIT, suggesting that LMO2-ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2-ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44+CD25- myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2-ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.
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Proteínas Adaptadoras Transductoras de Señales/genética , Transformación Celular Neoplásica/genética , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas con Dominio LIM/genética , Células Progenitoras Linfoides/metabolismo , Plásmidos/genética , Proteínas Proto-Oncogénicas/genética , Tetraciclinas/farmacología , Transgenes , Animales , Biomarcadores , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Células Progenitoras Linfoides/patología , Ratones , Ratones Transgénicos , Transducción GenéticaRESUMEN
The transcription factor B cell CLL/lymphoma 11B (BCL11B) is indispensable for T lineage development of lymphoid progenitors. Here, we show that chimeric antigen receptor (CAR) expression during early phases of ex vivo generation of lymphoid progenitors suppressed BCL11B, leading to suppression of T cell-associated gene expression and acquisition of NK cell-like properties. Upon adoptive transfer into hematopoietic stem cell transplant recipients, CAR-expressing lymphoid progenitors differentiated into CAR-induced killer (CARiK) cells that mediated potent antigen-directed antileukemic activity even across MHC barriers. CD28 and active immunoreceptor tyrosine-based activation motifs were critical for a functional CARiK phenotype. These results give important insights into differentiation of murine and human lymphoid progenitors driven by synthetic CAR transgene expression and encourage further evaluation of ex vivo-generated CARiK cells for targeted immunotherapy.
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Antígenos CD28/metabolismo , Células Asesinas Naturales/citología , Linfocitos/citología , Receptores Quiméricos de Antígenos/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T/citología , Proteínas Supresoras de Tumor/metabolismo , Animales , Antígenos CD19/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Separación Celular , Citotoxicidad Inmunológica , Citometría de Flujo , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunoterapia , Inmunoterapia Adoptiva , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Ingeniería de Proteínas , Células Madre/citología , TransgenesAsunto(s)
Neoplasias Primarias Secundarias , Neoplasias , Niño , Humanos , Factores de Riesgo , SobrevivientesRESUMEN
The clinical potential of transplantation is often reduced by T cell-mediated alloresponses that cause graft rejection or graft-versus-host disease. Integrin-mediated adhesion between alloreactive T cells and antigen-presenting cells is essential for allorejection. The identity of the signaling events needed for the activation of integrins such as LFA-1 is poorly understood. Here, we identified a novel role of the protein tyrosine phosphatase SHP-1 in the regulation of murine LFA-1-mediated adhesion in an allograft setting. Upon alloactivation, SHP-1 activity is reduced, resulting in an increase in LFA-1 adhesion compared to that for syngeneically activated T cells. The importance of these differential activation properties was further indicated by small interfering RNA (siRNA) knockdown of SHP-1 in syngeneically and allogeneically stimulated T cells. Mechanistically, SHP-1 modulated the binding of SLP-76 to ADAP by dephosphorylation of the YDGI tyrosine motif of ADAP, a known docking site for the Src family kinase Fyn. This novel key role of SHP-1 in the regulation of LFA-1-mediated adhesion may provide a new insight into T cell-mediated alloresponses and may pave the way to the development of new immunosuppressive pharmaceutical agents.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aloinjertos/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Fosfoproteínas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Linfocitos T/citología , Proteínas Adaptadoras Transductoras de Señales/química , Aloinjertos/metabolismo , Animales , Sitios de Unión , Adhesión Celular , Células Cultivadas , Regulación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Ratones , Fosforilación , Unión Proteica , Linfocitos T/metabolismoRESUMEN
Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer.