RESUMO
Patients with acute myeloid leukaemia (AML) have a five-year survival rate of 28·7%. Natural killer (NK)-cell have anti-leukaemic activity. Here, we report on a series of 13 patients with high-risk R/R AML, treated with repeated infusions of double-bright (CD56bright /CD16bright ) expanded NK cells at an academic centre in Brazil. NK cells from HLA-haploidentical donors were expanded using K562 feeder cells, modified to express membrane-bound interleukin-21. Patients received FLAG, after which cryopreserved NK cells were thawed and infused thrice weekly for six infusions in three dose cohorts (106 -107 cells/kg/infusion). Primary objectives were safety and feasibility. Secondary endpoints included overall response (OR) and complete response (CR) rates at 28-30 days after the first infusion. Patients received a median of five prior lines of therapy, seven with intermediate or adverse cytogenetics, three with concurrent central nervous system (CNS) leukaemia, and one with concurrent CNS mycetoma. No dose-limiting toxicities, infusion-related fever, or cytokine release syndrome were observed. An OR of 78·6% and CR of 50·0% were observed, including responses in three patients with CNS disease and clearance of a CNS mycetoma. Multiple infusions of expanded, cryopreserved NK cells were safely administered after intensive chemotherapy in high-risk patients with R/R AML and demonstrated encouraging outcomes.
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Antígeno CD56/análise , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/transplante , Leucemia Mieloide Aguda/terapia , Receptores de IgG/análise , Adolescente , Adulto , Brasil/epidemiologia , Antígeno CD56/imunologia , Criança , Feminino , Proteínas Ligadas por GPI/análise , Proteínas Ligadas por GPI/imunologia , Doença Enxerto-Hospedeiro/etiologia , Humanos , Imunoterapia Adotiva/efeitos adversos , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mieloide Aguda/imunologia , Masculino , Pessoa de Meia-Idade , Estudo de Prova de Conceito , Receptores de IgG/imunologia , Adulto JovemRESUMO
Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention.
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Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Resistencia a Medicamentos Antineoplásicos/imunologia , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Linfócitos T Citotóxicos/transplante , Animais , Apoptose , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/genética , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/genética , Proliferação de Células , Feminino , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/terapia , Camundongos , Prognóstico , Taxa de Sobrevida , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A distributed curvature sensor based on Brillouin optical time-domain reflectometry interrogation technique in a D-shaped 7-core fibre is presented. By comparing the relative Brillouin frequency shift between the central core and three of the outer cores of the 7-core fibre, the curvature of various spools with different diameters is measured with a deviation from the actual value ranging between 9% and 15%. The analysis and results presented in this study show the first demonstration of distributed bend sensing using a specially designed multicore D-shaped fibre, paving the way for fully distributed 3D shape sensing.
RESUMO
There was a typo in author Andrew Wahba's name in the initial online publication. The original article has been corrected.
RESUMO
PURPOSE: Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. METHODS: To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. RESULTS: The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F-labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. CONCLUSIONS: Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.
Assuntos
Neoplasias Cerebelares/prevenção & controle , Monitoramento de Medicamentos/métodos , Radioisótopos de Flúor/uso terapêutico , Células Matadoras Naturais/transplante , Imageamento por Ressonância Magnética/métodos , Meduloblastoma/prevenção & controle , Animais , Apoptose , Proliferação de Células , Neoplasias Cerebelares/imunologia , Neoplasias Cerebelares/patologia , Humanos , Meduloblastoma/imunologia , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.
Assuntos
Interleucina-15/metabolismo , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/fisiologia , Animais , Antígenos CD19/metabolismo , Humanos , Imunoterapia Adotiva , Ativação Linfocitária , Camundongos , Células Precursoras de Linfócitos T/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Myeloablative autologous hematopoietic stem cell transplantation (HSCT) is a mainstay of therapy for relapsed intermediate-grade B-cell non-Hodgkin lymphoma (NHL); however, relapse rates are high. In phase 1 studies designed to improve long-term remission rates, we administered adoptive T-cell immunotherapy after HSCT, using ex vivo-expanded autologous central memory-enriched T cells (TCM) transduced with lentivirus expressing CD19-specific chimeric antigen receptors (CARs). We present results from 2 safety/feasibility studies, NHL1 and NHL2, investigating different T-cell populations and CAR constructs. Engineered TCM-derived CD19 CAR T cells were infused 2 days after HSCT at doses of 25 to 200 × 10(6) in a single infusion. In NHL1, 8 patients safely received T-cell products engineered from enriched CD8(+) TCM subsets, expressing a first-generation CD19 CAR containing only the CD3ζ endodomain (CD19R:ζ). Four of 8 patients (50%; 95% confidence interval [CI]: 16-84%) were progression free at both 1 and 2 years. In NHL2, 8 patients safely received T-cell products engineered from enriched CD4(+) and CD8(+) TCM subsets and expressing a second-generation CD19 CAR containing the CD28 and CD3ζ endodomains (CD19R:28ζ). Six of 8 patients (75%; 95% CI: 35-97%) were progression free at 1 year. The CD4(+)/CD8(+) TCM-derived CD19 CAR T cells (NHL2) exhibited improvement in expansion; however, persistence was ≤28 days, similar to that seen by others using CD28 CARs. Neither cytokine release syndrome nor delayed hematopoietic engraftment was observed in either trial. These data demonstrate the safety and feasibility of CD19 CAR TCM therapy after HSCT. Trials were registered at www.clinicaltrials.gov as #NCT01318317 and #NCT01815749.
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Transplante de Células-Tronco Hematopoéticas , Memória Imunológica , Imunoterapia Adotiva/métodos , Linfoma de Células B/terapia , Linfócitos T/transplante , Adulto , Idoso , Antígenos CD19/metabolismo , Contagem de Células , Terapia Combinada/efeitos adversos , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Imunoterapia Adotiva/efeitos adversos , Linfoma de Células B/imunologia , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/terapia , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transplante Autólogo , Adulto JovemRESUMO
The advent of efficient approaches to the genetic modification of T cells has provided investigators with clinically appealing methods to improve the potency of tumor-specific clinical grade T cells. For example, gene therapy has been successfully used to enforce expression of chimeric antigen receptors (CARs) that provide T cells with ability to directly recognize tumor-associated antigens without the need for presentation by human leukocyte antigen. Gene transfer of CARs can be undertaken using viral-based and non-viral approaches. We have advanced DNA vectors derived from the Sleeping Beauty (SB) system to avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors. After electroporation, the transposon/transposase improves the efficiency of integration of plasmids used to express CAR and other transgenes in T cells. The SB system combined with artificial antigen-presenting cells (aAPC) can selectively propagate and thus retrieve CAR(+) T cells suitable for human application. This review describes the translation of the SB system and aAPC for use in clinical trials and highlights how a nimble and cost-effective approach to developing genetically modified T cells can be used to implement clinical trials infusing next-generation T cells with improved therapeutic potential.
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Antígenos CD19/imunologia , Terapia Genética , Vetores Genéticos/genética , Recombinação Homóloga , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Técnicas de Cultura de Células , Epitopos de Linfócito T/imunologia , Técnicas de Transferência de Genes , Engenharia Genética , Terapia Genética/métodos , Humanos , Imunoterapia Adotiva/métodos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Multiple myeloma (MM) is a disease with known immune dysregulation. Natural killer (NK) cells have shown preclinical activity in MM. We conducted a first-in-human study of umbilical cord blood-derived (CB) NK cells for MM patients undergoing high dose chemotherapy and autologous haematopoietic stem cell transplantation (auto-HCT). Patients received lenalidomide (10 mg) on days -8 to -2, melphalan 200 mg/m2 on day -7, CB-NK cells on day -5 and auto-HCT on day 0. Twelve patients were enrolled, three on each of four CB-NK cell dose levels: 5 × 106 , 1 × 107 , 5 × 107 and 1 × 108 CB-NK cells/kg. Ten patients had either high-risk chromosomal changes or a history of relapsed/progressed disease. There were no infusional toxicities and no graft-versus-host disease. One patient failed to engraft due to poor autologous graft quality and was rescued with a back-up autologous graft. Overall, 10 patients achieved at least a very good partial response as their best response, including eight with near complete response or better. With a median follow-up of 21 months, four patients have progressed or relapsed, two of whom have died. CB-NK cells were detected in vivo in six patients, with an activated phenotype (NKG2D+ /NKp30+ ). These data warrant further development of this novel cellular therapy.
Assuntos
Sangue Fetal/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células Matadoras Naturais/transplante , Mieloma Múltiplo/terapia , Transferência Adotiva/métodos , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Separação Celular/métodos , Terapia Combinada , Feminino , Citometria de Fluxo/métodos , Sobrevivência de Enxerto , Humanos , Lenalidomida , Masculino , Melfalan/administração & dosagem , Pessoa de Meia-Idade , Talidomida/administração & dosagem , Talidomida/análogos & derivados , Resultado do TratamentoRESUMO
Delayed engraftment is a major limitation of cord blood transplantation (CBT), due in part to a defect in the cord blood (CB) cells' ability to home to the bone marrow. Because this defect appears related to low levels of fucosylation of cell surface molecules that are responsible for binding to P- and E-selectins constitutively expressed by the marrow microvasculature, and thus for marrow homing, we conducted a first-in-humans clinical trial to correct this deficiency. Patients with high-risk hematologic malignancies received myeloablative therapy followed by transplantation with 2 CB units, one of which was treated ex vivo for 30 minutes with the enzyme fucosyltransferase-VI and guanosine diphosphate fucose to enhance the interaction of CD34(+) stem and early progenitor cells with microvessels. The results of enforced fucosylation for 22 patients enrolled in the trial were then compared with those for 31 historical controls who had undergone double unmanipulated CBT. The median time to neutrophil engraftment was 17 days (range, 12-34 days) compared with 26 days (range, 11-48 days) for controls (P = .0023). Platelet engraftment was also improved: median was 35 days (range, 18-100 days) compared with 45 days (range, 27-120 days) for controls (P = .0520). These findings support ex vivo fucosylation of multipotent CD34(+) CB cells as a clinically feasible means to improve engraftment efficiency in the double CBT setting. The trial is registered to www.clinicaltrials.gov as #NCT01471067.
Assuntos
Plaquetas/citologia , Sangue Fetal/citologia , Fucose/metabolismo , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Neutrófilos/transplante , Adolescente , Adulto , Idoso , Plaquetas/imunologia , Estudos de Coortes , Selectina E/metabolismo , Estudos de Viabilidade , Feminino , Sangue Fetal/imunologia , Fucosiltransferases/metabolismo , Doença Enxerto-Hospedeiro , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/mortalidade , Células-Tronco Hematopoéticas/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/citologia , Neutrófilos/imunologia , Selectina-P/metabolismo , Transfusão de Plaquetas , Prognóstico , Taxa de Sobrevida , Adulto JovemRESUMO
We conducted a retrospective analysis of outcomes for children and young adults with sAML/sMDS who underwent HSCT at our institution. Thirty-two patients (median age 20 years) with sAML (n=24) and sMDS (n=8) received HSCT between 1990 and 2013. The median time from sAML/sMDS diagnosis to HSCT was 4.1 months (range: 1.2-27.2 months). The transplant regimens were primarily busulfan based (n=19). BM was the primary donor source (n=15). Eleven recipients were transplanted with residual disease. At a median follow-up of 62.3 months (range: 0.4-250.9 months), 14 patients had disease recurrence. Acute GVHD, grade III/IV, occurred in three patients. Causes of death were as follows: disease relapse (n=12), infection (n=2), pneumonia (n=1), pulmonary hemorrhage (n=1), acute GVHD (n=1), and graft failure (n=1). A PS of ≥90% at the time of HSCT had a significant impact on PFS (P=.02). Patients achieving pretransplant primary CR (n=8) and those with sMDS and RA (n=6) had prolonged PFS (P=.04). On multivariate analysis, shorter time to transplantation (≤6 months from diagnosis of sAML/sMDS) was associated with superior OS (P=.0018) and PFS (P=.0005).
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Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda/terapia , Síndromes Mielodisplásicas/terapia , Adolescente , Adulto , Bussulfano/uso terapêutico , Criança , Feminino , Doença Enxerto-Hospedeiro , Humanos , Masculino , Análise Multivariada , Recidiva Local de Neoplasia , Recidiva , Estudos Retrospectivos , Fatores de Tempo , Condicionamento Pré-Transplante , Transplante Homólogo , Resultado do Tratamento , Adulto JovemRESUMO
INTRODUCTION: High rates of patients require readmission to the hospital within 6 months of hematopoietic stem cell transplantation (HSCT). We investigated the relationship between readmission rates and outcomes after HSCT in children, adolescents, and young adults (CAYA). MATERIALS AND METHODS: A retrospective analysis of patients (26 years or younger) treated with HSCT was conducted. RESULTS: A chart review of 435 CAYA who underwent HSCT from 2008 to 2015 revealed that 171 patients (39%) had at least 1 hospital readmission within 180 days of transplant; 87% received allogeneic and 13% received autologous HSCT. A total of 312 readmission events were reported. The median follow-up time was 31 months. Documented infection (n=99) and graft-versus-host disease complications (n=60) were the most common causes. Higher than 2 readmission rates were associated with lower overall survival (OS) (P=0.001) and disease-free survival (P<0.001) in patients who received allogeneic HSCT. These findings were not found in the autologous HSCT. In a multivariate analysis of those who received allogeneic HSCT, prior treatment with ≥2 chemotherapy regimens (P=0.03) was independent predictor of lower OS. There were also trends noted toward lower OS for patients with documented infections at index admission or subsequent readmissions (P=0.09). CONCLUSIONS: More than 2 hospital readmissions within 6 months of allogeneic HSCT in CAYA, who are either heavily pretreated or had documented infections at index admission or subsequent readmissions adversely affected the outcomes.
Assuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Readmissão do Paciente/estatística & dados numéricos , Adolescente , Adulto , Criança , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/epidemiologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/mortalidade , Pesquisas sobre Atenção à Saúde , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Infecções/epidemiologia , Infecções/etiologia , Infecções/mortalidade , Masculino , Estudos Retrospectivos , Análise de Sobrevida , Texas/epidemiologia , Fatores de Tempo , Transplante Autólogo , Transplante Homólogo , Adulto JovemRESUMO
Chimeric antigen receptor (CAR) endows specificity to T-cells independent of human leukocyte antigen (HLA). This enables one immunoreceptor to directly target the same surface antigen on different subsets of tumor cells from multiple HLA-disparate recipients. Most approaches manufacture individualized CAR(+)T-cells from the recipient or HLA-compatible donor, which are revealing promising clinical results. This is the impetus to broaden the number of patients eligible to benefit from adoptive immunotherapy such as to infuse third-party donor derived CAR(+)T-cells. This will overcome issues associated with (i) time to manufacture T-cells, (ii) cost to generate one product for one patient, (iii) inability to generate a product from lymphopenic patients or patient's immune cells fail to complete the manufacturing process, and (iv) heterogeneity of T-cell products produced for or from individual recipients. Establishing a biobank of allogeneic genetically modified immune cells from healthy third-party donors, which are cryopreserved and validated in advance of administration, will facilitate the centralizing manufacturing and widespread distribution of CAR(+)T-cells to multiple points-of-care in a timely manner. To achieve this, it is necessary to engineer an effective strategy to avoid deleterious allogeneic immune responses leading to toxicity and rejection. We review the strategies to establish "off-the-shelf" donor-derived biobanks for human application of CAR(+)T-cells as a drug.
Assuntos
Expressão Gênica , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão , Linfócitos T/imunologia , Linfócitos T/metabolismo , Pesquisa Translacional Biomédica , Animais , Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Neoplasias/imunologia , Neoplasias/terapiaRESUMO
Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAK(S2580F) or ERBB2(H473Y) or the HLA-DQB*0601-restricted neoantigen ERBB2IP(E805G) were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRß (mTCRß) expression. Rapid expansion of mTCRß(+) T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27(-)CD45RA(-)) and less-differentiated (CD27(+)CD45RA(+)) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens.
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Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/patologia , Transposases/metabolismo , Animais , Elementos de DNA Transponíveis , Engenharia Genética , Antígeno HLA-A2/metabolismo , Cadeias beta de HLA-DQ/metabolismo , Humanos , Proteínas de Membrana/imunologia , Camundongos , Receptor ErbB-2/imunologia , Linfócitos T/imunologiaRESUMO
Clinical-grade T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) to redirect their specificity to target tumor-associated antigens in vivo. We now have developed this molecular strategy to render cytotoxic T cells specific for fungi. We adapted the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-ζ (designated "D-CAR") upon binding with carbohydrate in the cell wall of Aspergillus germlings. T cells genetically modified with the Sleeping Beauty system to express D-CAR stably were propagated selectively on artificial activating and propagating cells using an approach similar to that approved by the Food and Drug Administration for manufacturing CD19-specific CAR(+) T cells for clinical trials. The D-CAR(+) T cells exhibited specificity for ß-glucan which led to damage and inhibition of hyphal growth of Aspergillus in vitro and in vivo. Treatment of D-CAR(+) T cells with steroids did not compromise antifungal activity significantly. These data support the targeting of carbohydrate antigens by CAR(+) T cells and provide a clinically appealing strategy to enhance immunity for opportunistic fungal infections using T-cell gene therapy.
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Aspergilose/imunologia , Aspergilose/terapia , Bioengenharia/métodos , Carboidratos/antagonistas & inibidores , Infecções Oportunistas/imunologia , Infecções Oportunistas/terapia , Linfócitos T/imunologia , Animais , Antígenos CD19/metabolismo , Aspergilose/microbiologia , Aspergilose/patologia , Aspergillus/efeitos dos fármacos , Aspergillus/fisiologia , Dexametasona/farmacologia , Humanos , Hifas/efeitos dos fármacos , Hifas/fisiologia , Imunofenotipagem , Lectinas Tipo C/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Infecções Oportunistas/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacosRESUMO
Methotrexate (MTX) is an anti-folate that inhibits de novo purine and thymidine nucleotide synthesis. MTX induces death in rapidly replicating cells and is used in the treatment of multiple cancers. MTX inhibits thymidine synthesis by targeting dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS). The use of MTX to treat cancer also causes bone marrow suppression and inhibits the immune system. This has led to the development of an MTX-resistant DHFR, DHFR L22F, F31S (DHFR(FS)), to rescue healthy cells. 5-Fluorouracil-resistant TYMS T51S, G52S (TYMS(SS)) is resistant to MTX and improves MTX resistance of DHFR(FS) in primary T cells. Here we find that a known mechanism of MTX-induced increase in DHFR expression persists with DHFR(FS) and cis-expressed transgenes. We also find that TYMS(SS) expression of cis-expressed transgenes is similarly decreased in an MTX-inducible manner. MTX-inducible changes in DHFR(FS) and TYMS(SS) expression changes are lost when both genes are expressed together. In fact, expression of the DHFR(FS) and TYMS(SS) cis-expressed transgenes becomes correlated. These findings provide the basis for an unrecognized post-transcriptional mechanism that functionally links expression of DHFR and TYMS. These findings were made in genetically modified primary human T cells and have a clear potential for use in clinical applications where gene expression needs to be regulated by drug or maintained at a specific expression level. We demonstrate a potential application of this system in the controlled expression of systemically toxic cytokine IL-12.
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Antimetabólitos Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Metotrexato/farmacologia , Mutação de Sentido Incorreto , Linfócitos T/enzimologia , Tetra-Hidrofolato Desidrogenase , Timidilato Sintase , Substituição de Aminoácidos , Humanos , Células Jurkat , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , TransgenesRESUMO
MOTIVATION: There is a need for effective automated methods for profiling dynamic cell-cell interactions with single-cell resolution from high-throughput time-lapse imaging data, especially, the interactions between immune effector cells and tumor cells in adoptive immunotherapy. RESULTS: Fluorescently labeled human T cells, natural killer cells (NK), and various target cells (NALM6, K562, EL4) were co-incubated on polydimethylsiloxane arrays of sub-nanoliter wells (nanowells), and imaged using multi-channel time-lapse microscopy. The proposed cell segmentation and tracking algorithms account for cell variability and exploit the nanowell confinement property to increase the yield of correctly analyzed nanowells from 45% (existing algorithms) to 98% for wells containing one effector and a single target, enabling automated quantification of cell locations, morphologies, movements, interactions, and deaths without the need for manual proofreading. Automated analysis of recordings from 12 different experiments demonstrated automated nanowell delineation accuracy >99%, automated cell segmentation accuracy >95%, and automated cell tracking accuracy of 90%, with default parameters, despite variations in illumination, staining, imaging noise, cell morphology, and cell clustering. An example analysis revealed that NK cells efficiently discriminate between live and dead targets by altering the duration of conjugation. The data also demonstrated that cytotoxic cells display higher motility than non-killers, both before and during contact. CONTACT: broysam@central.uh.edu or nvaradar@central.uh.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Comunicação Celular , Rastreamento de Células/métodos , Células Matadoras Naturais/citologia , Nanoestruturas/química , Linfócitos T/citologia , Imagem com Lapso de Tempo/métodos , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Ensaios de Triagem em Larga Escala/métodos , Humanos , Processamento de Imagem Assistida por Computador , Células K562RESUMO
Haploidentical transplantation can extend the opportunity for transplantation to almost all patients who lack an HLA-matched donor. Advances in the field of haploidentical transplantation have led to a marked decrease in treatment-related mortality, allowing investigators to focus on developing rationale pre- and peri-remission therapies aimed at preventing disease relapse after transplantation. Because of widespread availability, low treatment-related mortality, and cost, haploidentical donors may become the preferred "alternative" donors for allogeneic hematopoietic stem cell transplantation. One of the major advantages of using a related donor is the possibility of collecting or generating additional cellular products from the same immediately available donor, which will not be rejected. Infusion of these cells in the peri-transplantation period, derived from the same immune system, is opening the possibility of markedly enhancing the antitumor effects of the graft and hastening immunologic reconstitution after transplantation.
Assuntos
Antígenos HLA/imunologia , Haplótipos , Transplante de Células-Tronco Hematopoéticas/métodos , Histocompatibilidade , Imunoterapia Adotiva/métodos , Linfócitos T/transplante , Ciclofosfamida/administração & dosagem , Ciclofosfamida/uso terapêutico , Genes Transgênicos Suicidas , Antígenos HLA/genética , Humanos , Células Matadoras Naturais/transplante , Doadores Vivos/provisão & distribuição , Depleção Linfocítica/métodos , Fármacos Fotossensibilizantes/uso terapêutico , Receptores de Antígenos de Linfócitos T/uso terapêutico , Rodaminas/uso terapêutico , Imunologia de TransplantesRESUMO
BACKGROUND: Poor engraftment due to low cell doses restricts the usefulness of umbilical-cord-blood transplantation. We hypothesized that engraftment would be improved by transplanting cord blood that was expanded ex vivo with mesenchymal stromal cells. METHODS: We studied engraftment results in 31 adults with hematologic cancers who received transplants of 2 cord-blood units, 1 of which contained cord blood that was expanded ex vivo in cocultures with allogeneic mesenchymal stromal cells. The results in these patients were compared with those in 80 historical controls who received 2 units of unmanipulated cord blood. RESULTS: Coculture with mesenchymal stromal cells led to an expansion of total nucleated cells by a median factor of 12.2 and of CD34+ cells by a median factor of 30.1. With transplantation of 1 unit each of expanded and unmanipulated cord blood, patients received a median of 8.34×10(7) total nucleated cells per kilogram of body weight and 1.81×10(6) CD34+ cells per kilogram--doses higher than in our previous transplantations of 2 units of unmanipulated cord blood. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 15 days in the recipients of expanded cord blood, as compared with 24 days in controls who received unmanipulated cord blood only (P<0.001); the median time to platelet engraftment was 42 days and 49 days, respectively (P=0.03). On day 26, the cumulative incidence of neutrophil engraftment was 88% with expansion versus 53% without expansion (P<0.001); on day 60, the cumulative incidence of platelet engraftment was 71% and 31%, respectively (P<0.001). CONCLUSIONS: Transplantation of cord-blood cells expanded with mesenchymal stromal cells appeared to be safe and effective. Expanded cord blood in combination with unmanipulated cord blood significantly improved engraftment, as compared with unmanipulated cord blood only. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00498316.).
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Mesenquimais , Adolescente , Adulto , Contagem de Células Sanguíneas , Plaquetas , Causas de Morte , Técnicas de Cultura de Células , Facilitação Imunológica de Enxerto , Doença Enxerto-Hospedeiro , Neoplasias Hematológicas/mortalidade , Humanos , Células-Tronco Mesenquimais , Pessoa de Meia-Idade , Neutrófilos , Quimeras de Transplante , Transplante Homólogo , Adulto JovemRESUMO
Myelodysplastic syndromes are a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Chromosomal abnormalities and gene mutations have been shown to have essential roles in pathogenesis and correlate with prognosis. Molecular markers, however, are not integrated into currently used prognostic systems. The goal of this study is to identify plasma microRNAs useful for classification and risk stratification of myelodysplastic syndromes. We applied a novel, high-throughput digital quantification technology (NanoString) to profile microRNA expression in plasma samples of 72 patients with myelodysplastic syndromes and 12 healthy individuals. We correlated these results with overall survival. In patients with myelodysplastic syndromes associated with a diploid karyotype, we identified and validated a 7-microRNA signature as an independent predictor of survival with a predictive power of 75% accuracy (P=0.008), better than those of the International Prognostic Scoring Systems and the MD Anderson Prognostic Lower Risk Prognostic Model. We also identified differentially expressed plasma microRNAs in patients with myelodysplastic syndromes versus healthy individuals and between patients with myelodysplastic syndromes associated with different cytogenetic features. These results validate the utility of circulating-microRNA levels as noninvasive biomarkers that can inform the management of patients with myelodysplastic syndromes. Our findings also shed light on interactions of gene regulation pathways that are likely involved in the pathogenesis of myelodysplastic syndromes.