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1.
Front Immunol ; 11: 93, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32076420

RESUMO

Defensins represents an integral part of the innate immune system serving to ward off potential pathogens and to protect the intestinal barrier from microbial encroachment. In addition to their antimicrobial activities, defensins in general, and human ß-defensin 2 (hBD2) in particular, also exhibit immunomodulatory capabilities. In this report, we assessed the therapeutic efficacy of systemically administered recombinant hBD2 to ameliorate intestinal inflammation in three distinct animal models of inflammatory bowel disease; i.e., chemically induced mucosal injury (DSS), loss of mucosal tolerance (TNBS), and T-cell transfer into immunodeficient recipient mice. Treatment efficacy was confirmed in all tested models, where systemically administered hBD2 mitigated inflammation, improved disease activity index, and hindered colitis-induced body weight loss on par with anti-TNF-α and steroids. Treatment of lipopolysaccharide (LPS)-activated human peripheral blood mononuclear cells with rhBD2 confirmed the immunomodulatory capacity in the circulatory compartment. Subsequent analyzes revealed dendritic cells (DCs) as the main target population. Suppression of LPS-induced inflammation was dependent on chemokine receptor 2 (CCR2) expression. Mechanistically, hBD2 engaged with CCR2 on its DC target cell to decrease NF-κB, and increase CREB phosphorylation, hence curbing inflammation. To our knowledge, this is the first study showing in vivo efficacy of a systemically administered defensin in experimental disease.

2.
Arthritis Res Ther ; 21(1): 212, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615552

RESUMO

BACKGROUND: Systemic sclerosis (SSc) is a potentially fatal autoimmune disease that leads to extensive fibrosis of the skin and internal organs. Invariant natural killer T (iNKT) cells are potent immunoregulatory T lymphocytes being able to orchestrate dysregulated immune responses. The purpose of this study was to evaluate numbers and function of iNKT cells in patients with SSc and to analyze their correlation with disease parameters. METHODS: Human iNKT cells from 88 patients with SSc and 33 healthy controls were analyzed by flow cytometry. Their proliferative capacity and cytokine production were investigated following activation with CD1d ligand α-galactosylceramide (α-GalCer). RESULTS: We observed an absolute and relative decrease of iNKT cells in patients with SSc compared with healthy controls. Interestingly, the subtype of SSc, disease severity, or treatment with immunosuppressive drugs did not affect iNKT cell numbers. However, T helper (Th) cell immune polarization was biased towards a Th17 immunophenotype in SSc patients. Moreover, iNKT cells from patients with SSc showed a significantly decreased expansion capacity upon stimulation with α-GalCer. CONCLUSION: iNKT cells are deficient and functionally impaired in patients with SSc. Therefore, adoptive transfer strategies using culture-expanded iNKT cells could be a novel approach to treat SSc patients.

3.
Oncogene ; 38(46): 7181-7195, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31417187

RESUMO

MLL rearrangements play a crucial role in leukemogenesis and comprise a poor prognosis. Therefore, new treatment strategies are urgently needed. We used the CRISPR/Cas9 system to generate an innovative leukemia model based on 100% pure MLL-AF4 or -AF9 rearranged cells derived from umbilical cord blood with indefinite growth in cell culture systems. Our model shared phenotypical, morphological and molecular features of patient cells faithfully mimicking the nature of the disease. Thus, it serves as a fundamental basis for pharmacological studies: inhibition of histone methyltransferase disruptor of telomeric silencing 1-like (DOT1L) is one specific therapeutic approach currently tested in clinical trials. However, success was limited by restricted response warranting further investigation of drug combinations. Recently, it has been shown that the inhibition of protein arginine methyltransferase 5 (PRMT5) exhibits anti-tumoral activity against human cell lines and in MLL mouse models. Here, we used DOT1L and PRMT5 inhibitors in our human MLL-rearranged model demonstrating dose-dependent reduced proliferation, impairment of cell cycle, increasing differentiation, apoptosis, downregulation of target genes and sensitization to chemotherapy. Strikingly, the combination of both compounds led to synergistic anti-tumoral effects. Our study provides a strong rationale for novel targeted combination therapies to improve the outcome of MLL-rearranged leukemias.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia , Modelos Biológicos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/farmacologia , Sistemas CRISPR-Cas , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Edição de Genes/métodos , Células-Tronco Hematopoéticas , Humanos , Isoquinolinas/farmacologia , Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia
4.
Front Immunol ; 10: 1542, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354710

RESUMO

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for hematologic malignancies but relapse remains the most common cause of death. Infusion of donor lymphocytes (DLIs) can induce remission and prolong survival by exerting graft-vs.-leukemia (GVL) effects. However, sufficient tumor control cannot be established in all patients and occurrence of graft-vs.-host disease (GVHD) prevents further dose escalation. Previous data indicate that invariant natural killer T (iNKT) cells promote anti-tumor immunity without exacerbating GVHD. In the present study we investigated lysis of leukemic blasts through iNKT cells from donor-derived lymphocytes for leukemia control and found that iNKT cells constitute about 0.12% of cryopreserved donor T cells. Therefore, we established a 2-week cell culture protocol allowing for a robust expansion of iNKT cells from cryopreserved DLIs (DLI-iNKTs) that can be used for further preclinical and clinical applications. Such DLI-iNKTs efficiently lysed leukemia cell lines and primary patient AML blasts ex vivo in a dose- and CD1d-dependent manner. Furthermore, expression of CD1d on target cells was required to release proinflammatory cytokines and proapoptotic effector molecules. Our results suggest that iNKT cells from donor-derived lymphocytes are involved in anti-tumor immunity after allo-HCT and therefore may reduce the risk of relapse and improve progression-free and overall survival.

5.
Br J Haematol ; 186(1): 60-71, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30916396

RESUMO

Donor lymphocyte infusion (DLI) is an effective method to establish full donor chimerism or to prevent and treat relapse after allogeneic haematopoietic cell transplantation (allo-HCT). Usually, DLIs are collected from naïve donors as steady-state lymphocytes. When donor lymphocytes are collected during stem cell apheresis, donors are pre-treated with granulocyte colony-stimulating factor (G-CSF). However, the impact of G-CSF stimulation and the resulting composition of DLIs on beneficial anti-leukaemic responses and survival remain elusive. Therefore, we performed a retrospective analysis to evaluate the role of G-CSF-DLIs: 44 patients received either steady-state DLIs or G-CSF-DLIs to prevent and treat relapse or establish full donor chimerism after allo-HCT. The G-CSF-DLI patient cohort showed an improved conversion to full donor chimerism and a lower cumulative incidence of relapse or disease progression without a significantly increased cumulative incidence of graft-versus-host disease (GVHD). CD34+ cells, monocytic myeloid-derived suppressor cells and monocytes as well as donor age and the subsequent occurrence of chronic GVHD were identified as risk factors that significantly improve overall survival after DLI administration. In conclusion, our data suggest that administration of G-CSF-DLIs results in graft-versus-leukaemia effects without exacerbating GVHD, therefore, improving survival after DLIs.

6.
Ann Hematol ; 97(12): 2491-2500, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30066039

RESUMO

Allogeneic hematopoietic cell transplantation (HCT) is the treatment of choice for high-risk myeloid and lymphoid leukemias. Relapse after allogeneic HCT is associated with a dismal prognosis and further therapeutic options are limited. One potential curative approach is a second allogeneic HCT. However, there is no consensus about optimal transplant modalities, suitable patients, and entities. We performed a retrospective analysis of our institutional database to evaluate risk factors that influence survival after a second allogeneic HCT for the treatment of relapsed acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). We identified 40 patients (AML, n = 29; ALL, n = 11) that received a second allogeneic HCT at our institution. At time of second HCT, 48% of patients were in complete remission (CR). Current overall survival (OS) was 14/40 patients with a median follow-up of 64 months (range 4-140) of patients alive resulting in a Kaplan-Meier estimated 2-year event-free survival (EFS) and OS of 32%, respectively. Cumulative incidence of non-relapse mortality (NRM) and relapse at 2 years was 31 and 37%, respectively. We identified several independent risk factors influencing OS: > 6 months from first to second transplant (p = 0.02), complete remission prior to transplant (p = 0.003), and the subsequent occurrence of chronic graft-versus-host disease (p = 0.003) were associated with a significantly improved OS. In conclusion, our data suggest that a second allogeneic HCT is a curative treatment option for relapsed acute leukemias in selected patients.


Assuntos
Bases de Dados Factuais , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Adulto , Aloenxertos , Doença Crônica , Intervalo Livre de Doença , Feminino , Doença Enxerto-Hospedeiro/sangue , Doença Enxerto-Hospedeiro/mortalidade , Humanos , Leucemia Mieloide Aguda/sangue , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Recidiva , Estudos Retrospectivos , Fatores de Risco , Taxa de Sobrevida
7.
Front Immunol ; 9: 1817, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30127790

RESUMO

Graft-versus-host disease (GVHD) is a major cause of significant morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are potent regulators of immune responses, protect from lethal GVHD, and promote graft-versus-leukemia effects in murine studies. Since iNKT cells constitute less than 0.5% of human peripheral blood mononuclear cells (PBMCs), in vitro expansion with their glycolipid ligands is required before they can be used for cytotherapy and experimental purposes. Three weeks of cell culture and autologous restimulation with either KRN7000, PBS44, or PBS57 resulted in a robust proliferation of iNKT cells from human PBMCs. Next, iNKT cells were sorted to a purity higher than 90% being crucial for further experimental and clinical applications. These iNKT cells significantly decreased activation and proliferation of allogeneic CD3+ T lymphocytes. In addition, leukemia cell lines and primary leukemia cells were efficiently lysed by culture-expanded iNKT cells. Importantly, culture-expanded donor iNKT cells promoted robust antileukemia activity against HLA-matched allogeneic patient leukemia cells. Our data indicate that the adoptive transfer of culture-expanded iNKT cells could be a powerful cytotherapeutic approach to induce immune tolerance and prevent leukemia relapse after allogeneic HCT in humans.


Assuntos
Citotoxicidade Imunológica , Isoantígenos/imunologia , Leucemia/imunologia , Leucemia/metabolismo , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Técnicas de Cocultura , Citocinas/metabolismo , Relação Dose-Resposta Imunológica , Glicolipídeos/metabolismo , Humanos , Tolerância Imunológica , Imunofenotipagem , Leucemia/patologia , Leucemia/terapia
8.
Blood Adv ; 2(8): 832-845, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29650777

RESUMO

Genome editing provides a potential approach to model de novo leukemogenesis in primary human hematopoietic stem and progenitor cells (HSPCs) through induction of chromosomal translocations by targeted DNA double-strand breaks. However, very low efficiency of translocations and lack of markers for translocated cells serve as barriers to their characterization and model development. Here, we used transcription activator-like effector nucleases to generate t(9;11) chromosomal translocations encoding MLL-AF9 and reciprocal AF9-MLL fusion products in CD34+ human cord blood cells. Selected cytokine combinations enabled monoclonal outgrowth and immortalization of initially rare translocated cells, which were distinguished by elevated MLL target gene expression, high surface CD9 expression, and increased colony-forming ability. Subsequent transplantation into immune-compromised mice induced myeloid leukemias within 48 weeks, whose pathologic and molecular features extensively overlap with de novo patient MLL-rearranged leukemias. No secondary pathogenic mutations were revealed by targeted exome sequencing and whole genome RNA-sequencing analyses, suggesting the genetic sufficiency of t(9;11) translocation for leukemia development from human HSPCs. Thus, genome editing enables modeling of human acute MLL-rearranged leukemia in vivo, reflecting the genetic simplicity of this disease, and provides an experimental platform for biological and disease-modeling applications.


Assuntos
Edição de Genes , Células-Tronco Hematopoéticas/metabolismo , Histona-Lisina N-Metiltransferase/genética , Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Translocação Genética , Animais , Carcinogênese , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 9 , Humanos , Leucemia/etiologia , Camundongos , Proteínas de Fusão Oncogênica/genética
9.
Clin Transplant ; 31(7)2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28470884

RESUMO

BACKGROUND: The role of allogeneic hematopoietic cell transplantation (HCT) for the treatment of multiple myeloma is controversial. However, the introduction of proteasome inhibitors and immunomodulatory drugs might influence outcomes in case of relapse or refractory disease after allogeneic HCT. METHODS: We report 41 consecutive patients that underwent allogeneic HCT for the treatment of relapsed or refractory multiple myeloma. RESULTS: Three-year event-free survival (EFS) and overall survival (OS) of the whole cohort were 15% and 51%, respectively. In a subgroup analysis, allogeneic HCT after a second high-dose chemotherapy with autologous stem cell support was associated with a decreased 3-year EFS (6% vs 24%, P=.04) and OS (35% vs 64%, P=.09). In case of relapse or refractory disease after allogeneic HCT, the treatment with proteasome inhibitors or immunomodulatory drugs significantly improved survival (1-year OS 79% vs 29%, P=.001). CONCLUSION: The incorporation of proteasome inhibitors and immunomodulatory drugs into transplant protocols has the potential to improve outcomes and refine the role of allogeneic HCT for the treatment of multiple myeloma as a platform for long-term disease control.


Assuntos
Rejeição de Enxerto/tratamento farmacológico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Mieloma Múltiplo/terapia , Recidiva Local de Neoplasia/tratamento farmacológico , Adulto , Idoso , Feminino , Seguimentos , Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/etiologia , Humanos , Imunomodulação , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/complicações , Recidiva Local de Neoplasia/etiologia , Prognóstico , Inibidores de Proteassoma , Estudos Retrospectivos , Fatores de Risco , Condicionamento Pré-Transplante , Transplante Homólogo
10.
Blood ; 129(23): 3121-3125, 2017 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-28416503

RESUMO

Chronic graft-versus-host-disease (cGVHD) can cause multiorgan system disease, typically with autoimmune-like features, resulting in high mortality and morbidity caused by treatment limitations. Invariant natural killer T cells (iNKTs), a small population characterized by expression of a semi-invariant T-cell receptor, rapidly produce copious amounts of diverse cytokines on activation that exert potent immune regulatory function. Here, we show that iNKTs are significantly reduced in a cGVHD murine model that recapitulates several aspects of autoimmunity and organ fibrosis observed in patients with cGVHD. Low iNKT infused doses effectively prevented and, importantly, reversed established cGVHD, as did third-party iNKTs. iNKTs suppressed the autoimmune response by reducing the germinal center (GC) reaction, which was associated with an increase in total Tregs and follicular Tregs (Tfr) that control the GC reaction, along with pathogenic antibody production. Treg depletion during iNKT infusions completely abolished iNKT efficacy in treating cGVHD. iNKT cell interleukin 4 production and GC migration were critical to cGVHD reversal. In vivo stimulation of iNKT cells by α-galactosyl-ceramide was effective in both preventing and treating cGVHD. Together, this study demonstrates iNKT deficiency in cGVHD mice and highlights the key role of iNKTs in regulating cGVHD pathogenesis and as a potentially novel prophylactic and therapeutic option for patients with cGVHD.


Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Doença Enxerto-Hospedeiro/terapia , Células T Matadoras Naturais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Transplante de Medula Óssea/efeitos adversos , Toxina Diftérica/toxicidade , Modelos Animais de Doenças , Centro Germinativo/imunologia , Doença Enxerto-Hospedeiro/imunologia , Humanos , Imunossupressão/métodos , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/transplante , Linfócitos T Reguladores/efeitos dos fármacos , Doadores de Tecidos
11.
Blood ; 128(6): 866-71, 2016 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-27365424

RESUMO

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) have been shown to effectively prevent graft-versus-host disease (GVHD) when adoptively transferred in murine models of hematopoietic cell transplantation and in phase 1/2 clinical trials. Critical limitations to Treg clinical application are the paucity of cells and limited knowledge of the mechanisms of in vivo function. We hypothesized that inflammatory conditions in GVHD modify Treg characteristics and activity. We found that peripheral blood of recipient animals during acute GVHD (aGVHD) induces Treg activation and enhances their function. The serum contains high levels of tumor necrosis factor-α (TNF-α) that selectively activates Tregs without impacting CD4(+)FoxP3(-) T cells. TNF-α priming induces Treg in vivo proliferation, whereas it limits the ability of CD4 and CD8 conventional T cells (Tcons) to proliferate and induce GVHD. TNF-α-primed Tregs prolong animal survival as compared with unprimed Tregs when used at an unfavorable Treg:Tcon ratio, demonstrating enhanced in vivo efficacy of TNF-α-primed Tregs. Because TNF-α is produced by several immune cells during inflammation, our work elucidates aspects of the physiologic mechanisms of Treg function. Furthermore, TNF-α priming of Tregs provides a new tool to optimize Treg cellular therapies for GVHD prevention and treatment.


Assuntos
Transferência Adotiva/métodos , Antígenos CD4/imunologia , Fatores de Transcrição Forkhead/imunologia , Doença Enxerto-Hospedeiro/terapia , Linfócitos T Reguladores/imunologia , Fator de Necrose Tumoral alfa/uso terapêutico , Animais , Antígenos CD4/análise , Células Cultivadas , Fatores de Transcrição Forkhead/análise , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/transplante , Fator de Necrose Tumoral alfa/imunologia
12.
Cancer Discov ; 6(7): 770-83, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27154821

RESUMO

UNLABELLED: Numerous studies in multiple systems support that histone H3 lysine 36 dimethylation (H3K36me2) is associated with transcriptional activation; however, the underlying mechanisms are not well defined. Here, we show that the H3K36me2 chromatin mark written by the ASH1L histone methyltransferase is preferentially bound in vivo by LEDGF, a mixed-lineage leukemia (MLL)-associated protein that colocalizes with MLL, ASH1L, and H3K36me2 on chromatin genome wide. Furthermore, ASH1L facilitates recruitment of LEDGF and wild-type MLL proteins to chromatin at key leukemia target genes and is a crucial regulator of MLL-dependent transcription and leukemic transformation. Conversely, KDM2A, an H3K36me2 demethylase and Polycomb group silencing protein, antagonizes MLL-associated leukemogenesis. Our studies are the first to provide a basic mechanistic insight into epigenetic interactions wherein placement, interpretation, and removal of H3K36me2 contribute to the regulation of gene expression and MLL leukemia, and suggest ASH1L as a novel target for therapeutic intervention. SIGNIFICANCE: Epigenetic regulators play vital roles in cancer pathogenesis and represent a new frontier in therapeutic targeting. Our studies provide basic mechanistic insight into the role of H3K36me2 in transcription activation and MLL leukemia pathogenesis and implicate ASH1L histone methyltransferase as a promising target for novel molecular therapy. Cancer Discov; 6(7); 770-83. ©2016 AACR.See related commentary by Balbach and Orkin, p. 700This article is highlighted in the In This Issue feature, p. 681.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Leucemia/genética , Leucemia/metabolismo , Lisina/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Proteínas F-Box/metabolismo , Feminino , Regulação Leucêmica da Expressão Gênica , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Leucemia/patologia , Metilação , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica
13.
Ann Hematol ; 95(6): 973-83, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27021303

RESUMO

Myelofibrosis (MF) is a rare disease responsible for an increasing ineffective hematopoesis by a progressive fibrosing process in the bone marrow. The only curative treatment option is allogeneic hematopoietic cell transplantation (HCT). In this single-center analysis, we evaluated retrospectively 54 consecutive patients suffering from primary or secondary MF which underwent HCT from 1997 to 2014 after either myeloablative (MAC, n = 19) or reduced-intensity conditioning (RIC, n = 35). Overall survival (OS) and disease-free survival (DFS) after 3 years was 54/53 % for RIC versus 63/58 % for MAC (p = 0.8/0.97). Cumulative incidence of relapse was 34 % after RIC and 8 % after MAC (p = 0.16). Three-year non-relapse mortality (NRM) was 15 % after RIC and 34 % after MAC (p = 0.29). We found that RIC was associated with a lower incidence of acute graft versus host disease (GvHD; II-IV 26 vs. 0 %, p = 0.004). Evaluation of prognostic relevance of the Dynamic International Prognostic Scoring System (DIPSS) score showed a significant better OS in patient with risk score ≤3 versus >3 (after 3 years, 71 vs. 39 %, p = 0.008). While similar OS and DFS were observed with MAC or RIC, the use of RIC resulted in lower incidence of acute GvHD. RIC regimens may be therefore the preferred conditioning approach for allogeneic HCT in patients with MF.


Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/terapia , Condicionamento Pré-Transplante/métodos , Adulto , Idoso , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Mortalidade/tendências , Mielofibrose Primária/mortalidade , Estudos Retrospectivos , Condicionamento Pré-Transplante/mortalidade , Transplante Homólogo/métodos , Transplante Homólogo/mortalidade
14.
Ann Hematol ; 95(1): 115-124, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26411736

RESUMO

We report a retrospective single-center analysis of 112 consecutive patients that underwent allogeneic hematopoietic cell transplantation (HCT) after reduced-intensity conditioning (RIC) with fludarabine (FLU) and busulfan (BU) for the treatment of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative syndrome (MPS) from 2005 to 2014. Three-year event-free survival (EFS) and overall survival (OS) were 46 and 58 %, respectively. Patients ≥60 years of age showed a similar outcome compared to younger patients (3-year OS 55 vs. 61 %, p = 0.96; 3-year EFS 46 vs. 46 %, p = 0.82). Cumulative incidence of non-relapse mortality (NRM) at 3 years adjusted for relapse as competing risk was 25 % for patients aged <60 years and 15 % for older patients (p = 0.15). Infusions of higher CD34(+) blood stem cell doses were associated with a significantly better outcome in the elderly subgroup (3-year OS 82 vs. 39 %, p = 0.007). Moreover, complete donor chimerism at day +100 was associated with a significantly improved survival (3-year OS 69 vs. 23 %, p = 0.003). In conclusion, our data suggest that RIC with FLU/BU enables long-term disease-free survival even in an elderly patient population. Age has no negative impact on the outcome of allogeneic HCT, and decision for transplant should be based on disease risk and performance status rather than age alone.


Assuntos
Doenças da Medula Óssea/terapia , Bussulfano/administração & dosagem , Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia Mieloide Aguda/terapia , Condicionamento Pré-Transplante/métodos , Vidarabina/análogos & derivados , Adulto , Idoso , Doenças da Medula Óssea/diagnóstico , Doenças da Medula Óssea/mortalidade , Feminino , Seguimentos , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Taxa de Sobrevida/tendências , Transplante Homólogo/métodos , Vidarabina/administração & dosagem
15.
PLoS One ; 10(12): e0145763, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26693907

RESUMO

The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.


Assuntos
Criopreservação , Regulação da Expressão Gênica/imunologia , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Selectina L/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Aloenxertos , Animais , Moléculas de Adesão Celular/imunologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mucoproteínas/genética , Mucoproteínas/imunologia , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/transplante
16.
Blood ; 126(14): 1683-94, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26311362

RESUMO

Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease-mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia.


Assuntos
Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Células-Tronco Hematopoéticas , Histona-Lisina N-Metiltransferase/genética , Leucemia Aguda Bifenotípica/genética , Proteína de Leucina Linfoide-Mieloide/genética , Animais , Antígenos CD34/imunologia , Separação Celular , Técnicas de Introdução de Genes , Genoma Humano , Humanos , Camundongos , Microscopia Confocal , Mutagênese Sítio-Dirigida , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transdução Genética , Transfecção
17.
Cancer Cell ; 28(2): 198-209, 2015 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-26190263

RESUMO

The genetic programs that maintain leukemia stem cell (LSC) self-renewal and oncogenic potential have been well defined; however, the comprehensive epigenetic landscape that sustains LSC cellular identity and functionality is less well established. We report that LSCs in MLL-associated leukemia reside in an epigenetic state of relative genome-wide high-level H3K4me3 and low-level H3K79me2. LSC differentiation is associated with reversal of these broad epigenetic profiles, with concomitant downregulation of crucial MLL target genes and the LSC maintenance transcriptional program that is driven by the loss of H3K4me3, but not H3K79me2. The H3K4-specific demethylase KDM5B negatively regulates leukemogenesis in murine and human MLL-rearranged AML cells, demonstrating a crucial role for the H3K4 global methylome in determining LSC fate.


Assuntos
Transformação Celular Neoplásica/metabolismo , Epigênese Genética , Histonas/metabolismo , Leucemia/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Células Cultivadas , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Leucemia/genética , Leucemia/patologia , Lisina/metabolismo , Metilação , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo
18.
Blood ; 126(4): 546-57, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-26063163

RESUMO

The paucity of regulatory T cells (Tregs) limits clinical translation to control aberrant immune reactions including graft-versus-host disease (GVHD). Recent studies showed that the agonistic antibody to DR3 (αDR3) expanded CD4(+)FoxP3(+) Tregs in vivo. We investigated whether treating donor mice with a single dose of αDR3 could alleviate acute GVHD in a MHC-mismatched bone marrow transplantation model. αDR3 induced selective proliferation of functional Tregs. CD4(+) T cells isolated from αDR3-treated mice contained higher numbers of Tregs and were less proliferative to allogeneic stimuli. In vivo GVHD studies confirmed that Tregs from αDR3-treated donors expanded robustly and higher frequencies of Tregs within donor CD4(+) T cells were maintained, resulting in improved survival. Conventional T cells derived from αDR3-treated donors showed reduced activation and proliferation. Serum levels of proinflammatory cytokines (IFNγ, IL-1ß, and TNFα) and infiltration of donor T cells into GVHD target tissues (gastrointestinal tract and liver) were decreased. T cells from αDR3-treated donors retained graft-vs-tumor (GVT) effects. In conclusion, a single dose of αDR3 alleviates acute GVHD while preserving GVT effects by selectively expanding and maintaining donor Tregs. This novel strategy will facilitate the clinical application of Treg-based therapies.


Assuntos
Anticorpos/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Tumor/imunologia , Linfoma de Células B/prevenção & controle , Membro 25 de Receptores de Fatores de Necrose Tumoral/imunologia , Linfócitos T Reguladores/imunologia , Animais , Transplante de Medula Óssea , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Imunoglobulinas/administração & dosagem , Linfoma de Células B/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Doadores de Tecidos , Transplante Homólogo , Células Tumorais Cultivadas
19.
J Immunol ; 195(1): 347-55, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25994967

RESUMO

Adoptive transfer of freshly isolated natural occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) prevents graft-versus-host disease (GVHD) in several animal models and following hematopoietic cell transplantation (HCT) in clinical trials. Donor-derived Treg have been mainly used, as they share the same MHC with CD4(+) and CD8(+) conventional T cells (Tcon) that are primarily responsible for GVHD. Third party-derived Treg are a promising alternative for cellular therapy, as they can be prepared in advance, screened for pathogens and activity, and banked. We explored MHC disparities between Treg and Tcon in HCT to evaluate the impact of different Treg populations in GVHD prevention and survival. Third-party Treg and donor Treg are equally suppressive in ex vivo assays, whereas both donor and third-party but not host Treg protect from GVHD in allogeneic HCT, with donor Treg being the most effective. In an MHC minor mismatched transplantation model (C57BL/6 → BALB/b), donor and third-party Treg were equally effective in controlling GVHD. Furthermore, using an in vivo Treg depletion mouse model, we found that Treg exert their main suppressive activity in the first 2 d after transplantation. Third-party Treg survive for a shorter period of time after adoptive transfer, but despite the shorter survival, they control Tcon proliferation in the early phases of HCT. These studies provide relevant insights on the mechanisms of Treg-mediated protection from GVHD and support for the use of third-party Treg in clinical trials.


Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Biomarcadores/sangue , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Fatores de Transcrição Forkhead/sangue , Expressão Gênica , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Teste de Histocompatibilidade , Subunidade alfa de Receptor de Interleucina-2/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/transplante , Doadores de Tecidos , Transplante Homólogo , Irradiação Corporal Total
20.
Blood ; 125(22): 3491-500, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-25795920

RESUMO

Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are a potent immunoregulatory T-cell subset in both humans and mice. Here, we explored the role of adoptively transferred third-party CD4(+) iNKT cells for protection from lethal GVHD in a murine model of allogeneic HCT across major histocompatibility barriers. We found that low numbers of CD4(+) iNKT cells from third-party mice resulted in a significant survival benefit with retained graft-versus-tumor effects. In vivo expansion of alloreactive T cells was diminished while displaying a T helper cell 2-biased phenotype. Notably, CD4(+) iNKT cells from third-party mice were as protective as CD4(+) iNKT cells from donor mice although third-party CD4(+) iNKT cells were rejected early after allogeneic HCT. Adoptive transfer of third-party CD4(+) iNKT cells resulted in a robust expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) that were required for protection from lethal GVHD. However, in vivo depletion of myeloid-derived suppressor cells abrogated both Treg expansion and protection from lethal GVHD. Despite the fact that iNKT cells are a rare cell population, the almost unlimited third-party availability and feasibility of in vitro expansion provide the basis for clinical translation.


Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/transplante , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/prevenção & controle , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/fisiologia , Células Matadoras Naturais/transplante , Animais , Proliferação de Células , Células Cultivadas , Citoproteção/imunologia , Feminino , Doença Enxerto-Hospedeiro/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T Auxiliares-Indutores/fisiologia
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