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1.
Blood Adv ; 3(20): 3143-3156, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31648313

RESUMO

Survival of patients with pediatric acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-SCT) is mainly compromised by leukemia relapse, carrying dismal prognosis. As novel individualized therapeutic approaches are urgently needed, we performed whole-exome sequencing of leukemic blasts of 10 children with post-allo-SCT relapses with the aim of thoroughly characterizing the mutational landscape and identifying druggable mutations. We found that post-allo-SCT ALL relapses display highly diverse and mostly patient-individual genetic lesions. Moreover, mutational cluster analysis showed substantial clonal dynamics during leukemia progression from initial diagnosis to relapse after allo-SCT. Only very few alterations stayed constant over time. This dynamic clonality was exemplified by the detection of thiopurine resistance-mediating mutations in the nucleotidase NT5C2 in 3 patients' first relapses, which disappeared in the post-allo-SCT relapses on relief of selective pressure of maintenance chemotherapy. Moreover, we identified TP53 mutations in 4 of 10 patients after allo-SCT, reflecting acquired chemoresistance associated with selective pressure of prior antineoplastic treatment. Finally, in 9 of 10 children's post-allo-SCT relapse, we found alterations in genes for which targeted therapies with novel agents are readily available. We could show efficient targeting of leukemic blasts by APR-246 in 2 patients carrying TP53 mutations. Our findings shed light on the genetic basis of post-allo-SCT relapse and may pave the way for unraveling novel therapeutic strategies in this challenging situation.

2.
Bone Marrow Transplant ; 54(11): 1847-1858, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31089287

RESUMO

Although allogeneic hematopoietic stem-cell transplantation (HSCT) provides high cure rates for children with high-risk acute lymphoblastic leukaemia (ALL), relapses remain the main cause of treatment failure. Whereas donor killer cell immunoglobulin-like receptor (KIR) genotype was shown to impact on relapse incidence in adult myeloid leukaemia similar studies in paediatric ALL are largely missing. Effect of donor KIR genotype on transplant outcome was evaluated in 317 children receiving a first myeloablative HSCT from an HLA-matched unrelated donor or sibling within the prospective ALL-SCT-BFM-2003 trial. Analysis of donor KIR gene polymorphism revealed that centromeric presence and telomeric absence of KIR B haplotypes was associated with reduced relapse risk. A centromeric/telomeric KIR score (ct-KIR score) integrating these observations correlated with relapse risk (hazard ratio (HR) 0.58; P = 0.002) while it had no impact on graft-versus-host disease or non-relapse mortality. In multivariable analyses ct-KIR score was associated with reduced relapse risk (HR 0.58; P = 0.003) and a trend towards improved event-free survival (HR 0.76; P = 0.059). This effect proved independent of MRD level prior to HSCT. Our data suggest that in children with ALL undergoing HSCT after myeloablative conditioning, donor selection based on KIR genotyping holds promise to improve clinical outcome by decreasing relapse risk and prolonged event-free survival.

3.
J Allergy Clin Immunol Pract ; 7(3): 848-855, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30391550

RESUMO

BACKGROUND: Biallelic variations in the dedicator of cytokinesis 8 (DOCK8) gene cause a combined immunodeficiency with eczema, recurrent bacterial and viral infections, and malignancy. Natural disease outcome is dismal, but allogeneic hematopoietic stem cell transplantation (HSCT) can cure the disease. OBJECTIVE: To determine outcome of HSCT for DOCK8 deficiency and define possible outcome variables. METHODS: We performed a retrospective study of the results of HSCT in a large international cohort of DOCK8-deficient patients. RESULTS: We identified 81 patients from 22 centers transplanted at a median age of 9.7 years (range, 0.7-27.2 years) between 1995 and 2015. After median follow-up of 26 months (range, 3-135 months), 68 (84%) patients are alive. Severe acute (III-IV) or chronic graft versus host disease occurred in 11% and 10%, respectively. Causes of death were infections (n = 5), graft versus host disease (5), multiorgan failure (2), and preexistent lymphoma (1). Survival after matched related (n = 40) or unrelated (35) HSCT was 89% and 81%, respectively. Reduced-toxicity conditioning based on either treosulfan or reduced-dose busulfan resulted in superior survival compared with fully myeloablative busulfan-based regimens (97% vs 78%; P = .049). Ninety-six percent of patients younger than 8 years at HSCT survived, compared with 78% of those 8 years and older (P = .06). Of the 73 patients with chimerism data available, 65 (89%) had more than 90% donor T-cell chimerism at last follow-up. Not all disease manifestations responded equally well to HSCT: eczema, infections, and mollusca resolved quicker than food allergies or failure to thrive. CONCLUSIONS: HSCT is curative in most DOCK8-deficient patients, confirming this approach as the treatment of choice. HSCT using a reduced-toxicity regimen may offer the best chance for survival.

4.
Clin Infect Dis ; 68(8): 1406-1409, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30357314

RESUMO

Here, we report the case of severe adenoviremia in a 7-year-old boy with highly-resistant, acute leukemia. A combined approach of αßTCR-CD19-depleted stem cell transplantation, enabling immunosuppression-free post-transplant care, and early transfer of adenovirus-specific donor T cells during aplasia resulted in rapid and complete clearance of the treatment-refractory adenoviremia.

5.
Front Immunol ; 8: 773, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28713390

RESUMO

Immunodeficiency, centromeric instability, and facial anomaly (ICF) syndrome is a rare autosomal recessive genetic condition with severe immunodeficiency, which leads to lethal infections if not recognized and treated in early childhood. Up-to-date treatment regimens consist of prophylactic and supportive treatment of the recurrent infections. Here, we report the case of a 1-year-old boy of Moroccan consanguineous parents, who was diagnosed at 4 months of age with ICF syndrome with a homozygous missense mutation in the DNMT3B gene. He was initially admitted to the hospital with recurrent pulmonary infections from the opportunistic pathogen Pneumocystis jirovecii (PJ). Further immunological workup revealed agammaglobulinemia in the presence of B cells. After successful recovery from the PJ pneumonia, he underwent hematopoietic stem cell transplantation (HSCT) from the HLA-matched healthy sister using a chemotherapeutic conditioning regimen consisting of treosulfan, fludarabine, and thiotepa. Other than acute chemotherapy-associated side effects, no serious adverse events occurred. Six months after HSCT immune-reconstitution, he had a stable chimerism with 2.9% autologous portion in the peripheral blood and a normal differential blood cell count, including all immunoglobulin subtypes. This is one of the first cases of successful HSCT in ICF syndrome. Early diagnosis and subsequent HSCT can prevent severe opportunistic infections and cure the immunodeficiency. Centromeric instability and facial anomaly remain unaffected. Although the long-term patient outcome and the neurological development remain to be seen, this curative therapy for immunodeficiency improves life expectancy and quality of life. This case is meant to raise physicians awareness for ICF syndrome and highlight the consideration for HSCT in ICF syndrome early on.

6.
J Immunother ; 40(6): 224-248, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28557814

RESUMO

Invariant natural killer T (iNKT)/natural killer (NK)/cytokine-induced killer (CIK) cells are important for immune surveillance. (I) Novel combinations of antibody 6B11 (targeting the Vα24-Jα18-invariant T-cell receptor) with CD4/CD8/CD1d/Vα24 for iNKT subset detection and "T/NK cell-like"-iNKT subsets were defined. Compared with healthy peripheral blood mononuclear cells (MNC) (significantly) lower proportions of iNKT cells (6B11/6B11CD3/6B11CD161), NK cells (CD3CD56/CD3CD161), and CIK cells (CD3CD56/CD3CD161) were found in peripheral blood MNC from acute myeloid (AML)/acute myeloid, lymphoid (ALL)/chronic lymphoid leukemia (CLL) patients in acute disease stages. Subtyping of iNKT cells revealed (significantly) higher proportions of CD3 T cells and CD161 NK cells in AML/ALL/CLL expressing 6B11 compared with healthy MNC. Prognostic evaluations showed higher proportions of iNKT/NK/CIK cells in favorable AML subgroups (younger age, primary, no extramedullary disease, achievement/maintenance of complete remission) or adult ALL and CLL patients. (II) iNKT/NK/CIK cell frequencies increased after (vs. before) mixed lymphocyte cultures of T-cell-enriched immune reactive cells stimulated with MNC/whole blood with or without pretreatment with "cocktails" (dendritic cells generating methods/kits inducing blasts' conversion to leukemia-derived dendritic cells from AML patients). Individual "cocktails" leading to "highest" iNKT cell frequencies could be defined. Antileukemic blast lytic activity correlated significantly with frequencies of iNKT/NK/CIK cells. In summary healthy MNC show significantly more iNKT/NK/CIK cells compared with AML/ALL/CLL MNC, a shift in the iNKT cell composition is seen in healthy versus leukemic samples and iNKT/NK/CIK cell-proportions in AML/ALL/CLL MNC samples correlate with prognosis. "Cocktail"-treated AML blasts lead to higher iNKT/NK/CIK cell frequencies and samples with antileukemic activity show significantly higher frequencies of iNKT/NK/CIK cells. Proportions of iNKT/NK/CIK cells should regularly be evaluated in AML/ALL/CLL diagnosis panels for quantitative/prognostic estimation of individual patients' antileukemic potential and their role in dendritic cells/leukemia-derived dendritic cells triggered immune surveillance.


Assuntos
Células Matadoras Induzidas por Citocinas/imunologia , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Células T Matadoras Naturais/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/metabolismo , Antígenos CD/imunologia , Antígenos CD/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Doença Crônica , Citotoxicidade Imunológica , Feminino , Humanos , Vigilância Imunológica , Imunofenotipagem , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
8.
Mol Ther ; 24(9): 1634-43, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27380762

RESUMO

Prognosis of primary refractory and relapsed pediatric B-lineage acute lymphoblastic leukemia (ALL) is very poor. Relapse rates significantly correlate with persistent minimal residual disease (MRD). In MRD, favorable effector-target ratios prevail and thus this situation might be optimally suited for immunotherapy with antibodies recruiting immunological effector cells. We here report on the generation, preclinical characterization and first clinical application in B-lineage ALL of an Fc-optimized CD19 antibody. This third-generation antibody (4G7SDIE) mediated enhanced antibody-dependent cellular cytotoxicity (ADCC) against leukemic blasts with effector cells from healthy volunteers and B-lineage ALL patients. The antibody was produced in a university-owned production unit and was applied on a compassionate use basis to 14 pediatric patients with refractory and relapsed B-lineage ALL at the stage of MRD. In 10/14 patients, MRD was reduced by ≥ 1 log or below the patient-individual detection limit, and 5/14 patients have achieved ongoing complete molecular remission with a median leukemia-free survival of 428 days. Two additional patients died in complete molecular remission due to complications not related to antibody therapy. Besides profound in vivo B-cell depletion, side effects were negligible. A clinical phase 1/2 study to further assess the therapeutic activity of 4G7SDIE is in preparation.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD19 , Fragmentos Fc das Imunoglobulinas , Neoplasia Residual/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Adolescente , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Terapia Combinada , Monitoramento de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Estimativa de Kaplan-Meier , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Retratamento , Resultado do Tratamento , Adulto Jovem
9.
Clin Immunol ; 162: 27-30, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26529633

RESUMO

PIK3R1 (phosphoinositide-3-kinase, regulatory subunit 1) gain-of-function has recently been described in patients with recurrent sinopulmonary infections, chronic CMV-/EBV-infections, lymphoproliferation, and hypogammaglobulinemia. Here we report a 15-year-old boy with treatment refractory CMV lymphadenitis, severe combined immunodeficiency, microcephaly and a severe developmental defect of Th17 cells. To avoid poor outcome, hematopoietic stem cell transplantation (HSCT) was performed. Subsequently, whole exome sequencing revealed a de novo heterozygous G-to-C mutation (chr5: 5:67,589,663: G>C) at the splice donor site of the PIK3R1 gene. Our data suggest that PIK3R1 gain-of-function leads to developmental defects in helper and regulatory T-cell subsets, the latter expanding the immunological features of PIK3R1 gain-of-function. T-cell subsets play a critical role in the regulation of immune response against infectious agents and of autoimmunity and thus may be particularly accountable for the clinical phenotype of affected patients.


Assuntos
Infecções por Citomegalovirus , Linfadenite , Microcefalia , Fosfatidilinositol 3-Quinases , Infecções Respiratórias , Adolescente , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Humanos , Linfadenite/complicações , Linfadenite/genética , Linfadenite/imunologia , Masculino , Microcefalia/complicações , Microcefalia/genética , Seios Paranasais/fisiopatologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Recidiva , Infecções Respiratórias/complicações , Infecções Respiratórias/genética , Células Th17/imunologia
10.
Blood ; 125(12): 1986-94, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25617426

RESUMO

Hematopoietic stem cell transplantation (HSCT) has improved over the last few decades. However, viral infections are often refractory to pharmacologic treatment and require alternative treatment strategies such as immunotherapy. Adenovirus (AdV) is th predominant disease-causing pathogen in pediatric HSCT. In a clinical trial, we analyzed safety and efficacy of ex vivo adoptive T-cell transfer (ACT) with hexon-specific T cells, predominantly of the T-helper cell 1 (Th1) phenotype, in 30 patients with AdV disease or viremia. ACT was feasible with no acute toxicities or significant onset of graft-versus-host disease. ACT led to in vivo antiviral immunity for up to 6 months with viral control, resulting in complete clearance of viremia in 86% of patients with antigen-specific T-cell responses. After ACT and a follow-up of 6 months, overall survival was markedly increased in responders (mean, 122 days; 15 survivors) compared with nonresponders who all died shortly after ACT (mean, 24 days; no survivors). AdV-related mortality was 100% in nonresponders compared with 9.5% in responders (≥1 log reduction of DNA copies per milliliter after ACT). In summary, ex vivo ACT of AdV-specific Th1 cells was well tolerated and led to successful and sustained restoration of T-cell immunity correlated with virologic response and protection from virus-related mortality. This cellular immunotherapy is a short-term available and broadly applicable treatment. The study is registered at European Union Clinical Trials Register as 2005-001092-35.


Assuntos
Infecções por Adenoviridae/complicações , Proteínas do Capsídeo/metabolismo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Imunoterapia Adotiva/métodos , Linfócitos T/citologia , Células Th1/citologia , Infecções por Adenoviridae/etiologia , Adolescente , Transferência Adotiva , Adulto , Criança , Pré-Escolar , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fenótipo , Probabilidade , Linfócitos T/imunologia , Resultado do Tratamento , Adulto Jovem
11.
Cell Commun Adhes ; 22(2-6): 49-65, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-27602789

RESUMO

To enlighten interactions between autologous, allogeneic or T-cells from patients after stem cell transplantation with leukaemia-derived-dendritic-cells containing dendritic cells or blast containing mononuclear cells (n = 21, respectively), we determined cytokine-concentrations (interleukin 2, 4, 6, 10, tumor-necrosis-factor-α, interferon-γ) in supernatants of mixed-lymphocyte-culture and in serum (n = 16) of 20 patients with acute myeloid leukaemia and three patients with myelodysplastic syndromes by cytometric-bead-assay. We correlated our data with lytic capabilities of stimulated T-cells in a fluorolysis-assay and clinical data: Dendritic-cell-/mononuclear-cell-stimulation of T-cells resulted in increased cytokine-levels in culture-medium compared to serum. There were no significant differences between cytokine-patterns of cases with/without lytic T-cell-activity, response to immunotherapy (stem cell transplantation/donor-lymphocyte-infusion) or graft-versus-host-disease. However, some predictive cytokine-cut-off-values for antileukaemic T-cell-activity, patients' response to immunotherapy and graft-versus-host-disease could be defined. Cytokine-profiles alone, without functional assays, are no useful tool to predict antileukaemic T-cell-function, although they can indicate lytic T-cell-activity, patients' response to immunotherapy and graft-versus-host-disease.


Assuntos
Células Dendríticas/imunologia , Imunoterapia , Leucemia Mieloide Aguda/terapia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Criança , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Feminino , Humanos , Imunoterapia/métodos , Leucemia Mieloide Aguda/imunologia , Teste de Cultura Mista de Linfócitos , Masculino , Pessoa de Meia-Idade , Transplante de Células-Tronco/métodos , Adulto Jovem
12.
Br J Haematol ; 169(1): 90-102, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25495919

RESUMO

Children with B cell malignancies refractory to standard therapy are known to have a poor prognosis and very limited treatment options. Here, we report on the treatment and follow-up of ten patients diagnosed with relapsed or refractory mature B-cell Non Hodgkin Lymphoma (B-NHL), Burkitt leukaemia (B-AL) or pre B-acute lymphoblastic leukaemia (pre B-ALL). All children were treated with FBTA05 (now designated Lymphomun), an anti-CD3 x anti-CD20 trifunctional bispecific antibody (trAb) in compassionate use. Within individual treatment schedules, Lymphomun was applied (a) after allogeneic stem cell transplantation (allo-SCT, n = 6) to induce sustained long-term remission, or (b) stand alone prior to subsequent chemotherapy to eradicate residual disease before allo-SCT (n = 4). Nine of ten children displayed a clinical response: three stable diseases (SD), one partial remission (PR) and five induced or sustained complete remissions (CR). Five of these nine responders died during follow-up. The other patients still maintain CR with a current overall survival of 874-1424 days (median: 1150 days). In conclusion, despite the dismal clinical prognosis of children refractory to standard therapy, immunotherapy with Lymphomun resulted in a favourable clinical outcome in this cohort of refractory paediatric patients.


Assuntos
Anticorpos Biespecíficos/administração & dosagem , Linfoma de Burkitt/terapia , Imunoterapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Transplante de Células-Tronco , Adolescente , Aloenxertos , Anticorpos Biespecíficos/efeitos adversos , Linfoma de Burkitt/mortalidade , Linfoma de Burkitt/patologia , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Masculino , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Taxa de Sobrevida
13.
Blood ; 124(4): 628-37, 2014 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-24855206

RESUMO

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are threatened by potentially lethal viral manifestations like cytomegalovirus (CMV) reactivation. Because the success of today's virostatic treatment is limited by side effects and resistance development, adoptive transfer of virus-specific memory T cells derived from the stem cell donor has been proposed as an alternative therapeutic strategy. In this context, dose minimization of adoptively transferred T cells might be warranted for the avoidance of graft-versus-host disease (GVHD), in particular in prophylactic settings after T-cell-depleting allo-HSCT protocols. To establish a lower limit for successful adoptive T-cell therapy, we conducted low-dose CD8(+) T-cell transfers in the well-established murine Listeria monocytogenes (L.m.) infection model. Major histocompatibility complex-Streptamer-enriched antigen-specific CD62L(hi) but not CD62L(lo) CD8(+) memory T cells proliferated, differentiated, and protected against L.m. infections after prophylactic application. Even progenies derived from a single CD62L(hi) L.m.-specific CD8(+) T cell could be protective against bacterial challenge. In analogy, low-dose transfers of Streptamer-enriched human CMV-specific CD8(+) T cells into allo-HSCT recipients led to strong pathogen-specific T-cell expansion in a compassionate-use setting. In summary, low-dose adoptive T-cell transfer (ACT) could be a promising strategy, particularly for prophylactic treatment of infectious complications after allo-HSCT.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Doença Enxerto-Hospedeiro/imunologia , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Imunodeficiência Combinada Severa/imunologia , Adolescente , Animais , Diferenciação Celular , Proliferação de Células , Criança , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/terapia , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Hematopoéticas , Proteínas de Homeodomínio/fisiologia , Humanos , Imunização , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Imunodeficiência Combinada Severa/metabolismo , Imunodeficiência Combinada Severa/terapia , Transplante Homólogo , Ativação Viral
15.
Immunobiology ; 219(4): 247-60, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24315637

RESUMO

T-cells play an important role in the remission-maintenance in AML-patients (pts) after SCT, however the role of LAA- (WT1, PR1, PRAME) or minor-histocompatibility (mHag, HA1) antigen-specific CD4(+) and CD8(+)T-cells is not defined. A LAA/HA1-peptide/protein stimulation, cloning and monitoring strategy for specific CD8(+)/CD4(+)T-cells in AML-pts after SCT is given. Our results show that (1) LAA-peptide-specific CD8+T-cells are detectable in every AML-pt after SCT. CD8(+)T-cells, recognizing two different antigens detectable in 5 of 7 cases correlate with long-lasting remissions. Clonal TCR-Vß-restriction exemplarily proven by spectratyping in PRAME-specific CD8(+)T-cells; high PRAME-peptide-reactivity was CD4(+)-associated, as shown by IFN-γ-release. (2) Two types of antigen-presenting cells (APCs) were tested for presentation of LAA/HA1-proteins to CD4(+)T-cells: miniEBV-transduced lymphoblastoid cells (B-cell-source) and CD4-depleted MNC (source for B-cell/monocyte/DC). We provide a refined cloning-system for proliferating, CD40L(+)CD4(+)T-cells after LAA/HA1-stimulation. CD4(+)T-cells produced cytokines (GM-CSF, IFN-γ) upon exposure to LAA/HA1-stimulation until after at least 7 restimulations and demonstrated cytotoxic activity against naive blasts, but not fibroblasts. Antileukemic activity of unstimulated, stimulated or cloned CD4(+)T-cells correlated with defined T-cell-subtypes and the clinical course of the disease. In conclusion we provide immunological tools to enrich and monitor LAA/HA1-CD4(+)- and CD8(+)T-cells in AML-pts after SCT and generate data with relevant prognostic value. We were able to demonstrate the presence of LAA-peptide-specific CD8(+)T-cell clones in AML-pts after SCT. In addition, we were also able to enrich specific antileukemic reactive CD4(+)T-cells without GvH-reactivity upon repeated LAA/HA1-protein stimulation and limiting dilution cloning.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Transplante de Células-Tronco , Antígenos de Neoplasias/imunologia , Ligante de CD40/metabolismo , Técnicas de Cultura de Células , Células Clonais , Citotoxicidade Imunológica , Feminino , Efeito Enxerto vs Leucemia/imunologia , Humanos , Interferon gama/metabolismo , Ativação Linfocitária , Masculino , Antígenos de Histocompatibilidade Menor/imunologia , Estadiamento de Neoplasias , Fragmentos de Peptídeos/imunologia , Transplante Homólogo , Proteínas WT1/imunologia
16.
Br J Haematol ; 162(6): 802-7, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23829632

RESUMO

The treatment outcome of children with refractory acute leukaemia or relapse post-stem cell transplantation is dismal. We report 10 children (non-remission n = 7) who underwent a new haploidentical transplant approach utilizing unmanipulated bone marrow followed by CD6-depleted peripheral blood stem cells. Nine patients had successful engraftment and no evidence of leukaemia. Acute and chronic graft-versus-host-disease was observed in five and three patients, respectively; two patients died of treatment-related toxicity. Seven patients relapsed after 7 (range 3-34) months, however two patients are alive at 6·5 and 7·0 years. This approach provides anti-leukaemic activity even in heavily pre-treated children but long-term disease control requires further intervention.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Transplante de Medula Óssea/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/imunologia , Leucemia/cirurgia , Adolescente , Criança , Pré-Escolar , Doença Enxerto-Hospedeiro/imunologia , Haploidia , Humanos , Lactente , Leucemia/imunologia , Depleção Linfocítica/métodos , Recidiva Local de Neoplasia/cirurgia , Resultado do Tratamento
17.
Haematologica ; 98(9): 1388-96, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23716544

RESUMO

Current diagnostic approaches that characterize T-cell deficiency by analyzing diversity of T-cell receptor sequences effectuate limited informational gain about the actual restrictiveness. For deeper insight into T-cell receptor repertoires we developed next-generation-sequencing-spectratyping, which employs high coverage Roche/454 sequencing of T-cell receptor (ß)-chain amplicons. For automated analysis of high-throughput-sequencing data, we developed a freely available software, the TCR profiler. Gene usage, length, encoded amino acid sequence and sequence diversity of the complementarity determining region 3 were determined and comprehensively integrated into a novel complexity score. Repertoires of CD8(+) T cells from children with idiopathic or hepatitis-induced very severe aplastic anemia (n=7), children two months after bone marrow transplantation (n=7) and healthy controls (children n=5, adults n=5) were analyzed. Complexity scores clearly distinguished between healthy and diseased, and even between different immune deficiency states. The repertoire of aplastic anemia patients was dominated by public (i.e. present in more than one person) T-cell receptor clonotypes, whereas only 0.2% or 1.9% were public in normal children and adults, respectively. The CDR3 sequence ASSGVGFSGANVLT was highly prevalent in 3 cases of hepatitis-induced anemia (15-32% of all sequences), but was only low expressed in idiopathic aplastic anemia (2-5%, n=4) or healthy controls (<1%). Fifteen high frequent sequences were present exclusively in aplastic anemia patients. Next-generation-sequencing-spectratyping allows in-depth analysis of T-cell receptor repertoires and their restriction in clinical samples. A dominating clonotype was identified in hepatitis-induced anemia that may be associated with disease pathogenesis and several aplastic-anemia-associated, putatively autoreactive clonotypes were sequenced.


Assuntos
Anemia Aplástica/genética , Regiões Determinantes de Complementaridade/genética , Hepatite/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Receptores de Antígenos de Linfócitos T/genética , Índice de Gravidade de Doença , Adolescente , Adulto , Sequência de Aminoácidos , Anemia Aplástica/diagnóstico , Criança , Pré-Escolar , Estudos de Coortes , Regiões Determinantes de Complementaridade/química , Hepatite/diagnóstico , Humanos , Lactente , Dados de Sequência Molecular , Estudos Prospectivos
18.
Clin Exp Med ; 13(1): 29-48, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22441559

RESUMO

Myeloid leukemic cells can be induced to differentiate into leukemia-derived dendritic cells (DCleu) regaining the stimulatory capacity of professional DCs while presenting the leukemic antigen repertoire. But so far, the induced antileukemic T-cell responses are variable both in specificity and in efficacy. In an attempt to elucidate the underlying causes of different T-cell response patterns, T-cell receptor (TR) Vß chain rearrangements were correlated with the T cells corresponding immunophenotypic profile, as well as their proliferative response and cytolytic capacities. In three different settings, donor T cells, either human leukocyte antigen matched or mismatched (haploidentical), or autologous T cells were repeatedly stimulated with myeloid blasts or leukemia-derived DC/DCleus from the corresponding patients diseased from acute myeloid leukemia (AML). Although no significant differences in T-cell proliferation were observed, the T-cell-mediated cytolytic response pattern varied considerably and even caused blast proliferation in two cases. Spectratyping revealed a remarkable restriction (>75% of normal level) of the CD4+ or CD8+-TR repertoire of blast- or DC/DCleu-stimulated T cells. Although in absolute terms, DC/DCleu stimulation induced the highest grade of restriction in the CD8+ T-cell subset, the CD4+ T-cell compartment seemed to be relatively more affected. But most importantly, in vitro stimulation with DC/DCleu resulted into an identical TR restriction pattern (ß chain) that could be identified in vivo in a patient sample 3 months after allo-SCT. Thus, in vitro tests combining functional flow cytometry with spectratyping might provide predictive information about T cellular response patterns in vivo.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunofenotipagem , Leucemia Mieloide Aguda/imunologia , Receptores de Antígenos de Linfócitos T/análise , Proliferação de Células , Células Cultivadas , Técnicas Citológicas/métodos , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Citometria de Fluxo , Humanos
20.
Haematologica ; 97(9): 1304-11, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22315490

RESUMO

UNLABELLED: BACKGROUND Very severe aplastic anemia is characterized by a hypoplastic bone marrow due to destruction of CD34(+) stem cells by autoreactive T cells. Investigation of the pathomechanism by patient-specific gene expression analysis of the attacked stem cells has previously been impractical because of the scarcity of these cells at diagnosis. DESIGN AND METHODS: Employing unbiased RNA amplification, patient-specific gene expression profiling was carried out for CD34(+) cells from patients newly diagnosed with very severe aplastic anemia (n=13), refractory anemia (n=8) and healthy controls (n=10). These data were compared to profiles of myelodysplastic disease (n=55), including refractory anemia (n=18). To identify possible targets of autoimmune attack, presence of autoreactive antibodies was tested in pre-therapeutic sera of patients with very severe aplastic anemia (n=19). RESULTS: CD34(+) gene expression profiling distinguished between healthy controls, children with aplastic or refractory anemia and clonal disease. Interferon stimulated genes such as the apoptosis inducing death ligand TRAIL were strongly up-regulated in CD34(+) cells of patients with aplastic anemia, in particular in patients responding to immunosuppressive treatment. In contrast, mRNA expression of integrin GPVI and the integrin complexes GPIa/IIa, GPIIb/IIIa, GPIB/GPIX/GPV was significantly down-regulated and corresponding antibodies were detected in 7 of 11 profiled patients and in 11 of 19 aplastic anemia patients. CONCLUSIONS As a potential diagnostic tool, patient-specific gene expression profiling of CD34(+) stem cells made it possible to make the difficult differential diagnosis of most patients with aplastic and refractory anemia. Profiling indicated a prognostic correlation of TRAIL expression and patient benefit from immunosuppressive therapy. Downregulation of integrin expression and concurrent presence of autoreactive anti-integrin-antibodies suggested a previously unrecognized pathological role of integrins in aplastic anemia.


Assuntos
Anemia Aplástica/genética , Anemia Refratária/genética , Antígenos CD34/genética , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Integrinas/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Adolescente , Anemia Aplástica/tratamento farmacológico , Anemia Aplástica/patologia , Anemia Refratária/tratamento farmacológico , Anemia Refratária/patologia , Apoptose , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Imunossupressores/uso terapêutico , Masculino , Análise em Microsséries , Prognóstico
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