RESUMO
Little is known about the efficacy of COVID-19 vaccines during acute lymphoblastic leukemia therapy (ALL); data for COVID-19 vaccine immune responses in pediatric leukemia remain sparse. We conducted a single center study of patients aged 5-25 years undergoing ALL chemotherapy who received COVID-19 vaccination. Twenty-one patients were enrolled; efficacy was evaluable in 20. Twenty were vaccinated while receiving chemotherapy. Twenty received the BNT162b2 mRNA vaccine. Spike reactive antibodies (S-IgG) and/or T-cells (SRT) were detected in 16 of 20 (80%) vaccinated patients; 13 (65%) and 9 (45%) were positive for S-IgG and SRT, respectively. Six (30%) showed both spike reactive B and T-cell responses. Eleven of the 13 with S-IgG positivity were negative for anti-Nucleocapsid IgG, an antibody profile consistent with a vaccine induced immune response. All 13S-IgG+ patients showed neutralizing antibodies. SRT included CD4+ (7) and CD8+ (6) T-cells; both CD4+ and CD8+ SRT were seen in 4. SRT were multifunctional (producing multiple cytokines) in most patients (8 of 9); 4 showed SRT with triple cytokine and B-cell co-stimulatory responses, indicating a multimodal adaptive immune response. Immune responses were seen among patients vaccinated in the settings of lymphopenia (6 of 12) intensive chemotherapy (3 of 4), and Peg allergy (6 of 8). Sequencing revealed public CD4+ and CD8+ TCR sequences reactive to epitopes across the spike protein. In conclusion, COVID-19 vaccination induced B and/or T-cell responses in a majority of children and young adults undergoing ALL chemotherapy.
Assuntos
Idarubicina , Leucemia Mieloide Aguda , Criança , Humanos , Idarubicina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica , Citarabina/uso terapêutico , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Vidarabina/uso terapêuticoRESUMO
Pediatric oncology patients are at risk for poor outcomes with respiratory viral infections. Outcome data for COVID-19 in children and young adults with cancer are needed; data are sparse for obese/overweight and adolescent and young adult subgroups. We conducted a single center cohort study of COVID-19 outcomes in patients younger than 25 years with cancer. Candidate hospitalization risk factors were analyzed via univariable and multivariable analyses. Eighty-seven patients with cancer and COVID-19 were identified. Most were Hispanic/Latinx (n = 63, 72%). Forty-two (48%) were overweight/obese. Anticancer therapy included chemotherapy only (n = 64, 74%), chimeric antigen receptor T-cells (CAR-T, n = 7), hematopoietic stem cell transplantation (HSCT, n = 12), or CAR-T and HSCT (n = 4). There was no COVID-19 related mortality. Twenty-six patients (30%) required COVID-19 related hospitalization; 4 required multiple hospitalizations. Nine (10%) had severe/critical infection; 6 needed intensive care. COVID-19 resulted in anticancer therapy delays in 22 (34%) of 64 patients on active therapy (median delay = 14 days). Factors associated with hospitalization included steroids within 2 weeks prior to infection, lymphopenia, previous significant non-COVID infection, and low COVID-19 PCR cycle threshold value. CAR-T recipients with B-cell aplasia tended to have severe/critical infection (3 of 7 patients). A COVID-19 antibody response was detected in 14 of 32 patients (44%). A substantial proportion of COVID-19 infected children and young adults with cancer require inpatient management; morbidity may be high in B-cell immunodeficiency. However, a majority of patients can be taken through chemotherapy without prolonged therapy delays. Viral load is a potential outcome predictor in COVID-19 in pediatric cancer.
Assuntos
COVID-19 , Transplante de Células-Tronco Hematopoéticas , Neoplasias , Receptores de Antígenos Quiméricos , Adolescente , Criança , Estudos de Coortes , Humanos , Neoplasias/complicações , Neoplasias/terapia , Obesidade , Sobrepeso , Adulto JovemRESUMO
The PI3K/Akt pathway-and in particular PI3Kδ-is known for its role in drug resistant B-cell acute lymphoblastic leukemia (B-ALL) and it is often upregulated in refractory or relapsed B-ALL. Myc proteins are transcription factors responsible for transcribing pro-proliferative genes and c-Myc is often overexpressed in cancers. The chromatin regulator BRD4 is required for expression of c-Myc in hematologic malignancies including B-ALL. Previously, combination of BRD4 and PI3K inhibition with SF2523 was shown to successfully decrease Myc expression. However, the underlying mechanism and effect of dual inhibition of PI3Kδ/BRD4 in B-ALL remains unknown. To study this, we utilized SF2535, a novel small molecule dual inhibitor which can specifically target the PI3Kδ isoform and BRD4. We treated primary B-ALL cells with various concentrations of SF2535 and studied its effect on specific pharmacological on-target mechanisms such as apoptosis, cell cycle, cell proliferation, and adhesion molecules expression usingin vitro and in vivo models. SF2535 significantly downregulates both c-Myc mRNA and protein expression through inhibition of BRD4 at the c-Myc promoter site and decreases p-AKT expression through inhibition of the PI3Kδ/AKT pathway. SF2535 induced apoptosis in B-ALL by downregulation of BCL-2 and increased cleavage of caspase-3, caspase-7, and PARP. Moreover, SF2535 induced cell cycle arrest and decreased cell counts in B-ALL. Interestingly, SF2535 decreased the mean fluorescence intensity (MFI) of integrin α4, α5, α6, and ß1 while increasing MFI of CXCR4, indicating that SF2535 may work through inside-out signaling of integrins. Taken together, our data provide a rationale for the clinical evaluation of targeting PI3Kδ/BRD4 in refractory or relapsed B-ALL using SF2535.
RESUMO
Although metabolic pathways have been shown to control differentiation and activation in peripheral T cells, metabolic studies on thymic T cell development are still lacking, especially in human tissue. In this study, we use transcriptomics and extracellular flux analyses to investigate the metabolic profiles of primary thymic and in vitro-derived mouse and human thymocytes. Core metabolic pathways, specifically glycolysis and oxidative phosphorylation, undergo dramatic changes between the double-negative (DN), double-positive (DP), and mature single-positive (SP) stages in murine and human thymus. Remarkably, despite the absence of the complex multicellular thymic microenvironment, in vitro murine and human T cell development recapitulated the coordinated decrease in glycolytic and oxidative phosphorylation activity between the DN and DP stages seen in primary thymus. Moreover, by inducing in vitro T cell differentiation from Rag1-/- mouse bone marrow, we show that reduced metabolic activity at the DP stage is independent of TCR rearrangement. Thus, our findings suggest that highly conserved metabolic transitions are critical for thymic T cell development.
Assuntos
Diferenciação Celular , Metabolismo Energético , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Animais , Evolução Biológica , Biomarcadores , Linhagem Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfopoese , Metaboloma , Metabolômica/métodos , Camundongos , Organoides , Timócitos/imunologia , Técnicas de Cultura de TecidosRESUMO
Single cell RNA sequencing of human thymic cells is dependent on isolation of highly pure and viable cell populations. This protocol describes the isolation of CD34+ progenitor and more differentiated CD34- fractions from post-natal thymic tissue to study thymopoiesis. CD34+ cells represent <1% of thymic cells, so this protocol uses magnetic- followed by fluorescence-activated cell separation to isolate highly enriched CD34+ cells. For complete details on the use and execution of this protocol, please refer to Le et al. (2020).
Assuntos
Separação Celular/métodos , RNA-Seq/métodos , Análise de Célula Única/métodos , Antígenos CD34/imunologia , Antígenos CD34/isolamento & purificação , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Humanos , RNA/genética , RNA/isolamento & purificação , Timo/metabolismo , Timo/fisiologiaRESUMO
Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Deleção de Genes , Integrina alfa6 , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Pirimidinas/farmacologia , Animais , Anticorpos Antineoplásicos/farmacologia , Anticorpos Neutralizantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapiaRESUMO
While early studies reported superior survival for cancer patients enrolled on clinical trials, recent findings are inconclusive. We investigated the association between enrollment on contemporary trials and event-free survival (EFS) in pediatric B-cell acute lymphoblastic leukemia (B-ALL). In a retrospective cohort of 274 children (1-21 years) treated for B-ALL from 2008 to 2015, 55.5% enrolled with no disparity in enrollment by age, sex, or ethnicity. Three-year EFS was similar for enrolled and not enrolled patients (90.1% [95% CI, 82.5-94.5] versus 86.5% [95% CI, 77.7-92.0]). Clinical trial enrollment did not affect pediatric B-ALL survival, albeit in a limited-size cohort treated at a single academic institution.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Adulto , Criança , Pré-Escolar , Ensaios Clínicos como Assunto , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Masculino , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
The quest continues for targeted therapies to reduce the morbidity of chemotherapy and to improve the response of resistant leukemia. Adhesion of acute lymphoblastic leukemia (ALL) cells to bone marrow stromal cells triggers intracellular signals that promote cell-adhesion-mediated drug resistance (CAM-DR). Idelalisib, an U.S. Food and Drug Administration (FDA)-approved PI3Kδ-specific inhibitor has been shown to be effective in CLL in down-regulating p-Akt and prolonging survival in combination with Rituximab; herein we explore the possibility of its use in B ALL and probe the mechanism of action. Primary B ALL in contact with OP9 stromal cells showed increased p-Aktser473. Idelalisib decreased p-Akt in patient samples of ALL with diverse genetic lesions. Addition of idelalisib to vincristine inhibited proliferation when compared to vincristine monotherapy in a subset of samples tested. Idelalisib inhibited ALL migration to SDF-1α in vitro and blocked homing of ALL cells to the bone marrow in vivo. This report tests PI3Kδ inhibitors in a more diverse group of ALL than has been previously reported and is the first published report of idelalisib inhibiting homing of ALL cells to bone marrow. Our data support further pre-clinical evaluation of idelalisib for the therapy of B ALL.
RESUMO
To elucidate the transcriptional 'landscape' that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitor cells spanning the earliest stages of B lymphoid and T lymphoid specification. Over 3,000 genes encoding previously unknown long non-coding RNAs (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage specific and were more lineage specific than those of protein-coding genes. Protein-coding genes co-expressed with neighboring lncRNA genes showed enrichment for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships among the earliest progenitor cells in the human bone marrow and thymus.
Assuntos
Linfócitos B/metabolismo , Linhagem da Célula/genética , Células Progenitoras Linfoides/metabolismo , RNA Longo não Codificante/genética , Linfócitos T/metabolismo , Transcriptoma , Teorema de Bayes , Células da Medula Óssea/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA/métodos , Timo/citologia , Timo/metabolismoRESUMO
The role of end of induction minimal residual disease (MRD) as determined by flow cytometry for treatment assignment in pediatric T-cell acute lymphoblastic leukemia (T-ALL) is not well defined. We studied 33 children with newly diagnosed T-ALL. Thirty-two of 33 patients remain in continuous complete remission at a median of 4 years. Nineteen patients were MRD positive at the end of induction and all remain in remission with augmented Berlin Frankfurt Münster-based therapy. One patient underwent hematopoietic stem cell transplant for rising MRD. Persistent end of induction MRD alone is not an indication to alter therapy in pediatric T-ALL.
Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Indução de Remissão , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células T Precursoras/sangue , Medição de RiscoRESUMO
A system that allows manipulation of the human thymic microenvironment is needed both to elucidate the extrinsic mechanisms that control human thymopoiesis and to develop potential cell therapies for thymic insufficiency. In this report, we developed an implantable thymic microenvironment composed of two human thymic stroma populations critical for thymopoiesis; thymic epithelial cells (TECs) and thymic mesenchyme (TM). TECs and TM from postnatal human thymi were cultured in specific conditions, allowing cell expansion and manipulation of gene expression, before reaggregation into a functional thymic unit. Human CD34+ hematopoietic stem and progenitor cells (HSPC) differentiated into T cells in the aggregates in vitro and in vivo following inguinal implantation of aggregates in immune deficient mice. Cord blood HSPC previously engrafted into murine bone marrow (BM), migrated to implants, and differentiated into human T cells with a broad T cell receptor repertoire. Furthermore, lentiviral-mediated expression of vascular endothelial growth factor in TM enhanced implant size and function and significantly increased thymocyte production. These results demonstrate an in vivo system for the generation of T cells from human HSPC and represent the first model to allow manipulation of gene expression and cell composition in the microenvironment of the human thymus.
Assuntos
Timo/citologia , Engenharia Tecidual/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Proliferação de Células/fisiologia , Microambiente Celular/fisiologia , Expressão Gênica , Humanos , Linfopoese/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Timo/efeitos dos fármacosRESUMO
Unlimited self renewal capacity and differentiation potential make human pluripotent stem cells (PSC) a promising source for the ex vivo manufacture of red blood cells (RBCs) for safe transfusion. Current methods to induce erythropoiesis from PSC suffer from low yields of RBCs, most of which are immature and contain embryonic and fetal rather than adult hemoglobins. We have previously shown that homodimerization of the intracellular component of MPL (ic-MPL) induces erythropoiesis from human cord blood progenitors. The goal of this study was to investigate the potential of ic-MPL dimerization to induce erythropoiesis from human embryonic stem cells (hESCs) and to identify the signaling pathways activated by this strategy. We present here the evidence that ic-MPL dimerization induces erythropoietin (EPO)-independent erythroid differentiation from hESC by inducing the generation of erythroid progenitors and by promoting more efficient erythroid maturation with increased RBC enucleation as well as increased gamma:epsilon globin ratio and production of beta-globin protein. ic-MPL dimerization is significantly more potent than EPO in inducing erythropoiesis, and its effect is additive to EPO. Signaling studies show that dimerization of ic-MPL, unlike stimulation of the wild type MPL receptor, activates AKT in the absence of JAK2/STAT5 signaling. AKT activation upregulates GATA-1 and FOXO3 transcriptional pathways with resulting inhibition of apoptosis, modulation of cell cycle, and enhanced maturation of erythroid cells. These findings open up potential new targets for the generation of therapeutically relevant RBC products from hPSC.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Eritropoese , Eritropoetina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Ciclo Celular , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Humanos , Megacariócitos/citologia , Megacariócitos/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Receptores de Trombopoetina/química , Receptores de Trombopoetina/metabolismoRESUMO
Hematopoietic stem and progenitor cells (HSPCs) emerge and develop adjacent to blood vessel walls in the yolk sac, aorta-gonad-mesonephros region, embryonic liver, and fetal bone marrow. In adult mouse bone marrow, perivascular cells shape a "niche" for HSPCs. Mesenchymal stem/stromal cells (MSCs), which support hematopoiesis in culture, are themselves derived in part from perivascular cells. In order to define their direct role in hematopoiesis, we tested the ability of purified human CD146(+) perivascular cells, as compared with unfractionated MSCs and CD146(-) cells, to sustain human HSPCs in coculture. CD146(+) perivascular cells support the long-term persistence, through cell-to-cell contact and at least partly via Notch activation, of human myelolymphoid HSPCs able to engraft primary and secondary immunodeficient mice. Conversely, unfractionated MSCs and CD146(-) cells induce differentiation and compromise ex vivo maintenance of HSPCs. Moreover, CD146(+) perivascular cells express, natively and in culture, molecular markers of the vascular hematopoietic niche. Unexpectedly, this dramatic, previously undocumented ability to support hematopoietic stem cells is present in CD146(+) perivascular cells extracted from the nonhematopoietic adipose tissue.
Assuntos
Vasos Sanguíneos/fisiologia , Antígeno CD146/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Adulto , Animais , Antígenos CD34/metabolismo , Vasos Sanguíneos/citologia , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Comunicação Celular , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Técnicas de Cocultura , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Receptores Notch/genética , Receptores Notch/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Serrate-JaggedRESUMO
During the last five decades, elegant mouse models of hematopoiesis have yielded most of the seminal insights into this complex biological system of self-renewal and lineage commitment. More recent advances in assays to measure human stem and progenitor cells as well as high resolution RNA profiling have revealed that although the basic roadmap of blood development is generally conserved across mammals, evolutionary pressures have generated many differences between the species that have important biological and translational implications. To enhance the utility of the mouse as a model organism, it is more important than ever that research data are presented with regard to how they might be influenced by the species of origin as well as the developmental source of the hematopoietic tissue.