RESUMO
Cyclic neutropenia (CyN) is a hematologic disorder in which peripheral blood absolute neutrophil counts (ANCs) show cycles of approximately 21-day intervals. The majority of CyN patients harbor ELANE mutations, but the mechanism of ANC cycling is unclear. We performed analysis of bone marrow (BM) subpopulations in CyN patients at the peak and the nadir of the ANC cycle and detected high proportions of BM hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) at the nadir of the ANC cycle, as compared with the peak. BM HSPCs produced fewer granulocyte colony-forming unit colonies at the ANC peak. To investigate the mechanism of cycling, we found that mRNA expression levels of ELANE and unfolded protein response (UPR)-related genes (ATF6, BiP (HSPA5), CHOP (DDIT3), and PERK (EIF2AK3)) were elevated, but antiapoptotic genes (Bcl-2 (BCL2) and bcl-xL (BCL2L1)) were reduced in CD34+ cells tested at the ANC nadir. Moreover, HSPCs revealed increased levels of reactive oxygen species and gH2AX at the ANC nadir. We suggest that in CyN patients, some HSPCs escape the UPR-induced endoplasmic reticulum (ER) stress and proliferate in response to granulocyte colony-stimulating factor (G-CSF) to a certain threshold at which UPR again affects the majority of HSPCs. There is a cyclic balance between ER stress-induced apoptosis of HSPCs and compensatory G-CSF-stimulated HSPC proliferation followed by granulocytic differentiation.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Elastase de Leucócito/genética , Neutropenia/etiologia , Resposta a Proteínas não Dobradas/fisiologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Seguimentos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Elastase de Leucócito/fisiologia , Mutação , Neutropenia/tratamento farmacológico , Neutropenia/metabolismo , Neutropenia/patologia , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genéticaRESUMO
A Autosomal-dominant ELANE mutations are the most common cause of severe congenital neutropenia. Although the majority of congenital neutropenia patients respond to daily granulocyte colony stimulating factor, approximately 15 % do not respond to this cytokine at doses up to 50 µg/kg/day and approximately 15 % of patients will develop myelodysplasia or acute myeloid leukemia. "Maturation arrest," the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated congenital neutropenia. As mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and "maturation arrest" corrected by a CRISPR/Cas9-sgRNA ribonucleoprotein mediated ELANE knockout. To examine this hypothesis, we used induced pluripotent stem cells from two congenital neutropenia patients and primary hematopoietic stem and progenitor cells from four congenital neutropenia patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE We observed that granulocytic differentiation of ELANE knockout induced pluripotent stem cells and primary hematopoietic stem and progenitor cells were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO (knockout) neutrophils were also normal. Knockdown of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils. These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients' primary hematopoietic stem and progenitor cells followed by autologous transplantation may be an alternative therapy for congenital neutropenia.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Neutropenia , Sistemas CRISPR-Cas , Síndrome Congênita de Insuficiência da Medula Óssea , Humanos , Mutação , Neutropenia/congênito , Neutropenia/genéticaRESUMO
Hematopoietic transcription factor LIM domain only 2 (LMO2), a member of the TAL1 transcriptional complex, plays an essential role during early hematopoiesis and is frequently activated in T-cell acute lymphoblastic leukemia (T-ALL) patients. Here, we demonstrate that LMO2 is activated by deacetylation on lysine 74 and 78 via the nicotinamide phosphoribosyltransferase (NAMPT)/sirtuin 2 (SIRT2) pathway. LMO2 deacetylation enables LMO2 to interact with LIM domain binding 1 and activate the TAL1 complex. NAMPT/SIRT2-mediated activation of LMO2 by deacetylation appears to be important for hematopoietic differentiation of induced pluripotent stem cells and blood formation in zebrafish embryos. In T-ALL, deacetylated LMO2 induces expression of TAL1 complex target genes HHEX and NKX3.1 as well as LMO2 autoregulation. Consistent with this, inhibition of NAMPT or SIRT2 suppressed the in vitro growth and in vivo engraftment of T-ALL cells via diminished LMO2 deacetylation. This new molecular mechanism may provide new therapeutic possibilities in T-ALL and may contribute to the development of new methods for in vitro generation of blood cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hematopoese , Proteínas com Domínio LIM/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Acetilação , Animais , Células Cultivadas , Células HEK293 , Humanos , Leucopoese , Camundongos , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Peixe-ZebraRESUMO
Influenza remains a major pathogen in older people. Infection with CMV and the accumulation of late-differentiated T cells associated with it have been implicated in poor Ab responsiveness to influenza vaccination in the elderly, most of whom are CMV positive. However, whether CMV infection also affects memory T cell responses to influenza remains unknown. To investigate this, we assessed T cell responses to influenza A matrix protein and nucleoprotein ex vivo in 166 Dutch individuals (mean age 62.2 y, range 42-82) and validated the results in a second cohort from North America (mean age 73.1 y, range 65-81, n = 28). We found that less than half of the CMV-infected older subjects mounted a CD4 T cell response to influenza Ags, whereas â¼80% of uninfected elderly did so. A similar proportion of younger subjects possessed influenza A virus-responsive CD4 T cells, and, interestingly, this was the case whether they were CMV-infected. Thus, the effect of CMV was only seen in the older donors, who may have been exposed to the virus for decades. The percentage of donors with CD8 responses to influenza A virus was lower than those with CD4; this was not influenced by whether the subjects were CMV seropositive or seronegative. CMV-seropositive responders had significantly higher frequencies of late-differentiated CD4 T-cells (CD45RA(+/-)CCR7(-)CD27(-)CD28(-)) compared with CMV-infected nonresponders. These data add to the accumulating evidence that infection with CMV has profound but heterogeneous effects on responses to the products of other viruses and have implications for the design of influenza vaccines, especially in the elderly.
Assuntos
Envelhecimento/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Memória Imunológica , Vírus da Influenza A/imunologia , Proteínas do Core Viral/imunologia , Latência Viral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Estudos de Coortes , Feminino , Humanos , Vacinas contra Influenza/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Influenza vaccination is less effective in the elderly compared to the young. Studies that have attempted to identify immune parameters correlating with satisfactory vaccine responses have yielded inconclusive results. Here, we correlate the distribution of different circulating CD4+ and CD8+ T-cell phenotypes with the humoral response to vaccination with Intanza, an intradermal seasonal vaccine, in 54 individuals of different ages. Subjects were stratified according to age (below or over 60) and presence of a latent infection with Cytomegalovirus (CMV). CMV-seropositivity was significantly associated with a lower response rate to the vaccine in people over but not below 60 yr of age. Unlike reported data, late-differentiated (CD45RA+CCR7-CD27-CD28-) CD4+, but not CD8+ T-cells associated with a poorer vaccine response. Thus, latent CMV infection has a deleterious effect on influenza antibody responses in the elderly, which might be mediated through CD4 T-cells lacking CCR7, CD27 and CD28 and re-expressing CD45RA.
Assuntos
Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Vacinas contra Influenza , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Formação de Anticorpos , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Infecções por Citomegalovirus/virologia , Feminino , Humanos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Antígenos Comuns de Leucócito/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores CCR7/metabolismo , Subpopulações de Linfócitos T/imunologia , Resultado do Tratamento , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Vacinação , Adulto JovemRESUMO
Human cytomegalovirus (CMV) establishes persistent, usually asymptomatic, infection in healthy people. Because CMV infection is associated with the presence of lower proportions of peripheral naïve CD8+ T-cells and a higher fraction of late-differentiated CD8+ cells, commonly taken as biomarkers of age-associated compromised adaptive immunity ('immunosenescence'), we asked whether chronic exposure to any persistent virus mediates these effects. Herpes simplex virus (HSV) is also a widespread herpesvirus that establishes lifelong persistence, but, unlike CMV, its impact on the distribution of T-cell subsets has not been established. Here, we analysed T-cell subsets in 93 healthy people aged 42-81 years infected or not infected with CMV and/or HSV. Individuals harbouring CMV were confirmed to possess lower frequencies of naïve CD8+ T-cells (defined as CD45RA+CCR7+CD27+CD28+) and greater proportions of late-differentiated effector memory (CD45RA-CCR7-CD27-CD28-) and so-called TEMRA (CD45RA+CCR7-CD27-CD28-) CD4 and CD8 subsets, independent of HSV seropositivity. In CMV-seronegative donors, HSV did not affect T-cell subset distribution significantly. We conclude that these hallmarks of age-associated alterations to immune signatures are indeed observed in the general population in people infected with CMV and not those infected with a different persistent herpesvirus.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/crescimento & desenvolvimento , Simplexvirus/crescimento & desenvolvimento , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD28/análise , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Diferenciação Celular , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Feminino , Citometria de Fluxo , Humanos , Antígenos Comuns de Leucócito/análise , Masculino , Pessoa de Meia-Idade , Receptores CCR7/metabolismo , Subpopulações de Linfócitos T/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análiseRESUMO
The aim of this study was to determine the prognostic implications of the pretreatment level of Th17 cells compared with regulatory T-cell status in prostate cancer patients receiving active whole cell immunotherapy. Ten-color flow cytometry was used to analyze IL-17-producing CD4(+) T-cells in the peripheral blood of hormone-resistant non-bone metastatic prostate cancer patients prior to immunotherapy with an allogeneic whole-cell vaccine. Surface expression of the chemokine receptors CCR4 and CCR6 was used to further subdivide IL-17-producing cells into subsets with distinct homing properties. The frequency of circulating regulatory T-cells (Tregs), defined as CD3(+)CD4(+)CD127(lo)Foxp3(+)CD25(+) was compared in the same patients. The frequency of CCR4(-)IL-17(+)CD4(+) T-cells prevaccination inversely correlated with time to disease progression (TTP) in 23 prostate cancer patients. Furthermore, responder (R) patients with statistically significant reductions in PSA velocity (PSAV) in response to the immunotherapy (n = 9), showed a Th17 profile similar to healthy male controls and significantly different from non-responder (NR) patients (n = 14) (i.e., those without any significant reduction in PSAV). In contrast, the frequency of Tregs in peripheral blood in PSA-R (n = 11) and -NR (n = 14) patients was similar (but in both cases, significantly higher than in age-matched healthy men). Accordingly, there was no significant correlation between frequency of Tregs and TTP in these late-stage prostate cancer patients undergoing active immunotherapy. These data imply an important role for IL-17-producing helper T-cells in cancer immunology and highlight their potential use as a pretreatment screen to ensure appropriate treatment is offered to hormone-resistant prostate cancer patients.