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1.
Cell Mol Life Sci ; 81(1): 199, 2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38683377

RESUMO

Tyrosine kinase 2 (TYK2) is involved in type I interferon (IFN-I) signaling through IFN receptor 1 (IFNAR1). This signaling pathway is crucial in the early antiviral response and remains incompletely understood on B cells. Therefore, to understand the role of TYK2 in B cells, we studied these cells under homeostatic conditions and following in vitro activation using Tyk2-deficient (Tyk2-/-) mice. Splenic B cell subpopulations were altered in Tyk2-/- compared to wild type (WT) mice. Marginal zone (MZ) cells were decreased and aged B cells (ABC) were increased, whereas follicular (FO) cells remained unchanged. Likewise, there was an imbalance in transitional B cells in juvenile Tyk2-/- mice. RNA sequencing analysis of adult MZ and FO cells isolated from Tyk2-/- and WT mice in homeostasis revealed altered expression of IFN-I and Toll-like receptor 7 (TLR7) signaling pathway genes. Flow cytometry assays corroborated a lower expression of TLR7 in MZ B cells from Tyk2-/- mice. Splenic B cell cultures showed reduced proliferation and differentiation responses after activation with TLR7 ligands in Tyk2-/- compared to WT mice, with a similar response to lipopolysaccharide (LPS) or anti-CD40 + IL-4. IgM, IgG, IL-10 and IL-6 secretion was also decreased in Tyk2-/- B cell cultures. This reduced response of the TLR7 pathway in Tyk2-/- mice was partially restored by IFNα addition. In conclusion, there is a crosstalk between TYK2 and TLR7 mediated by an IFN-I feedback loop, which contributes to the establishment of MZ B cells and to B cell proliferation and differentiation.


Assuntos
Linfócitos B , Interferon Tipo I , Transdução de Sinais , Baço , TYK2 Quinase , Receptor 7 Toll-Like , Animais , Camundongos , Linfócitos B/metabolismo , Linfócitos B/imunologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Interferon Tipo I/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/citologia , Baço/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 7 Toll-Like/genética , TYK2 Quinase/metabolismo , TYK2 Quinase/genética
2.
Br J Cancer ; 117(2): 256-265, 2017 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-28557976

RESUMO

BACKGROUND: In B-cell precursor acute lymphoblastic leukaemia (B-ALL), the identification of additional genetic alterations associated with poor prognosis is still of importance. We determined the frequency and prognostic impact of somatic mutations in children and adult cases with B-ALL treated with Spanish PETHEMA and SEHOP protocols. METHODS: Mutational status of hotspot regions of TP53, JAK2, PAX5, LEF1, CRLF2 and IL7R genes was determined by next-generation deep sequencing in 340 B-ALL patients (211 children and 129 adults). The associations between mutation status and clinicopathological features at the time of diagnosis, treatment outcome and survival were assessed. Univariate and multivariate survival analyses were performed to identify independent prognostic factors associated with overall survival (OS), event-free survival (EFS) and relapse rate (RR). RESULTS: A mutation rate of 12.4% was identified. The frequency of adult mutations was higher (20.2% vs 7.6%, P=0.001). TP53 was the most frequently mutated gene (4.1%), followed by JAK2 (3.8%), CRLF2 (2.9%), PAX5 (2.4%), LEF1 (0.6%) and IL7R (0.3%). All mutations were observed in B-ALL without ETV6-RUNX1 (P=0.047) or BCR-ABL1 fusions (P<0.0001). In children, TP53mut was associated with lower OS (5-year OS: 50% vs 86%, P=0.002) and EFS rates (5-year EFS: 50% vs 78.3%, P=0.009) and higher RR (5-year RR: 33.3% vs 18.6% P=0.037), and was independently associated with higher RR (hazard ratio (HR)=4.5; P=0.04). In adults, TP53mut was associated with a lower OS (5-year OS: 0% vs 43.3%, P=0.019) and a higher RR (5-year RR: 100% vs 61.4%, P=0.029), whereas JAK2mut was associated with a lower EFS (5-year EFS: 0% vs 30.6%, P=0.035) and a higher RR (5-year RR: 100% vs 60.4%, P=0.002). TP53mut was an independent risk factor for shorter OS (HR=2.3; P=0.035) and, together with JAK2mut, also were independent markers of poor prognosis for RR (TP53mut: HR=5.9; P=0.027 and JAK2mut: HR=5.6; P=0.036). CONCLUSIONS: TP53mut and JAK2mut are potential biomarkers associated with poor prognosis in B-ALL patients.


Assuntos
Biomarcadores Tumorais/genética , Janus Quinase 2/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/patologia , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Receptores de Citocinas/biossíntese , Resultado do Tratamento
3.
J Pathol ; 236(2): 219-28, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25664618

RESUMO

BCR-JAK2 is an infrequent gene fusion found in chronic/acute, myeloid/lymphoid Philadelphia chromosome-negative leukaemia. In this study, we demonstrated that in vivo expression of BCR-JAK2 in mice induces neoplasia, with fatal consequences. Transplantation of BCR-JAK2 bone marrow progenitors promoted splenomegaly, with megakaryocyte infiltration and elevated leukocytosis of myeloid origin. Analysis of peripheral blood revealed the presence of immature myeloid cells, platelet aggregates and ineffective erythropoiesis. A possible molecular mechanism for these observations involved inhibition of apoptosis by deregulated expression of the anti-apoptotic mediator Bcl-xL and the serine/threonine kinase Pim1. Together, these data provide a suitable in vivo molecular mechanism for leukaemia induction by BCR-JAK2 that validates the use of this model as a relevant preclinical tool for the design of new targeted therapies in Philadelphia chromosome-negative leukaemia involving BCR-JAK2-driven activation of the JAK2 pathway.


Assuntos
Janus Quinase 2/fisiologia , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , Proteínas Proto-Oncogênicas c-bcr/fisiologia , Animais , Feminino , Rearranjo Gênico , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/fisiologia , Janus Quinase 2/genética , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/mortalidade , Leucocitose/etiologia , Masculino , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-bcr/genética , Retroviridae , Fator de Transcrição STAT5/metabolismo , Esplenomegalia/etiologia , Transdução Genética/métodos , Transgenes
4.
Nat Biotechnol ; 40(11): 1680-1689, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35697804

RESUMO

Fast, high-throughput methods for measuring the level and duration of protective immune responses to SARS-CoV-2 are needed to anticipate the risk of breakthrough infections. Here we report the development of two quantitative PCR assays for SARS-CoV-2-specific T cell activation. The assays are rapid, internally normalized and probe-based: qTACT requires RNA extraction and dqTACT avoids sample preparation steps. Both assays rely on the quantification of CXCL10 messenger RNA, a chemokine whose expression is strongly correlated with activation of antigen-specific T cells. On restimulation of whole-blood cells with SARS-CoV-2 viral antigens, viral-specific T cells secrete IFN-γ, which stimulates monocytes to produce CXCL10. CXCL10 mRNA can thus serve as a proxy to quantify cellular immunity. Our assays may allow large-scale monitoring of the magnitude and duration of functional T cell immunity to SARS-CoV-2, thus helping to prioritize revaccination strategies in vulnerable populations.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Imunidade Celular , Reação em Cadeia da Polimerase , Linfócitos T
5.
Cell Rep ; 36(8): 109570, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34390647

RESUMO

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.


Assuntos
COVID-19/prevenção & controle , Linfócitos T/imunologia , Vacinas Sintéticas/administração & dosagem , Anticorpos Antivirais/sangue , Ligante de CD40/metabolismo , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-2/metabolismo , Peptídeos/imunologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Vacinação , Vacinas Sintéticas/imunologia , Vacinas de mRNA
6.
Genes (Basel) ; 11(12)2020 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-33260630

RESUMO

B-cell precursor acute lymphoblastic leukaemia (B-ALL) is a malignancy of lymphoid progenitor cells with altered genes including the Janus kinase (JAK) gene family. Among them, tyrosine kinase 2 (TYK2) is involved in signal transduction of cytokines such as interferon (IFN) α/ß through IFN-α/ß receptor alpha chain (IFNAR1). To search for disease-associated TYK2 variants, bone marrow samples from 62 B-ALL patients at diagnosis were analysed by next-generation sequencing. TYK2 variants were found in 16 patients (25.8%): one patient had a novel mutation at the four-point-one, ezrin, radixin, moesin (FERM) domain (S431G) and two patients had the rare variants rs150601734 or rs55882956 (R425H or R832W). To functionally characterise them, they were generated by direct mutagenesis, cloned in expression vectors, and transfected in TYK2-deficient cells. Under high-IFNα doses, the three variants were competent to phosphorylate STAT1/2. While R425H and R832W induced STAT1/2-target genes measured by qPCR, S431G behaved as the kinase-dead form of the protein. None of these variants phosphorylated STAT3 in in vitro kinase assays. Molecular dynamics simulation showed that TYK2/IFNAR1 interaction is not affected by these variants. Finally, qPCR analysis revealed diminished expression of TYK2 in B-ALL patients at diagnosis compared to that in healthy donors, further stressing the tumour immune surveillance role of TYK2.


Assuntos
Simulação de Dinâmica Molecular , Mutação , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras B , TYK2 Quinase , Adolescente , Adulto , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , TYK2 Quinase/química , TYK2 Quinase/genética , TYK2 Quinase/metabolismo
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