Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 970
Filtrar
1.
Nat Commun ; 12(1): 865, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558546

RESUMO

Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.


Assuntos
Antígenos CD19/metabolismo , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Análise de Célula Única , Criança , Células Clonais , Humanos , Recidiva
2.
Methods Mol Biol ; 2270: 77-90, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33479894

RESUMO

Regulatory B cells (Bregs) have immunosuppressive capacity, primarily via the production of IL-10. IL-10 expression and immunosuppression have been described in a number of human B cell subsets, two of which include the CD19+CD24hiCD38hi and CD19+CD24hiCD27+ populations. In this chapter, we describe how to identify and isolate these subsets from peripheral blood B cells via flow cytometry. We also explain how to expand Bregs in culture and how to identify them based on intracellular expression of IL-10.


Assuntos
Subpopulações de Linfócitos B/citologia , Separação Celular/métodos , Imunofenotipagem/métodos , Antígenos CD/metabolismo , Antígenos CD19/metabolismo , Subpopulações de Linfócitos B/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B Reguladores/citologia , Linfócitos B Reguladores/imunologia , Antígeno CD24/metabolismo , Técnicas de Cultura de Células/métodos , Citometria de Fluxo/métodos , Humanos , Interleucina-10/metabolismo
3.
Methods Mol Biol ; 2270: 93-111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33479895

RESUMO

With the ever-increasing understanding of the roles of B cells in immune response and autoimmune pathogenesis, various techniques have been optimized for the detection of IL-10 production in B cells. In this chapter, we describe several commonly used methods for the effective detection of IL-10 in B cells at both mRNA and protein levels, including quantitative PCR analysis, intracellular staining of IL-10 in live B cells by flow cytometry, ELISA for secreted IL-10 detection, and ELISPOT assay for enumerating IL-10-producing B cells. We have further co-stained IL-10 with other cytokines and examined the staining efficiency. Moreover, we provide a detailed protocol for the detection of IL-10-producing B cells in situ by immunofluorescence microscopy. Since emerging evidence has suggested the promising strategy of cell therapy, we also provide a protocol to determine CD19+CD1dhiCD5+ B-cell distribution upon adoptive transfer using tile-scan imaging. Together, the application of the described methods for the detection of IL-10 will facilitate the characterization of B-cell subsets with regulatory functions and enhance our current understanding of the critical roles of B cells in immune response and autoimmune development.


Assuntos
Citometria de Fluxo/métodos , Interleucina-10/análise , Interleucina-10/isolamento & purificação , Transferência Adotiva/métodos , Animais , Antígenos CD19/metabolismo , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B Reguladores/citologia , Linfócitos B Reguladores/imunologia , Antígenos CD5/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos
4.
PLoS One ; 15(9): e0238819, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32976541

RESUMO

Adoptive cell transfer of Chimeric Antigen Receptor (CAR)-T cells showed promising results in patients with B cell malignancies. However, the detailed mechanism of CAR-T cell interaction with the target tumor cells is still not well understood. This work provides a systematic method for analyzing the activation and degranulation of second-generation CAR-T cells utilizing antigen-presenting cell surfaces. Antigen-presenting cell surfaces composed of circular micropatterns of CAR-specific anti-idiotype antibodies have been developed to mimic the interaction of CAR-T cells with target tumor cells using micro-contact printing. The levels of activation and degranulation of fixed non-transduced T cells (NT), CD19.CAR-T cells, and GD2.CAR-T cells on the antigen-presenting cell surfaces were quantified and compared by measuring the intensity of the CD3ζ chain phosphorylation and the Lysosome-Associated Membrane Protein 1 (LAMP-1), respectively. The size and morphology of the cells were also measured. The intracellular Ca2+ flux of NT and CAR-T cells upon engagement with the antigen-presenting cell surface was reported. Results suggest that NT and CD19.CAR-T cells have comparable activation levels, while NT have higher degranulation levels than CD19.CAR-T cells and GD2.CAR-T cells. The findings show that antigen-presenting cell surfaces allow a quantitative analysis of the molecules involved in synapse formation in different CAR-T cells in a systematic, reproducible manner.


Assuntos
Antígenos de Superfície/metabolismo , Linfoma de Células B/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Transferência Adotiva/métodos , Células Apresentadoras de Antígenos/imunologia , Antígenos CD19/metabolismo , Linfócitos B/imunologia , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva/métodos , Linfoma de Células B/terapia , Linfócitos T/imunologia
5.
PLoS One ; 15(9): e0238493, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32886698

RESUMO

To better understand anti-malaria protective immune responses, we examined the cellular mechanisms that govern protective immunity in a murine Plasmodium yoelii 17X NL (PyNL) re-infection model. Initially, we confirmed that immune B cells generated during a primary PyNL infection were largely responsible for protection from a second PyNL infection. Using the previously identified memory B cell markers CD80, PD-L2, and CD73, we found an increase in the frequency of CD80-PD-L2-CD73+ B cells up to 55 days after a primary PyNL infection and at 4-6 days following a second PyNL infection. Moreover, injection of enriched immune CD19+CD73+ B cells into nonimmune mice were significantly more protective against a PyNL infection than CD73- B cells. Interestingly, a substantial fraction of these CD73+ B cells also expressed IgM and granzyme B, a biomolecule that has been increasingly associated with protective responses against malaria.


Assuntos
5'-Nucleotidase/metabolismo , Granzimas/metabolismo , Malária/prevenção & controle , 5'-Nucleotidase/imunologia , Animais , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Linfócitos B/imunologia , Feminino , Imunidade , Imunoglobulina M , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium yoelii/imunologia , Plasmodium yoelii/patogenicidade
6.
Nat Commun ; 11(1): 3412, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641742

RESUMO

Regulatory B cells restrict immune and inflammatory responses across a number of contexts. This capacity is mediated primarily through the production of IL-10. Here we demonstrate that the induction of a regulatory program in human B cells is dependent on a metabolic priming event driven by cholesterol metabolism. Synthesis of the metabolic intermediate geranylgeranyl pyrophosphate (GGPP) is required to specifically drive IL-10 production, and to attenuate Th1 responses. Furthermore, GGPP-dependent protein modifications control signaling through PI3Kδ-AKT-GSK3, which in turn promote BLIMP1-dependent IL-10 production. Inherited gene mutations in cholesterol metabolism result in a severe autoinflammatory syndrome termed mevalonate kinase deficiency (MKD). Consistent with our findings, B cells from MKD patients induce poor IL-10 responses and are functionally impaired. Moreover, metabolic supplementation with GGPP is able to reverse this defect. Collectively, our data define cholesterol metabolism as an integral metabolic pathway for the optimal functioning of human IL-10 producing regulatory B cells.


Assuntos
Linfócitos B Reguladores/metabolismo , Colesterol/metabolismo , Interleucina-10/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Animais , Antígenos CD19/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Técnicas de Cocultura , Doenças Hereditárias Autoinflamatórias/metabolismo , Humanos , Macrófagos/metabolismo , Síndrome Metabólica/metabolismo , Deficiência de Mevalonato Quinase/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Análise de Componente Principal , Transdução de Sinais , Células Th1/metabolismo , Receptor Toll-Like 9/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
7.
Am J Med Sci ; 359(6): 347-353, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32354596

RESUMO

BACKGROUND: CD19+IL-10+B cells are considered as a particular subset of immunosuppressive cells by producing interleukin 10 (IL-10), which plays an important role in infectious and autoimmune diseases. The aim of this study was to determine the number of CD19+IL-10+B cells in Helicobacter pylori (H. pylori) positive patients in comparison with H. pylori negative patients, and to determine the association with different clinical outcomes, such as gastritis and peptic ulcer disease (PUD), in infected patients. METHODS AND MATERIALS: We studied 25 infected patients with gastritis, 25 infected patients with PUD, and 25 patients negative for H. pylori. The number of CD19+IL-10+B cells was determined by immunofluorescence. RESULTS: The number of CD19+IL-10+B cells in patients infected with H. pylori was significantly 2.5-fold higher than uninfected patients (P < 0.0001). Also, the number of CD19+IL-10+B cells in infected patients with gastritis was significantly 1.45-fold elevated compared to infected patients with PUD (P = 0.001). CONCLUSIONS: These results demonstrate that the increased number of CD19+IL-10+B cells in infected patients and its association with other cells may play an important role in the pathogenesis of H. pylori infection.


Assuntos
Antígenos CD19/metabolismo , Linfócitos B/microbiologia , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/imunologia , Interleucina-10/metabolismo , Adulto , Idoso , Feminino , Mucosa Gástrica/microbiologia , Gastrite/sangue , Gastrite/microbiologia , Helicobacter pylori , Humanos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Úlcera Péptica/sangue , Úlcera Péptica/microbiologia , Resultado do Tratamento
8.
J Immunol Res ; 2020: 4098235, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32149157

RESUMO

Objectives: To explore effects of Epstein-Barr virus (EBV) infection on CD19+ B lymphocytes in patients with immunorelated pancytopenia (IRP). Methods: An enzyme-linked immunosorbent assay (ELISA) in vitro diagnostic kit was used to detect EBV capsid antigen- (CA-) IgG and VCA-IgM antibodies in the serum. We analyzed the EBV-DNA copies of CD19+ B lymphocyte by using real-time quantitative polymerase chain reaction (RT-qPCR). CD21, CD23, CD5, CD80, and CD86 receptors on the surfaces of CD19+ B cells were detected by flow cytometry (FCM). The correlation between these receptors and EBV-DNA copies were evaluated. Results: The results revealed that the positive rate of EBVCA-IgM and CD19+ B lymphocyte EBV-DNA copy in the IRP group were significantly higher than those in the control group (P < 0.05). CD19+ B lymphocyte EBV-DNA copies were also more abundant in IRP patients than in control subjects (P < 0.05). CD19+ B lymphocyte EBV-DNA copies were also more abundant in IRP patients than in control subjects (P < 0.05). CD19+ B lymphocyte EBV-DNA copies were also more abundant in IRP patients than in control subjects (. Conclusions: EBV infection may activate CD19+ B lymphocytes and further disrupt bone marrow hematopoiesis in IRP patients.


Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4 , Pancitopenia/etiologia , Adolescente , Adulto , Idoso , Antígenos CD19/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/virologia , Feminino , Humanos , Imunoglobulina M/sangue , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Pancitopenia/sangue , Pancitopenia/metabolismo , Prognóstico , Adulto Jovem
9.
J Immunother Cancer ; 8(1)2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32152221

RESUMO

BACKGROUND: CD19 chimeric antigen receptor T (CAR-T) cells demonstrate remarkable remission rates in pediatric and adult patients with refractory or relapsed (r/r) acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). In 2016, we initiated a clinical trial with in-house produced CD19 CAR-T cells with a CD28 co-stimulatory domain. We analyzed, for the first time, differences in production features and phenotype between ALL and NHL patients. METHODS: Non-cryopreserved CAR-T cells were produced from patients' peripheral blood mononuclear cells within 9 to 10 days. 93 patients with r/r ALL and NHL were enrolled under the same study. CAR-T cells of ALL and NHL patients were produced simultaneously, allowing the head-to-head comparison. RESULTS: All patients were heavily pretreated. Three patients dropped out from the study due to clinical deterioration (n=2) or production failure (n=1). Cells of ALL patients (n=37) expanded significantly better and contained more CAR-T cells than of NHL patients (n=53). Young age had a positive impact on the proliferation capacity. The infusion products from ALL patients contained significantly more naïve CAR-T cells and a significantly higher expression of the chemokine receptor CXCR3. PD-1, LAG-3, TIM-3, and CD28 were equally expressed. 100% of ALL patients and 94% of NHL patients received the target dose of 1×10e6 CAR-T/kg. The overall response rate was 84% (30/36) in ALL and 62% (32/52) in NHL. We further compared CAR-T cell infusion products to tumor infiltrating lymphocytes (TIL), another common type of T cell therapy, mainly clinically effective in solid tumors. CAR-T cells contained significantly more naïve T cells and central memory T cells and significantly less CCR5 compared to TIL infusion products. CONCLUSIONS: The in-house production of CAR-T cells is highly efficient and fast. Clinical response rate is high. CAR-T cells can be successfully produced for 99% of patients in just 9 to 10 days. Cells derived from ALL patients demonstrate a higher proliferation rate and contain higher frequencies of CAR-T cells and naïve T cells than of NHL patients. In addition, understanding the differences between CAR-T and TIL infusion products, may provide an angle to develop CAR-T cells for the treatment of solid tumors in the future. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov; CAR-T: NCT02772198, First posted: May 13, 2016; TIL: NCT00287131, First posted: February 6, 2006.


Assuntos
Antígenos CD19/imunologia , Imunoterapia Adotiva/métodos , Leucócitos Mononucleares/imunologia , Linfoma não Hodgkin/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos Quiméricos/imunologia , Adolescente , Adulto , Fatores Etários , Antígenos CD19/genética , Antígenos CD19/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/imunologia , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/patologia , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores de Antígenos Quiméricos/genética , Resultado do Tratamento , Adulto Jovem
10.
Mediators Inflamm ; 2020: 3019378, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104147

RESUMO

CD19+CD24hiCD38hi B cells are immature transitional B cells that, in normal individuals, exert suppressive effects by IL-10 production but are quantitatively altered and/or functionally impaired in individuals with various autoimmune diseases. Primary biliary cholangitis (PBC), an autoimmune disease, clinically presents as chronic cholestasis and nonsuppurative destructive cholangitis. A role for CD19+CD24hiCD38hi B cells in PBC is unknown. This study investigated the frequency and functional variation of circulating CD19+CD24hiCD38hi B cells in PBC patients. Flow cytometry was employed to quantify the percentage of CD19+CD24hiCD38hi B cells in peripheral blood samples. Correlations between CD19+CD24hiCD38hi B cells and routine laboratory parameters were assessed. Levels of IL-10, TNF-α, IL-6 and IL-12, and Tim-1 in CD19+CD24hiCD38hi B cells from PBC patients were analyzed. The effect of CD19+CD24hiCD38hi B cells on CD4+T cell differentiation was evaluated. The percentage of CD19+CD24hiCD38hi B cells in PBC patients was significantly higher than in healthy controls and was positively correlated with liver cholestasis. After activation by anti-B cell receptor and CpG, the production of IL-10 was decreased and the production of IL-6 and IL-12 was increased in CD19+CD24hiCD38hi B cells from PBC patients. Moreover, Tim-1 levels were significantly downregulated in CD19+CD24hiCD38hi B cells from PBC patients. Coculture showed that PBC-derived CD19+CD24hiCD38hi B cells were less capable of CD4+T cell inhibition, but promoted Th1 cell differentiation. In conclusion, PBC patients have expanded percentages, but impaired CD19+CD24hiCD38hi B cells, which correlate with disease damage. In PBC patients, this B cell subset has a skewed proinflammatory cytokine profile and a decreased capacity to suppress immune function, which may contribute to the pathogenesis of PBC.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Antígeno CD24/metabolismo , Cirrose Hepática Biliar/imunologia , Cirrose Hepática Biliar/patologia , Idoso , Diferenciação Celular/fisiologia , Feminino , Citometria de Fluxo , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Cirrose Hepática Biliar/metabolismo , Masculino , Pessoa de Meia-Idade , Células-Tronco de Sangue Periférico/imunologia , Células-Tronco de Sangue Periférico/metabolismo , Células-Tronco de Sangue Periférico/patologia , Fator de Necrose Tumoral alfa/metabolismo
12.
Gene ; 732: 144336, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31935514

RESUMO

In the present study, we aimed to evaluate effects of autologous mesenchymal stem cells (MSCs) intravenous administration on the response of B cells, BAFF, APRIL, and their receptors on the surface of B cells at 1, 6, and 12 month follow-up periods in refractory rheumatoid arthritis (RA) patients. Thirteen patients with refractory RA received autologous MSCs. Plasma levels of BAFF and APRIL were measured employing ELISA method, followed by estimating B cell population and BAFFRs evaluation by flow cytometry technique. Gene expression of BAFF, APRIL, and their receptors on B cell surface in PBMCs was evaluated by SYBR Green real-time PCR technique. Plasma concentration of BAFF significantly decreased 1 and 6 months after the MSCT (MSCs Transplantation). Plasma concentration of APRIL significantly decreased 1 month after the MSCT. Percentages of CD19 + B cells in the PBMC population significantly decreased 12 months after the MSCT. Percentages of BR3 + CD19 + B cells and BCMA + CD19 + B cells significantly decreased at the 12th month after the MSCT. The gene expression of BAFF in the PBMC population significantly decreased during 6, and 12 months after the MSCT. The gene expression of APRIL significantly decreased on month 6 after the MSCT. The gene expression of BR3 significantly decreased during 1, 6, and 12 months after the MSCT. The MSCT seems to decrease B cells response because of the reduced production of BAFF and APRIL cytokines and decrease the expression of their receptors on the surface of B cells.


Assuntos
Artrite Reumatoide/terapia , Fator Ativador de Células B/metabolismo , Receptor do Fator Ativador de Células B/metabolismo , Regulação para Baixo , Transplante de Células-Tronco Mesenquimais/métodos , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Administração Intravenosa , Adulto , Antígenos CD19/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/genética , Linfócitos B/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Resultado do Tratamento , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética
13.
Nat Commun ; 11(1): 439, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974357

RESUMO

Regulation of membrane receptor mobility tunes cellular response to external signals, such as in binding of B cell receptors (BCR) to antigen, which initiates signaling. However, whether BCR signaling is regulated by BCR mobility, and what factors mediate this regulation, are not well understood. Here we use single molecule imaging to examine BCR movement during signaling activation and a novel machine learning method to classify BCR trajectories into distinct diffusive states. Inhibition of actin dynamics downstream of the actin nucleating factors, Arp2/3 and formin, decreases BCR mobility. Constitutive loss or acute inhibition of the Arp2/3 regulator, N-WASP, which is associated with enhanced signaling, increases the proportion of BCR trajectories with lower diffusivity. Furthermore, loss of N-WASP reduces the diffusivity of CD19, a stimulatory co-receptor, but not that of FcγRIIB, an inhibitory co-receptor. Our results implicate a dynamic actin network in fine-tuning receptor mobility and receptor-ligand interactions for modulating B cell signaling.


Assuntos
Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Antígenos CD19/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genética , Receptores de IgG/metabolismo , Transdução de Sinais , Imagem Individual de Molécula , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética
15.
Biochem Biophys Res Commun ; 524(1): 96-102, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980173

RESUMO

Mesothelin (MSLN) has been reported to be overexpressed in ovarian cancer and may be an ideal target for immunotherapy. Recent studies have suggested that natural killer (NK) cells may be better chimeric antigen receptor (CAR) drivers because of their favorable innate characteristics, such as directly recognizing and killing tumor cells, resulting in a graft-versus-tumor effect but irresponsible for graft-versus-host disease (GVHD). The therapeutic effects of CAR-engineered NK cells targeting MSLN in ovarian cancer have not been evaluated. In this study, MSLN- and CD19-targeted CAR NK-92 (MSLN- and CD19-CAR NK) cells were constructed. Both MSLN- and CD19-CAR molecules were highly expressed on the surface of NK-92 cells following lentiviral gene transduction. MSLN-CAR NK cells specifically killed MSLN-positive ovarian cancer cells (OVCAR-3 and SK-OV-3), rather than MSLN-negative cells (SK-HEP-1), in vitro. Moreover, compared with parental NK-92 cells and CD19-CAR NK cells, stronger cytokine secretion was detected in MSLN-CAR NK cells cocultured with OVCAR-3 and SK-OV-3. Furthermore, MSLN-CAR NK cells effectively eliminated ovarian cancer cells in both subcutaneous and intraperitoneal tumor models; these cells also significantly prolonged the survival of intraperitoneally tumor-bearing mice. These results demonstrate that MSLN-CAR NK cells have robust specific antitumor activity, both in vitro and in vivo, suggesting that mesothelin could be a potential target for CAR NK cells and could be applied in the treatment of ovarian cancer.


Assuntos
Carcinoma Epitelial do Ovário/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Neoplasias Ovarianas/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Antígenos CD19/metabolismo , Apoptose , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Humanos , Imunoterapia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/metabolismo , Lentivirus/genética , Camundongos , Modelos Biológicos , Neoplasias Experimentais , Transfecção
16.
Eur J Immunol ; 50(4): 558-567, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31803941

RESUMO

The transcription factor STAT6 regulates gene expression in response to IL-4 and IL-13. To further investigate how activated STAT6 modulates B cells development and function in vivo, we characterized mice that express a constitutively active version specifically in B cells. CD19Cre_STAT6vt mice show spontaneous phosphorylation and nuclear translocation of STAT6 in B cells. About 80 genes were more than twofold up- or downregulated in splenic B cells from CD19Cre_STAT6vt as compared to control mice. B cell development, tissue localization, and populations of follicular and marginal zone B cells, B1 B cells, GC B cells, and plasma cells (PCs) appeared to be normal. However, the number of IgE+ and IgG1+ GC B cells and PCs as well as serum IgE and IgG1 levels were increased in CD19Cre_STAT6vt mice. Infection with Lymphocytic choriomeningitis virus associated with high levels of TNF and IFN-γ did not prevent the development of a significantly increased IgE and IgG1 response against the virus in CD19Cre_STAT6vt mice. These results suggest that prolonged STAT6 activation during chronic allergic inflammation may result in IgE responses during subsequent viral or bacterial infection that could further stimulate mast cell activation even in the absence of the initial allergic response.


Assuntos
Infecções por Arenaviridae/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Hipersensibilidade/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Fator de Transcrição STAT6/metabolismo , Animais , Anticorpos Antivirais/sangue , Antígenos CD19/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Imunidade Humoral , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Interferon gama/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Transcrição STAT6/genética , Fator de Necrose Tumoral alfa/genética
18.
Scand J Immunol ; 91(2): e12836, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31598989

RESUMO

PD-1/PD-L1 pathway is crucial to immune regulation by controlling the balance between T cell tolerance and activation. However, the association between PD-1/PD-L1 pathway and regulatory B cells has not been fully investigated in allergic rhinitis. In this study, we detected the number of peripheral CD19+ CD25+ Bregs and the expression of IL-10 on this cell subset in healthy control and patients with allergic rhinitis using flow cytometry. Then, we evaluated the level of PD-L1 in CD19+ CD25+ Bregs and investigated the correlation between PD-L1 and CD4+ follicular T helper cells. Finally, we studied the effects of anti-PD-L1 on the apoptosis of Bregs and the production of IL-10. Comparing with healthy controls, the percentage of CD19+ CD25+ Bregs and the expression of IL-10 were both significantly decreased in AR group. In addition, the expression of PD-L1 on CD19+ CD25+ Bregs was also lower in allergic rhinitis patients. Interestingly, a negative correlation was found between the expression of PD-L1+ Bregs and CD4+ CXCR5+ follicular T helper cells. In vitro assay revealed that anti-PD-L1 promoted Bregs apoptosis and inhibited the expression of IL-10 in CD19+ CD25+ Bregs. Collectively, these results suggest that PD-L1 expressed on CD19+ CD25+ Bregs may be a potential regulator in the treatment of allergic rhinitis. Blockade of PD-1/PD-L1 pathway might be a valuable pathogenic target for allergic rhinitis through inhibiting the secretion of immunosuppressive cytokine and promoting CD19+ CD25+ Bregs apoptosis.


Assuntos
Linfócitos B Reguladores/imunologia , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/imunologia , Centro Germinativo/imunologia , Interleucina-10/sangue , Receptor de Morte Celular Programada 1/metabolismo , Rinite Alérgica/imunologia , Adulto , Antígenos CD19/metabolismo , Apoptose , Feminino , Humanos , Tolerância Imunológica , Subunidade alfa de Receptor de Interleucina-2 , Masculino , Pessoa de Meia-Idade , Transdução de Sinais
19.
Clin Nephrol ; 93(1): 42-46, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31670650

RESUMO

BACKGROUND: Various studies have demonstrated that interleukin-6 (IL-6) activates the central magnocellular arginine vasopressin (AVP)-secreting neurons in the brain to produce non-osmotic, non-volume-mediated increases in AVP. The most common toxicity of CD19+ chimeric antigen receptor (CAR) T-cells is cytokine release syndrome, which is related to increased levels of IL-6. This study will evaluate the correlation of IL-6 levels with hyponatremia in patients receiving CD19+ CAR T-cells. MATERIALS AND METHODS: This is a single-center retrospective analysis of adult patients who received CD19+ CAR T-cells for the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL). RESULTS: Hyponatremia, defined as a serum sodium (Na) ≤ 135 mEq/L, occurred in 31 (61%) patients. A change in Na > 7 mEq occurred in 32 (63%) patients, and the median lowest Na was 133 mEq/L (interquartile range (IQR): 131 - 136)). There was an inverse linear relationship between IL-6 levels and lowest Na (p = 0.001). Overall, per 10-fold increase in IL-6, Na decreased by an average of 2.68 mEq/L. CONCLUSION: Hyponatremia is common in patients who received CD19+ CAR T-cells. There is an inverse linear relationship between IL-6 levels and nadir Na (p = 0.001). Further studies will be needed to confirm a causative relationship between IL-6 levels and hyponatremia following CD19+ CAR T-cell infusion.


Assuntos
Hiponatremia/sangue , Hiponatremia/etiologia , Imunoterapia Adotiva/efeitos adversos , Interleucina-6/sangue , Sódio/sangue , Adulto , Idoso , Antígenos CD19/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos Quiméricos , Estudos Retrospectivos , Linfócitos T/metabolismo , Adulto Jovem
20.
Kurume Med J ; 65(4): 169-176, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31723081

RESUMO

The maternal immune system needs to be tolerant of allogeneic fetal tissue for reproductive success. The regulatory immune cell network plays an essential role in maintaining maternal tolerance to the fetus. We herein demonstrate in a green fluorescent protein (GFP)/IL-10 reporter mouse system that unique IL-10-expressing cells exist presumably in chorionic villi within the placenta. Flow cytometric analysis revealed that these IL-10- expressing cells exhibit a unique CD19 negative, CD3 negative, and B220 positive phenotype. Interestingly, these cells were enriched during in vitro culture, but well-known stimuli for T cells and B cells failed to enhance their growth, suggesting that the CD19- CD3- B220+ cells were self renewing. Unexpectedly, in an adoptive cell trans fer experiment, IL-10 production was detected in Sca-1+ CD4+ CD25+ regulatory T cells (Treg). To our knowledge, this is the first report to identify IL-10-producing CD19- CD3- B220+ cells in the fetus. These cells may rep resent a potential progenitor of Sca-1+ Treg or pluripotent precursor cells for immune tolerance.


Assuntos
Interleucina-10/metabolismo , Placenta/metabolismo , Células Precursoras de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Animais , Antígenos CD19/metabolismo , Antígenos Ly/metabolismo , Complexo CD3/metabolismo , Linhagem da Célula , Células Cultivadas , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Hospedeiro Imunocomprometido , Interleucina-10/genética , Antígenos Comuns de Leucócito/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Fenótipo , Placenta/citologia , Placenta/imunologia , Células Precursoras de Linfócitos T/imunologia , Células Precursoras de Linfócitos T/transplante , Gravidez , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...