Flow Cytometry Analysis of mTOR Signaling in Antigen-Specific B Cells

2018 ◽  
pp. 95-109
Author(s):  
Qizhao Huang ◽  
Haoqiang Wang ◽  
Lifan Xu ◽  
Jianjun Hu ◽  
Pengcheng Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Mtor Signaling ◽  
Flow Cytometry Analysis

scholarly journals Pirfenidone Reverses Murine Chronic Graft Versus Host Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1158-1158
Author(s):  
Jing Du ◽  
Ryan P Flynn ◽  
Katelyn Paz ◽  
Ante Vulic ◽  
Tara M. Robinson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Graft Versus Host Disease ◽  
Flow Cytometry Analysis ◽  
T Follicular Helper Cells ◽  
Hematopoietic Stem ◽  
Host Disease ◽  
Graft Versus Host ◽  
Helper Cells ◽  
Follicular Helper

Abstract Allogeneic hematopoietic stem cell transplantation (aHSCT) is hampered by chronic graft-versus-host disease (cGVHD), which results in multi-organ fibrosis and loss of function. In particular, bronchiolitis obliterans (BO) and scleroderma resulting from fibrotic bronchiolar and cutaneous response, respectively, are two devastating outcomes for cGVHD patients. Fibrotic manifestations often are considered irreversible and progressive. Therefore, new therapies targeting fibrosis are urgently needed. Pirfenidone (5-methyl-1-phenyl-2- (1H)-pyridone) exhibits a well-documented anti-inflammatory and anti-fibrosis function in multiple pre-clinical models and is the first and only FDA-approved drug for idiopathic pulmonary fibrosis. For this study, Pirfenidone was synthesized as a crystalline solid and found to be pure both by melting point and NMR spectroscopy. We evaluated Pirfenidone's anti-fibrosis function in 2 pathophysiologically distinct cGVHD murine models: 1. a major mismatched multi-organ system model (C57BL/6 to B10.BR) that induces BO as a result of a cGVHD-induced germinal center (GC) reaction, antibody deposition and fibrosis in the lung; and 2. a minor antigen mismatched model (B10.D2 to BALB/c) in which severe scleroderma is the major disease manifestation. In the BO model, pulmonary function loss in cGVHD mice (as reflected by increased resistance, elastance and decreased compliance of the lung) was restored by Pirfenidone treatment (400mg/kg) during both the early (day28-56) (Fig A, representative of 3 experiments with 5-8 mice per group) and late stages (day56-84) of the disease. Pathologic changes in the lung, such as collagen deposition and narrowing of bronchioles, were significantly reduced by Pirfenidone. The size and frequency of GCs in the spleen, and the frequency of GC B cells (Fig B, representative of 2 experiments with 5-8 mice per group) and T follicular helper cells were all significantly reduced in Pirfenidone- treated groups. To determine whether GCs were directly affected by Pirfenidone, we evaluated Pirfenidone in C57BL/6 mice immunized with sheep red blood cells (SRBC) to induce GCs. Interestingly, Pirfenidone did not reduce the SRBC-induced GC reaction (Fig C) (comparable frequencies of splenic GC B cells, T follicular helper cells and serum IgG levels were seen between Pirfenidone and vehicle-treated groups). These results suggested that Pirfenidone suppresses the GC reaction through a cGVHD-specific mechanism, rather than through immune regulation. Mechanistically, Pirfenidone administration attenuated the sequestration of pro-fibrogenic F4/80+ macrophages (Fig D, representative of 2 experiments) and TGF-β (Fig E, representative of 2 experiments) production within the lung. These results have led us to elucidate a potential mechanism of cGVHD: antibody deposition in the lung results in the activation of macrophages and TGF-β that drive fibrotic change and tissue damage, resulting in the exposure of auto- and allo- antigens to the immune system that support and sustain pathologic GC reactions. In the B10.D2 to BALB/c sclerodermatous cGVHD model, Pirfenidone treatment (400mg/kg, day21-55) improved clinical signs of scleroderma and reduced macrophage infiltration in the skin (Fig F). In summary, this is the first study evaluating a commercially available anti-fibrosis drug on pathologically distinct pre-clinical cGVHD models. Our data suggests Prifenidone reversed cGVHD in the BO model and, to a lesser extent, in the scleroderma model. Thus, Pirfenidone is a novel therapeutic agent for treating cGVHD patients with fibrosis that have been typically refractory to therapies. A. Resistance of lungs was measured on day56 of transplantation; Elastance and compliance correlated with resistance but were not shown here. B. Flow cytometry analysis of GC B cells of no cGVHD vs cGVHD mice treated with Pirfenidone or vehicle; C. Flow cytometry analysis of GC B cells from SRBC-immunized mice treated with Pirfenidone or vehicle; D and E. Macrophage F4/80 and TGF-β quantification of day56 lungs of no cGVHD vs cGVHD mice treated as indicated; F. Skin GVHD scores were recorded on indicated dates of irradiated BALB/c mice transplanted with B10.D2 donor BM alone or with T cells and treated as indicated. Unpaired student T test was used for statistical analysis. ****:P<0.0001; ***: P<0.001; **: P<0.01; *: P<0.05; ns: not significant. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Human Meprin Alfa Metalloprotease Is Differentially Expressed in Peripheral Blood Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3855-3855
Author(s):  
Kimberly Dunham ◽  
Renee Yura ◽  
Lei Bao ◽  
Judith Bond ◽  
Qi Sun
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Human Peripheral Blood ◽  
Pcr Amplification ◽  
Peripheral Blood Cells ◽  
Flow Cytometry Analysis

Abstract Meprin metaloproteases are highly expressed in the apical brush-border of the intestinal and renal proximal tubule epithelia. However, the expression and function of these metaloproteinease in other tissues have not been well established. Using a specific antisera raised against rat but cross-reactive with human Meprin Alfa, we examined the expression of this protein in human peripheral blood cells. Flow cytometry analysis showed that the post-, but not the pre-, immune sera stained strongly for the CD14+ monocytes and moderately for CD56+ NK cells, but not for CD3+ T and CD19+ B cells. To confirm Meprin Alfa expression at the RNA level, peripheral blood cells were fractionated based on the expression of the above surface markers, followed by detection of Meprin Alfa message RNA by semi-quantitative RT-PCR. Consistent with the flow cytometry data, Meprin Alfa messages were detected only in cell fractions containing monocytes and NK cells but not T and B cells. Subsequent sequencing of the PCR amplified fragments confirmed the specificity of the PCR amplification. Interestingly, flow cytometry analysis further revealed that while the expression of Meprin Alfa was maintained in the macrophages differentiated in vitro from isolated monocytes, its expression was reduced in monocytes-derived immature dendritic cells (DC) and diminished in mature DC. The differential expression of Meprin Alfa in the peripheral blood cells suggests important physiological function of this protein in the immune system.

Methylene Blue Selectively Inhibits B Cell Development and E2A Activity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4848-4848
Author(s):  
Eroica Soans ◽  
John K Choi
Keyword(s):  
Flow Cytometry ◽  
Methylene Blue ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Luciferase Reporter ◽  
Flow Cytometry Analysis ◽  
Murine Bone Marrow

Abstract Introduction E2A, a basic helix loop helix transcription factor, is essential for B lymphocyte proliferation, survival and differentiation. Mutations in E2A are associated with B cell leukemia and lymphomas. Our preliminary data suggests that E2A activity is altered by HSP70 and since HSP70 ATPase activity is inhibited by methylene blue (MB), we hypothesized that MB will alter E2A activity and thus alter B cell development. Methods We studied the effects of MB on normal and leukemic B cells compared to its effect on myeloid cells. The cell lines used were REH, NALM-6 and BAF3-p185 cells compared to myeloid cell lines, CCRF and CMK for the proliferation assay. To study MB effect on normal B and myeloid progenitors, methylcellulose colony formation assays with murine bone marrow cells (strain 129S1/SVImJ, 8-12 weeks old mice) were performed. We also treated mice with MB in their drinking water (1000 μM) up to 16 weeks to study the in-vivo effect on B cells as compared to granulocyte, macrophages, T cells and NK cells in the peripheral blood by flow cytometry analysis. To understand the mechanisms by which MB alters B cell proliferation, we examined the effects of MB on cell cycle progression, apoptosis and E2A activity in B cells.  For cell progression NALM-6 cells were treated with 4 μM MB for 0, 6, 12 and 24 hrs and assayed using BRDU staining by flow cytometry analysis. Further, apoptosis was measured using Annexin V staining by flow cytometry analysis in NALM-6 cells up to 48hrs post treatment with MB. Lastly, E2A activity was measured using an E2A-specific luciferase reporter assay in 293T cells. Results MB decreased the proliferation of precursor B cell lines, REH, NALM-6 and BAF3-p185 cells compared to myeloid cell lines, CCRF and CMK. Similar effects of MB were also seen with normal B and myeloid progenitors as assayed by methylcellulose colony formation assays with murine bone marrow cells. Similarly, there is a significant decrease in peripheral B cells as compared to granulocytes, macrophages, T cells and NK cells when the mice were treated with 1000 μM of methylene blue in their drinking water as compared to those that remained untreated. MB caused apoptosis and decreased the S phase fraction of treated NALM-6 cells. In addition, we found that MB inhibited E2A activity using an E2A-specific luciferase reporter assay. Conclusion Our findings suggest that MB is a specific inhibitor of B cell proliferation, survival and development. This effect may be mediated by the inhibition of E2A activity by methylene blue. Disclosures: No relevant conflicts of interest to declare.

scholarly journals THU0231 IL-2 DRIVES THE CONVERSION OF T FOLLICULAR HELPER TO T FOLLICULAR REGULATORY CELLS THROUGH EPIGENETIC MODIFICATION IN SYSTEMIC LUPUS ERYTHEMATOSUS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 343.2-343
Author(s):  
H. Hao ◽  
S. Nakayamada ◽  
Y. Kaoru ◽  
N. Ohkubo ◽  
S. Iwata ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Flow Cytometry ◽  
B Cells ◽  
Histone Modifications ◽  
Lupus Erythematosus ◽  
Regulatory Cells ◽  
Systemic Lupus ◽  
Research Support ◽  
Tfh Cells ◽  
Follicular Helper

Background:Systemic lupus erythematosus (SLE) is a complex polygenic autoimmune disease characterized by immune-system aberrations. Among several types of immune cells, T follicular helper (Tfh) cells promote autoantibody production, whereas T follicular regulatory (Tfr) cells suppress Tfh-mediated antibody responses.(1)Objectives:To identify the characteristics of Tfr cells and to elucidate the mechanisms of conversion of Tfh cells to Tfr cells, we probed the phenotype of T helper cells in patients with SLE and underlying epigenetic modifications by cytokine-induced signal transducer and activators of transcription (STAT) family factors.Methods:Peripheral blood mononuclear cells from SLE patients (n=44) and healthy donors (HD; n=26) were analyzed by flow cytometry. Memory Tfh cells were sorted and cultured under stimulation with T cell receptor and various cytokines. Expression of characteristic markers and phosphorylation of STATs (p-STATs) were analyzed by flow cytometry and quantitation PCR. Histone modifications were evaluated by chromatin immunoprecipitation.Results:The proportion of CXCR5+FoxP3+Tfr cells in CD4+T cells tended to increase (2.1% vs 1.7%, p=0.17); however, that of CD4+CD45RA-FoxP3hiactivated Tfr cells in Tfr cells was decreased (4.8% vs 7.1%, p<0.05), while CD4+CD45RA-FoxP3lownon-suppressive Tfr cells was increased (50.1% vs 38.2%, p<0.01) in SLE compared to HD. The percentage of PD-1hiactivated Tfh cells was significantly higher in SLE compared to HD (15.7% vs 5.9%, p<0.01). Furthermore, active patients had a higher ratio of activated Tfh/Tfr cells compared to inactive patients. In vitro study showed that IL-2, but not other cytokines such as TGF-β1, IL-12, IL-27, and IL-35, induced the conversion of memory Tfh cells to functional Tfr cells characterized by CXCR5+Bcl6+Foxp3hipSTAT3+pSTAT5+cells. The loci ofFOXP3at STAT binding sites were marked by bivalent histone modifications. After IL-2 stimulation, STAT5 directly bound on FOXP3 gene loci accompanied by suppressing H3K27me3. Finally, we found that serum level of IL-2 was decreased in SLE and that stimulation with IL-2 suppressed the generation of CD38+CD27+B cells by ex vivo coculture assay using Tfh cells and B cells isolated from human blood.Conclusion:Our findings indicated that the regulatory function of Tfr cells is impaired due to the low ability of IL-2 production and that IL-2 restores the function of Tfr cells through conversion of Tfh cells to Tfr cells in SLE. Thus, the reinstatement of the balance between Tfh and Tfr cells will provide important therapeutic approaches for SLE.References:[1]Deng J, Wei Y, Fonseca VR, et al. T follicular helper cells and T follicular regulatory cells in rheumatic diseases. Nat Rev Rheumatol. 2019; 15 (8): 475-90.Disclosure of Interests: :He Hao: None declared, Shingo Nakayamada Grant/research support from: Mitsubishi-Tanabe, Takeda, Novartis and MSD, Speakers bureau: Bristol-Myers, Sanofi, Abbvie, Eisai, Eli Lilly, Chugai, Asahi-kasei and Pfizer, Yamagata Kaoru: None declared, Naoaki Ohkubo: None declared, Shigeru Iwata: None declared, Yoshiya Tanaka Grant/research support from: Asahi-kasei, Astellas, Mitsubishi-Tanabe, Chugai, Takeda, Sanofi, Bristol-Myers, UCB, Daiichi-Sankyo, Eisai, Pfizer, and Ono, Consultant of: Abbvie, Astellas, Bristol-Myers Squibb, Eli Lilly, Pfizer, Speakers bureau: Daiichi-Sankyo, Astellas, Chugai, Eli Lilly, Pfizer, AbbVie, YL Biologics, Bristol-Myers, Takeda, Mitsubishi-Tanabe, Novartis, Eisai, Janssen, Sanofi, UCB, and Teijin

scholarly journals A Synergic Potential of Antimicrobial Peptides against Pseudomonas syringae pv. actinidiae

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1461
Author(s):  
Nuno Mariz-Ponte ◽  
Laura Regalado ◽  
Emil Gimranov ◽  
Natália Tassi ◽  
Luísa Moura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antimicrobial Peptides ◽  
Pseudomonas Syringae ◽  
Pathogenic Bacteria ◽  
High Efficiency ◽  
Bacterial Canker ◽  
Membrane Permeation ◽  
Flow Cytometry Analysis ◽  
Pathogenic Agent ◽  
Peptide Mixtures

Pseudomonas syringae pv. actinidiae (Psa) is the pathogenic agent responsible for the bacterial canker of kiwifruit (BCK) leading to major losses in kiwifruit productions. No effective treatments and measures have yet been found to control this disease. Despite antimicrobial peptides (AMPs) having been successfully used for the control of several pathogenic bacteria, few studies have focused on the use of AMPs against Psa. In this study, the potential of six AMPs (BP100, RW-BP100, CA-M, 3.1, D4E1, and Dhvar-5) to control Psa was investigated. The minimal inhibitory and bactericidal concentrations (MIC and MBC) were determined and membrane damaging capacity was evaluated by flow cytometry analysis. Among the tested AMPs, the higher inhibitory and bactericidal capacity was observed for BP100 and CA-M with MIC of 3.4 and 3.4–6.2 µM, respectively and MBC 3.4–10 µM for both. Flow cytometry assays suggested a faster membrane permeation for peptide 3.1, in comparison with the other AMPs studied. Peptide mixtures were also tested, disclosing the high efficiency of BP100:3.1 at low concentration to reduce Psa viability. These results highlight the potential interest of AMP mixtures against Psa, and 3.1 as an antimicrobial molecule that can improve other treatments in synergic action.

scholarly journals OP0186 CHANGES IN CIRCULATING B CELL LEVELS AND IMMUNOPHENOTYPE ARE ASSOCIATED WITH DEVELOPMENT OF ARTHRITIS FOLLOWING TREATMENT WITH CHECKPOINT INHIBITORS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 112.2-113
Author(s):  
M. Gatto ◽  
S. Bjursten ◽  
C. Jonell ◽  
C. Jonsson ◽  
S. Mcgrath ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Adverse Events ◽  
B Cell ◽  
Mononuclear Cells ◽  
Checkpoint Inhibitors ◽  
Therapy Monitoring ◽  
Cell Subset ◽  
Peripheral Blood Mononuclear ◽  
Transitional B Cells

Background:Inflammatory arthritis (IA) is frequent among rheumatic side effects induced by checkpoint inhibitor (CPI) therapy for metastatic malignancies1. While T cells are likely to sustain the inflammatory process2, fewer data are available concerning the role of B cells3.Objectives:To investigate the phenotype of circulating B cells in patients who develop CPI-induced IA (CPI-IA) and to compare it with features of B cells in patients not developing immune-related adverse events (irAE) upon CPI treatment.Methods:B cell subsets at baseline (before CPI initiation) and during CPI treatment were analyzed in CPI-IA patients and in patients receiving CPI but who did not develop irAE (non-irAE). Peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry and B cells were identified as CD19+ and divided into naïve (CD27-IgD+), memory (CD27+IgD+/-), double negative (CD27-IgD-) and transitional (CD10+CD24+CD38+/hi) B cells. Levels of CD21, an activation marker on transitional B cells, were also analyzed. Non-parametric tests were used for analysis of differences between groups.Results:Six CPI-IA and 7 non-irAE patients matched for age, gender and CPI treatment were included, who had received CPI treatment due to metastatic melanoma. Flow cytometry revealed a significant increase of circulating B cells (p=0.002) (Figure 1A) and especially of transitional B cells in CPI-IA patients vs. non-irAE (median %, range: 7.8 (4.5-11.4) vs. 3.2 (1.6-4.3),p=0.007) (Figure 1B), while no remarkable changes were seen across other subsets. Transitional B cell levels significantly decreased from active to quiescent CPI-IA in all patients (p=0.008). In two CPI-IA patients for whom baseline sampling was available, the increase of transitional levels occurred early after CPI treatment and before CPI-IA onset. Levels of expression of CD21 on transitional B cells were increased in CPI-IA vs. non-irAE (p=0.01).Conclusion:Transitional B cells are expanded in CPI-IA patients and seem to increase early after start of CPI therapy. Monitoring this B cell subset might lead to closer follow-up and earlier diagnosis of CPI-IA.References:[1]Ramos-Casals M, Brahmer JR, Callahan MK, et al. Immune-related adverse events of checkpoint inhibitors. Nat Rev Dis Primers 2020;6:38[2]Murray-Brown W, Wilsdon TD, Weedon H, et al. Nivolumab-induced synovitis is characterized by florid T cell infiltration and rapid resolution with synovial biopsy-guided therapy. J Immunother Cancer 2020;8:e000281[3]Das R, Bar N, Ferreira M, et al. Early B cell changes predict autoimmunity following combination immune checkpoint blockade. J Clin Invest. 2018;128:715-2Disclosure of Interests:None declared

scholarly journals Flow cytometry analysis of anti-polyethylene glycol antibodies in human plasma

2021 ◽  
Vol 8 ◽  
pp. 148-154
Author(s):  
Jia-Long Fang ◽  
Frederick A. Beland ◽  
Yangshun Tang ◽  
Steve R. Roffler
Keyword(s):  
Flow Cytometry ◽  
Polyethylene Glycol ◽  
Human Plasma ◽  
Flow Cytometry Analysis
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