scholarly journals Multiplexed detection and isolation of viable low-frequency cytokine-secreting human B cells using cytokine secretion assay and flow cytometry (CSA-Flow)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ayman Rezk ◽  
Rui Li ◽  
Amit Bar-Or
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Immune Cells ◽  
High Frequency ◽  
Intracellular Cytokine Staining ◽  
Simultaneous Detection ◽  
Low Frequency ◽  
Cytokine Secretion ◽  
Human B Cells ◽  
Secretion Assay

Abstract The ability to functionally characterize cytokine-secreting immune cells has broad implications in both health and a range of immune-mediated and auto-immune diseases. Low-frequency cytokine-defined immune-cell subsets can play key immune-regulatory roles, yet their detailed study is often hampered by limited clinical sample availability. Commonly used techniques including intracellular cytokine staining require cell fixation, precluding subsequent functional interrogation. The cytokine-secretion assay (CSA) can overcome this limitation, though has mostly been used for detection of relatively high-frequency, single-cytokine secreting cells. We examined how adaptation of the CSA in combination with multiparametric flow-cytometry (CSA-Flow) may enable simultaneous isolation of multiple, low-frequency, cytokine-secreting cells. Focusing on human B cells (traditionally recognized as harder to assay than T cells), we show that single-capture CSA-Flow allows for isolation of highly-purified populations of both low-frequency (IL-10+; GM-CSF+) and high-frequency (TNF+) cytokine-defined B cells. Simultaneous detection and isolation of up to three viable and highly-purified cytokine-secreting B-cell subpopulations is feasible, albeit with some signal loss, with fractions subsequently amenable to gene expression analysis and in vitro cell culture. This multiplexing CSA-Flow approach will be of interest in many human cellular immunology contexts aiming to functionally characterize cytokine-secreting immune cells, especially when sample volumes and cell numbers are limited.

scholarly journals Highly Sensitive Flow Cytometry Allows Monitoring of Changes in Circulating Immune Cells in Blood After Tdap Booster Vaccination

2021 ◽  
Vol 12 ◽  
Author(s):  
Annieck M. Diks ◽  
Indu Khatri ◽  
Liesbeth E.M. Oosten ◽  
Bas de Mooij ◽  
Rick J. Groenland ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Immune Cells ◽  
Plasma Cells ◽  
Booster Vaccination ◽  
Added Value ◽  
Maximum Expansion ◽  
Specific Serum ◽  
Circulating T Cells ◽  
Time Changes

Antigen-specific serum immunoglobulin (Ag-specific Ig) levels are broadly used as correlates of protection. However, in several disease and vaccination models these fail to predict immunity. In these models, in-depth knowledge of cellular processes associated with protective versus poor responses may bring added value. We applied high-throughput multicolor flow cytometry to track over-time changes in circulating immune cells in 10 individuals following pertussis booster vaccination (Tdap, Boostrix®, GlaxoSmithKline). Next, we applied correlation network analysis to extensively investigate how changes in individual cell populations correlate with each other and with Ag-specific Ig levels. We further determined the most informative cell subsets and analysis time points for future studies. Expansion and maturation of total IgG1 plasma cells, which peaked at day 7 post-vaccination, was the most prominent cellular change. Although these cells preceded the increase in Ag-specific serum Ig levels, they did not correlate with the increase of Ig levels. In contrast, strong correlation was observed between Ag-specific IgGs and maximum expansion of total IgG1 and IgA1 memory B cells at days 7 to 28. Changes in circulating T cells were limited, implying the need for a more sensitive approach. Early changes in innate immune cells, i.e. expansion of neutrophils, and expansion and maturation of monocytes up to day 5, most likely reflected their responses to local damage and adjuvant. Here we show that simultaneous monitoring of multiple circulating immune subsets in blood by flow cytometry is feasible. B cells seem to be the best candidates for vaccine monitoring.

scholarly journals The ultrastructural localization of immunoglobulins in human b cells of immunoproliferative diseases

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1132-1140 ◽  
Author(s):  
MF Gourdin ◽  
JP Farcet ◽  
F Reyes
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cells ◽  
Plasma Cells ◽  
Simultaneous Detection ◽  
Ultrastructural Localization ◽  
Lymphocytic Leukemia ◽  
Cellular Distribution ◽  
Complex Plasma ◽  
Perinuclear Space ◽  
Human B Cells

Abstract The cellular distribution of immunoglobulins in human malignant and normal B cells was investigated by immunoelectron microscopy by direct incubation of fixed cells with electron microscopy by direct incubation of fixed cells with peroxidase-coupled antibody. These conjugates penetrated into the cell, resulting in the simultaneous detection of surface and cytoplasmic immunoglobulins. The latter were seen as specific intracisternal staining of the perinuclear space and endoplasmic reticulum and occasionally of the Golgi complex. Plasma cells were frequently characterized by a heterogeneity of reactivity of the endoplasmic reticulum. Minute amounts of cytoplasmic immunoglobulin were demonstrated in cells without developed secretory organelles, such as lymphoma cells and lymphocytes from chronic lymphocytic leukemia (CLL). The method allowed us to define several subsets of cells according to the expression of surface and cytoplasmic immunoglobulins and thus to determine the stage of maturation of cells involved in monoclonal proliferation.

scholarly journals Simultaneous analysis of antigen-specific B and T cells after SARS-CoV-2 infection and vaccination

2021 ◽  
Author(s):  
Krista L Newell ◽  
Mitchell J Waldran ◽  
Stephen J Thomas ◽  
Timothy P Endy ◽  
Adam Tully Waickman
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
B Cells ◽  
Simultaneous Detection ◽  
T Cell Subsets ◽  
Cell Populations ◽  
T And B Cells ◽  
Simultaneous Identification ◽  
Immune Profiling ◽  
Insight Into

Conventional methods for quantifying and phenotyping antigen-specific lymphocytes can rapidly deplete irreplaceable specimens. This is due to the fact that antigen-specific T and B cells have historically been analyzed in independent assays each requiring millions of cells. A technique that facilitates the simultaneous detection of antigen-specific T and B cells would allow for more thorough immune profiling with significantly reduced sample requirements. To this end, we developed the B And T cell Tandem Lymphocyte Evaluation (BATTLE) assay, which allows for the simultaneous identification of SARS-CoV-2 Spike reactive T and B cells using an optimized Activation Induced Marker (AIM) T cell assay and dual-color B cell antigen probes. Using this assay, we demonstrate that antigen-specific B and T cell subsets can be identified simultaneously using conventional flow cytometry platforms and provide insight into the differential effects of mRNA vaccination on B and T cell populations following natural SARS-CoV-2 infection.

scholarly journals The ultrastructural localization of immunoglobulins in human b cells of immunoproliferative diseases

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1132-1140
Author(s):  
MF Gourdin ◽  
JP Farcet ◽  
F Reyes
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cells ◽  
Plasma Cells ◽  
Simultaneous Detection ◽  
Ultrastructural Localization ◽  
Lymphocytic Leukemia ◽  
Cellular Distribution ◽  
Complex Plasma ◽  
Perinuclear Space ◽  
Human B Cells

The cellular distribution of immunoglobulins in human malignant and normal B cells was investigated by immunoelectron microscopy by direct incubation of fixed cells with electron microscopy by direct incubation of fixed cells with peroxidase-coupled antibody. These conjugates penetrated into the cell, resulting in the simultaneous detection of surface and cytoplasmic immunoglobulins. The latter were seen as specific intracisternal staining of the perinuclear space and endoplasmic reticulum and occasionally of the Golgi complex. Plasma cells were frequently characterized by a heterogeneity of reactivity of the endoplasmic reticulum. Minute amounts of cytoplasmic immunoglobulin were demonstrated in cells without developed secretory organelles, such as lymphoma cells and lymphocytes from chronic lymphocytic leukemia (CLL). The method allowed us to define several subsets of cells according to the expression of surface and cytoplasmic immunoglobulins and thus to determine the stage of maturation of cells involved in monoclonal proliferation.

scholarly journals AB0029 INHIBITION OF EFFECTOR B CELLS BY IBRUTINIB IN SYSTEMIC SCLEROSIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1317.1-1318
Author(s):  
J. Einhaus ◽  
A. C. Pecher ◽  
E. Asteriti ◽  
H. Schmid ◽  
K. A. Secker ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Transcription Factor ◽  
Systemic Sclerosis ◽  
B Cells ◽  
B Cell ◽  
Therapeutic Potential ◽  
Controlled Trial ◽  
Intracellular Cytokine Staining ◽  
Disease Onset ◽  
Innate Immune

Background:Systemic sclerosis (SSc) is a connective tissue disease with significant morbidity and mortality. Effective treatment is still missing, and clinical control of the disease remains challenging. In particular, the development of pulmonary and cardiac fibrosis and pulmonary hypertension are severe complications responsible for excessive mortality. Currently available treatment strategies – besides aggressive autologous stem cell transplantation which is an option only for selected patients – only alleviate symptoms and slow disease progression. Previous attempts of immunomodulating therapies addressing B cell pathology like rituximab and tocilizumab in SSc showed mixed efficacy1,2Objectives:Here, we investigated the therapeutic potential of ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor used in B cell malignancies, to alter B cell pathology in SSc in anin vitromodel of autoimmunity.Methods:PBMCs and sorted B cells of 24 patients with SSc were used for functional testing after stimulation with hypomethylated DNA fragments (CpG) to induce an innate immune response. The effects of ibrutinib on cytokine production, autoantibody release and activation of the transcription factor NFκB were evaluated via multiplex cytokine assay, ELISA and flow cytometry.Results:Ibrutinib was able to reduce the production of the profibrotic hallmark cytokines IL-6 and TNF-α, which are mainly released by the effector B cell population, in response to TLR9-stimulation. Importantly, small doses of ibrutinib (0.1 µM) preserved the production of immunoregulatory IL-10 and IFN-γ while effectively inhibiting the cardinal cytokines of hyperactivated profibrotic effector B cells in SSc. Intracellular cytokine staining of IL-6 in B cell subsets further endorsed the potential of ibrutinib to inhibit B cells in a subset-specific manner, reducing IL-6+naïve B cells significantly but not IL-6+memory B cells. The subset specificity was abolished when high doses of ibrutinib (10 µM) were applied. In a flow cytometry analysis of phosphorylated NFκB, an important transcription factor in the induction of innate immune responses in B cells, significantly less activation was observed with ibrutinib treatment (0.1 µM). Higher doses of ibrutinib were unable to further reduce the abundance of pNFκB.Conclusion:Our data could pave the avenue for a clinical application of ibrutinib for patients with SSc as a novel treatment option for the underlying pathogenetic immune imbalance contributing to disease onset and progression.References:[1]Khanna, D.et al.Efficacy and Safety of Tocilizumab for the Treatment of Systemic Sclerosis: Results from a Phase 3 Randomized Controlled Trial [abstract]. Arthritis Rheumatol. 2018; 70 (suppl 10).[2]Jordan, S.et al.Effects and safety of rituximab in systemic sclerosis: an analysis from the European Scleroderma Trial and Research (EUSTAR) group.Ann. Rheum. Dis.74, 1188–1194 (2015).Disclosure of Interests:Jakob Einhaus: None declared, Ann-Christin Pecher: None declared, Elisa Asteriti: None declared, Hannes Schmid: None declared, Kathy-Ann Secker: None declared, Silke Duerr-Stoerzer: None declared, Hildegard Keppeler: None declared, Reinhild Klein: None declared, Corina Schneidawind: None declared, Jörg Henes Grant/research support from: Novartis, Roche-Chugai, Consultant of: Novartis, Roche, Celgene, Pfizer, Abbvie, Sanofi, Boehringer-Ingelheim,, Dominik Schneidawind: None declared

scholarly journals Characteristic pancreatic and splenic immune cell infiltration patterns in mouse acute pancreatitis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Baibing Yang ◽  
Joy M. Davis ◽  
Thomas H. Gomez ◽  
Mamoun Younes ◽  
Xiurong Zhao ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Pancreatitis ◽  
B Cells ◽  
Immune Cells ◽  
Immune Cell ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Immune Cell Infiltration ◽  
Computerized Analysis ◽  
Dimensional Flow

Abstract Background A systemic evaluation of immune cell infiltration patterns in experimental acute pancreatitis (AP) is lacking. Using multi-dimensional flow cytometry, this study profiled infiltrating immune cell types in multiple AP mouse models. Methods Three AP models were generated in C57BL/6 mice via cerulein (CAE) injection, alcohol and palmitoleic acid (EtOH + POA) injection, and alcohol diet feeding and cerulein (EtOH + CAE) injection. Primary pancreatic cells and splenocytes were prepared, and multi-dimensional flow cytometry was performed and analyzed by manual gating and computerized PhenoGraph, followed by visualization with t-distributed stochastic neighbor embedding (t-SNE). Results CAE treatment induced a time-dependent increase of major innate immune cells and a decrease of follicular B cells, and TCD4+ cells and the subtypes in the pancreas, whereas elicited a reversed pattern in the spleen. EtOH + POA treatment resulted in weaker effects than CAE treatment. EtOH feeding enhanced CAE-induced amylase secretion, but unexpectedly attenuated CAE-induced immune cell regulation. In comparison with manual gating analysis, computerized analysis demonstrated a remarkable time efficiency and reproducibility on the innate immune cells and B cells. Conclusions The reverse pattern of increased innate and decreased adaptive immune cells was consistent in the pancreas in CAE and EtOH + POA treatments. Alcohol feeding opposed the CAE effect on immune cell regulation. Together, the immune profiling approach utilized in this study provides a better understanding of overall immune responses in AP, which may facilitate the identification of intervention windows and new therapeutic strategies. Computerized analysis is superior to manual gating by dramatically reducing analysis time.

scholarly journals A High-Resolution Landscape of Mutations in the BCL6 Super-Enhancer in Normal Human B-Cells

2019 ◽  
Author(s):  
Jiang-Cheng Shen ◽  
Ashwini S. Kamath-Loeb ◽  
Brendan F. Kohrn ◽  
Keith R. Loeb ◽  
Bradley D. Preston ◽  
...  
Keyword(s):  
B Cells ◽  
High Resolution ◽  
Early Detection ◽  
B Cell ◽  
Low Frequency ◽  
Human B Cells ◽  
Super Enhancer ◽  
Normal Individuals ◽  
Normal Human ◽  
Duplex Sequencing

AbstractThe super-enhancers (SE) of lineage-specific genes in B-cells are off-target sites of somatic hypermutation. However, the inability to detect sufficient numbers of mutations in normal human B-cells has precluded the generation of a high-resolution mutational landscape of SEs. Here, we captured and sequenced 12 B-cell SEs at single-nucleotide resolution from ten healthy individuals across diverse ethnicities. We detected a total of ∼9000 subclonal mutations (allele frequencies <0.1%); of these, ∼8000 are present in the BCL6 SE alone. Within the BCL6 SE, we identified three regions of clustered mutations where the mutation frequency is ∼7X10-4. Mutational spectra show a predominance of C>T/G>A and A>G/T>C substitutions, consistent with the activities of activation-induced-cytidine deaminase (AID) and the A-T mutator, DNA Polymerase η, respectively, in mutagenesis in normal B-cells. Analyses of mutational signatures further corroborate the participation of these factors in this process. Single base substitution signature SBS85, SBS37, and SBS39 were found in the BCL6 SE. While SBS85 is a denoted signature of AID in lymphoid cells, the etiologies of SBS37 and SBS39 are still unknown. Our analysis suggests the contribution of error-prone DNA polymerases to the latter signatures. The high-resolution mutation landscape has enabled accurate profiling of subclonal mutations in B-cell SEs in normal individuals. By virtue of the fact that subclonal SE mutations are clonally expanded in B-cell lymphomas, our studies also offer the potential for early detection of neoplastic alterations.SignificanceWe used Duplex Sequencing to detect low-frequency mutations in the BCL6 super-enhancer locus in normal human B-cells. The landscape of pre-existing mutations is remarkably conserved across different ethnicities and reveals clustered mutational hotspots that correlate with reported sites of clonal mutations and translocation breakpoints in human B-cell lymphomas. This high-resolution genomic landscape revealed by Duplex Sequencing offers accurate and thorough profiling of low frequency, pre-existing mutations in normal individuals, and the potential for early detection of neoplastic alterations.

scholarly journals Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Cheri L. Lamb ◽  
Emily Price ◽  
Kevin P. Field ◽  
Christopher Dayton ◽  
Eric R. McIndoo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
B Cells ◽  
Batch Culture ◽  
Group A Streptococcus ◽  
Direct Method ◽  
Low Frequency ◽  
Memory B Cells ◽  
Specific Memory ◽  
Group A

ABSTRACT The low frequency of circulating antigen-specific memory B cells is a considerable obstacle in the discovery and development of human monoclonal antibodies for therapeutic application. Here, we evaluate two solid-phase isolation methods to enrich the number of antigen-specific B cells from individuals naturally immunized against streptolysin O (SLO), a key virulence factor and known immunogen of group A streptococcus (GAS). Class-switched B cells obtained from individuals with a history of GAS infection were separated from peripheral blood mononuclear cells (PBMCs) by immunomagnetic methods. SLO-specific B cells were further enriched directly by binding to SLO monomers and captured by streptavidin-coated magnetic microbeads or indirectly by binding a fluorescently labeled SLO-streptavidin tetramer and captured by anti-fluorophore immunomagnetic microbeads. SLO-bound B cells were quantitated by flow cytometry and/or expanded in batch culture to determine IgG specificity. From individuals who have suffered a GAS infection ≥2 years prior, only the direct method enriched SLO-specific B cells, as determined by flow cytometry. Likewise, in batch culture, B cells isolated by the direct method resulted in an average of 375-fold enrichment in anti-SLO IgG, while no enrichment was observed for B cells isolated by the indirect method. The direct method established here provides a simple approach to increase low-frequency antigen-specific B cell populations supporting many downstream applications, such as immortalization of B cells, cloning of immunoglobulin genes, or purification of antibodies from supernatant for future study. Overall, this process is efficient, is inexpensive, and can be applied to many naturally immunogenic antigens. IMPORTANCE Bacteria called group A streptococci can cause a variety of skin and soft tissue infections ranging from mild pharyngitis (“strep throat”) to deadly necrotizing fasciitis (sometimes called “flesh-eating” disease). In each case, the development of disease and the degree of tissue damage are mediated by toxins released from the bacteria during infection. Consequently, novel therapies aimed at clearing bacterial toxins are greatly needed. One promising new treatment is the utilization of monoclonal antibodies delivered as an immunotherapeutic for toxin neutralization. However, current methods of antibody development are laborious and costly. Here, we report a method to enrich and increase the detection of highly desirable antigen-specific memory B cells from individuals previously exposed to GAS using a cost-effective and less-time-intensive strategy. We envision that this method will be incorporated into many applications supporting the development of immunotherapeutics.

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