A new staining protocol for detection of murine antibody‐secreting plasma cell subsets by flow cytometry

Pracht, Katharina; Meinzinger, Julia; Daum, Patrick; Schulz, Sebastian; Reimer, Dorothea; Hauke, Manuela; Roth, Edith; Mielenz, Dirk; Berek, Claudia; Côrte‐Real, Joana; Jäck, Hans‐Martin; Schuh, Wolfgang

doi:10.1002/eji.201747019

Cited by 120 publications

(143 citation statements)

References 19 publications

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“…B). Plasma cell differentiation negatively correlates with B220 expression . Notably, we observed that the ratio of B220 negative to B220 intermediate NP + CD138 + TACI + cells in BM were ∼2‐fold decreased in anti‐CD4 Ab‐treated mice (Fig.…”

Section: Resultsmentioning

confidence: 76%

Residual LCMV antigen in transiently CD4⁺ T cell‐depleted mice induces high levels of virus‐specific antibodies but only limited B‐cell memory

et al. 2019

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Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8+ T cells independently of CD4+ T cell help. Residual viral antigen may, however, persist for a prolonged time. Here, we demonstrate that mice that had been transiently depleted of CD4+ T cells during acute LCMV Arm infection generated high levels of virus‐specific IgG antibodies (Ab) after viral clearance. Robust induction of LCMV‐specific IgG after transient CD4+ T cell depletion was dependent on Fcγ receptors but not on the complement receptors CD21/CD35. In contrast to the potent production of LCMV‐specific IgG, the generation of LCMV‐specific isotype‐switched memory B cells after transient CD4+ T cell depletion was considerably reduced. Moreover, mice depleted of CD4+ T cells during acute infection were strongly impaired in generating a secondary LCMV‐specific B cell response upon LCMV rechallenge. In conclusion, our data indicate that LCMV antigen depots after viral clearance were capable of inducing high levels of virus‐specific IgG. They failed, however, to induce robust virus‐specific B cell memory revealing a previously unappreciated dichotomy of specific Ab production and memory cell formation after priming with residual antigen.

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Section: Resultsmentioning

confidence: 76%

Residual LCMV antigen in transiently CD4⁺ T cell‐depleted mice induces high levels of virus‐specific antibodies but only limited B‐cell memory

et al. 2019

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“…2B) . B220 is expressed on the surface of B cells and downregulated during plasma cell differentiation (Pracht et al, 2017). We therefore defined three cell populations based on the levels of GFP fluorescence and B220 surface expression: GFP low B220 high , GFP int B220 int , GFP high B220 low , and a population negative for both markers (GFP neg B220 neg ; Fig.…”

Section: Resultsmentioning

confidence: 99%

Genetic timestamping of plasma cellsin vivoreveals homeostatic population turnover

Barbosa

Calado

2020

Preprint

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Plasma cells (PC)s are essential for protection from infection, and at the origin of incurable cancers. Current studies do not circumvent limitations of removing PCs from their microenvironment and confound formation and maintenance. This is in part due to the lack of tools to perform specific genetic manipulation in vivo. Also, studies of PC population dynamics have mostly relied on the use of nucleotide analog incorporation that does not label quiescent cells, a property of most PCs. Here we characterize in detail a genetic tool (JchaincreERT2) that permits first-ever specific genetic manipulation in PC in vivo, across immunoglobulin isotypes. Using this tool we found that PC numbers remained constant over-time and that PC decay was compensated by the emergence of new cells, supporting an homeostatic turnover of the population. The JchaincreERT2 genetic tool paves the way for in-depth mechanistic understanding of PC biology and pathology in vivo, in their microenvironment.HighlightsJchain expression occurs in most plasma cells across immunoglobulin isotypesJchaincreERT2 mediated genetic manipulation is effective only in plasma cellsGenetic timestamping of plasma cells reveals homeostatic regulation

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“…Plasma cells were identified using the surface markers TACI and CD138 as described in Pracht et al. . For detection of intracellular APRIL and IL6, we fixed the cells for 30 min at room temperature with 3% PFA and permeabilized with 1% Triton X‐100 (Sigma) before adding Anti‐APRIL‐PE (A3D8, Biolegend) or Anti‐IL‐6‐PE (MP5‐20F3, Biolegend).…”

Section: Methodsmentioning

confidence: 99%

Eosinophils are not essential for maintenance of murine plasma cells in the bone marrow

Ackermann

Ipseiz

et al. 2018

Eur J Immunol

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Eosinophils were reported to serve as an essential component of the plasma cell niche within the bone marrow. As the potential contribution of eosinophils to humoral immunity has remained incompletely understood, we aimed to further characterize their role during antibody responses and to additionally investigate their role in autoimmune disease. Contrary to our expectations and the currently prevailing paradigm, we found that eosinophils are fully dispensable for the survival of murine bone marrow plasma cells and accordingly do not contribute to antibody production and autoantibody-mediated disease. Littermate wild type and eosinophil-deficient ΔdblGATA-1 animals showed similar numbers and frequencies of plasma cells and did not differ in steady state levels of immunoglobulins or their ability to raise antigen-specific antibody responses. Eosinophils were likewise dispensable for autoantibody production or autoantibody-induced disease in a mouse model of systemic lupus erythematosus. Our findings thus argue against a role of eosinophils during the maintenance of the plasma cell pool and challenge the hitherto postulated concept of an eosinophil-sustained bone marrow niche.

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A new staining protocol for detection of murine antibody‐secreting plasma cell subsets by flow cytometry

Abstract: We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice.

Cited by 120 publications

References 19 publications

Residual LCMV antigen in transiently CD4⁺ T cell‐depleted mice induces high levels of virus‐specific antibodies but only limited B‐cell memory

Residual LCMV antigen in transiently CD4⁺ T cell‐depleted mice induces high levels of virus‐specific antibodies but only limited B‐cell memory

Genetic timestamping of plasma cellsin vivoreveals homeostatic population turnover

Eosinophils are not essential for maintenance of murine plasma cells in the bone marrow

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A new staining protocol for detection of murine antibody‐secreting plasma cell subsets by flow cytometry

Abstract: We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice.

Cited by 120 publications

References 19 publications

Residual LCMV antigen in transiently CD4+ T cell‐depleted mice induces high levels of virus‐specific antibodies but only limited B‐cell memory

Residual LCMV antigen in transiently CD4+ T cell‐depleted mice induces high levels of virus‐specific antibodies but only limited B‐cell memory

Genetic timestamping of plasma cellsin vivoreveals homeostatic population turnover

Eosinophils are not essential for maintenance of murine plasma cells in the bone marrow

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Product

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Residual LCMV antigen in transiently CD4⁺ T cell‐depleted mice induces high levels of virus‐specific antibodies but only limited B‐cell memory

Residual LCMV antigen in transiently CD4⁺ T cell‐depleted mice induces high levels of virus‐specific antibodies but only limited B‐cell memory