Single-cell analysis of germinal-center B cells informs on lymphoma cell of origin and outcome

In response to T cell–dependent antigens, mature B cells are stimulated to form germinal centers (GCs), the sites of B cell affinity maturation and the cell of origin (COO) of most B cell lymphomas. To explore the dynamics of GC B cell development beyond the known dark zone and light zone compartments, we performed single-cell (sc) transcriptomic analysis on human GC B cells and identified multiple functionally linked subpopulations, including the distinct precursors of memory B cells and plasma cells. The gene expression signatures associated with these GC subpopulations were effective in providing a sc-COO for ∼80% of diffuse large B cell lymphomas (DLBCLs) and identified novel prognostic subgroups of DLBCL.

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High-Throughput Single-Cell Analysis of B Cell Receptor Usage among Autoantigen-Specific Plasma Cells in Celiac Disease

The Journal of Immunology ◽

10.4049/jimmunol.1700169 ◽

2017 ◽

Vol 199 (2) ◽

pp. 782-791 ◽

Cited By ~ 26

Author(s):

Bishnudeo Roy ◽

Ralf S. Neumann ◽

Omri Snir ◽

Rasmus Iversen ◽

Geir Kjetil Sandve ◽

...

Keyword(s):

Celiac Disease ◽

B Cell ◽

Single Cell ◽

High Throughput ◽

Plasma Cells ◽

Single Cell Analysis ◽

Cell Receptor ◽

B Cell Receptor ◽

Cell Analysis ◽

Receptor Usage

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Single-Cell Analysis of FVIII-Specific Memory B Cells in Murine Hemophilia A.

Blood ◽

10.1182/blood.v110.11.1157.1157 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1157-1157 ◽

Cited By ~ 1

Author(s):

Christina Hausl ◽

Rafi U. Ahmad ◽

Bernhard Baumgartner ◽

Hans Peter Schwarz ◽

Hartmut Ehrlich ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Single Cell ◽

Cd4 T Cells ◽

Single Cell Analysis ◽

Memory B Cells ◽

Cell Analysis ◽

Memory B Cell ◽

Specific Memory

Abstract The elimination of FVIII-specific memory B cells is an essential step in the design of new therapeutic strategies for the induction of immune tolerance in hemophilia A with FVIII inhibitors. Using a mouse model of hemophilia A we recently reported that low dose FVIII stimulates the differentiation of FVIII-specific memory B cells into antibody-secreting plasma cells whereas high dose FVIII inhibits this process. The inhibition of memory-B-cell re-stimulation is irreversible and seems to be due to an induction of apoptosis. Further understanding of the complex interactions that lead to either re-stimulation and differentiation of memory B cells or inhibition and eradication of these cells requires appropriate technologies for single-cell analysis and functional studies. We established a new technology for single-cell analysis and cell sorting of FVIII-specific murine memory B cells. A combination of magnetic bead separation and multi-color flow cytometry enabled us to analyze and purify FVIII-specific memory B cells obtained from hemophilic mice treated with FVIII. In a first step, we depleted undesirable cell populations (IgM+, IgD+, CD11c+, F4/80+, Gr1+ and CD49b+ cells) from total spleen cells by magnetic bead separation. In a second step, we used multicolor flow cytometry to exclude CD4+ T cells and analyze the FVIII-specific memory B cell compartment. This compartment was specified by staining the specific B-cell receptor with FVIII and anti-IgG antibodies. Frequencies of cells in this compartment ranged from 0.1–0.5% of total spleen cells in animals treated with 4 intravenous doses of FVIII, given at weekly intervals. We could not detect any FVIII-specific memory B cells in naïve mice. By means of single cell sorting we isolated FVIII-specific memory B cells for further functional studies. We were able to cultivate FVIII-specific memory B cells in microwell cultures in vitro and differentiate them into antibody-secreting plasma cells. The re-stimulation and differentiation of single-cell sorted memory B cells was strictly dependent on the presence of activated CD4+ T cells. CD4+ T cells obtained from naïve mice did not support the memory response. Furthermore, the re-stimulation and differentiation of memory B cells in the presence of activated CD4+ T cells did not require additional dendritic cells for antigen presentation. Obviously, memory B cells provide sufficient antigen presentation to CD4+ T cells to enable them to trigger the memory response. Our approach for single-cell analysis and purification of FVIII-specific memory B cells provides a new tool for tracking memory B cell populations in vivo and for directly analyzing the regulation of memory B cell function. It opens the field for future studies which should elucidate signals and molecules involved in activation or inhibition and eradication of FVIII-specific memory B cells. These activities will eventually lead to the identification of targets for the design of new treatment strategies for patients with FVIII inhibitors.

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Single cell analysis of B lymphocytes from Wegener's granulomatosis: B cell receptors display affinity maturation within the granulomatous lesions

Clinical & Experimental Immunology ◽

10.1111/j.1365-2249.2008.03775.x ◽

2008 ◽

Vol 154 (3) ◽

pp. 339-345 ◽

Cited By ~ 23

Author(s):

J. Voswinkel ◽

G. Assmann ◽

G. Held ◽

S. Pitann ◽

W. L. Gross ◽

...

Keyword(s):

B Cell ◽

Single Cell ◽

B Lymphocytes ◽

Single Cell Analysis ◽

Wegener's Granulomatosis ◽

Affinity Maturation ◽

B Cell Receptors ◽

Cell Analysis ◽

Cell Receptors ◽

Granulomatous Lesions

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Signatures of B Cell Receptor Repertoire Following Pneumocystis Infection

Frontiers in Microbiology ◽

10.3389/fmicb.2021.636250 ◽

2021 ◽

Vol 12 ◽

Author(s):

Han Sun ◽

Hu-Qin Yang ◽

Kan Zhai ◽

Zhao-Hui Tong

Keyword(s):

B Cells ◽

B Cell ◽

Single Cell ◽

Plasma Cells ◽

Somatic Hypermutation ◽

Cell Receptor ◽

B Cell Receptor ◽

Receptor Repertoire ◽

Elevated Expression ◽

Pneumocystis Infection

B cells play vital roles in host defense against Pneumocystis infection. However, the features of the B cell receptor (BCR) repertoire in disease progression remain unclear. Here, we integrated single-cell RNA sequencing and single-cell BCR sequencing of immune cells from mouse lungs in an uninfected state and 1–4 weeks post-infection in order to illustrate the dynamic nature of B cell responses during Pneumocystis infection. We identified continuously increased plasma cells and an elevated ratio of (IgA + IgG) to (IgD + IgM) after infection. Moreover, Pneumocystis infection was associated with an increasing naïve B subset characterized by elevated expression of the transcription factor ATF3. The proportion of clonal expanded cells progressively increased, while BCR diversity decreased. Plasma cells exhibited higher levels of somatic hypermutation than naïve B cells. Biased usage of V(D)J genes was observed, and the usage frequency of IGHV9-3 rose. Overall, these results present a detailed atlas of B cell transcriptional changes and BCR repertoire features in the context of Pneumocystis infection, which provides valuable information for finding diagnostic biomarkers and developing potential immunotherapeutic targets.

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IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses

Journal of Experimental Medicine ◽

10.1084/jem.20091738 ◽

2010 ◽

Vol 207 (2) ◽

pp. 353-363 ◽

Cited By ~ 496

Author(s):

Michelle A. Linterman ◽

Laura Beaton ◽

Di Yu ◽

Roybel R. Ramiscal ◽

Monika Srivastava ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Plasma Cells ◽

Cell Formation ◽

Affinity Maturation ◽

Tfh Cells ◽

Follicular Helper ◽

Early Memory ◽

Bone Marrow Chimeras ◽

Follicular Response

During T cell–dependent responses, B cells can either differentiate extrafollicularly into short-lived plasma cells or enter follicles to form germinal centers (GCs). Interactions with T follicular helper (Tfh) cells are required for GC formation and for selection of somatically mutated GC B cells. Interleukin (IL)-21 has been reported to play a role in Tfh cell formation and in B cell growth, survival, and isotype switching. To date, it is unclear whether the effect of IL-21 on GC formation is predominantly a consequence of this cytokine acting directly on the Tfh cells or if IL-21 directly influences GC B cells. We show that IL-21 acts in a B cell–intrinsic fashion to control GC B cell formation. Mixed bone marrow chimeras identified a significant B cell–autonomous effect of IL-21 receptor (R) signaling throughout all stages of the GC response. IL-21 deficiency profoundly impaired affinity maturation and reduced the proportion of IgG1+ GC B cells but did not affect formation of early memory B cells. IL-21R was required on GC B cells for maximal expression of Bcl-6. In contrast to the requirement for IL-21 in the follicular response to sheep red blood cells, a purely extrafollicular antibody response to Salmonella dominated by IgG2a was intact in the absence of IL-21.

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024 Single cell analysis reveals the autoantigen-reactive B cell cytokine production in systemic sclerosis

Journal of Investigative Dermatology ◽

10.1016/j.jid.2017.02.037 ◽

2017 ◽

Vol 137 (5) ◽

pp. S4

Author(s):

A. Yoshizaki ◽

T. Fukasawa ◽

S. Ebata ◽

T. Kitamori ◽

S. Sato

Keyword(s):

Systemic Sclerosis ◽

B Cell ◽

Single Cell ◽

Cytokine Production ◽

Single Cell Analysis ◽

Cell Analysis ◽

Cell Cytokine Production

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Hyperproliferation of B Cells Specific for a Weakly Immunogenic PorA in a Meningococcal Vaccine Model

Clinical and Vaccine Immunology ◽

10.1128/cvi.00192-08 ◽

2008 ◽

Vol 15 (10) ◽

pp. 1598-1605 ◽

Cited By ~ 6

Author(s):

Thomas A. Luijkx ◽

Jacqueline A. M. van Gaans-van den Brink ◽

Harry H. van Dijken ◽

Germie P. J. M. van den Dobbelsteen ◽

Cécile A. C. M. van Els

Keyword(s):

B Cells ◽

B Cell ◽

Bactericidal Activity ◽

Plasma Cells ◽

Affinity Maturation ◽

Memory B Cells ◽

Vaccine Antigen ◽

Cell Subpopulation ◽

Cell Subpopulations ◽

Group B

ABSTRACT Highly homologous meningococcal porin A (PorA) proteins induce protective humoral immunity against Neisseria meningitidis group B infection but with large and consistent differences in the levels of serum bactericidal activity achieved. We investigated whether a poor PorA-specific serological outcome is associated with a limited size of the specific B-cell subpopulation involved. The numbers of PorA-specific splenic plasma cells, bone marrow (BM) plasma cells, and splenic memory B cells were compared between mice that received priming and boosting with the weakly immunogenic PorA (P1.7-2,4) protein and those that received priming and boosting with the highly immunogenic PorA (P1.5-1,2-2) protein. Immunoglobulin G (IgG) titers (except at day 42), bactericidal activity, and the avidity of IgG produced against P1.7-2,4 were significantly lower at all time points after priming and boosting than against P1.5-1,2-2. These differences, however, were not associated with a lack of P1.7-2,4-specific plasma cells. Instead, priming with both of the PorAs resulted in the initial expansion of comparable numbers of splenic and BM plasma cells. Moreover, P1.7-2,4-specific BM plasma cells, but not P1.5-1,2-2-specific plasma cells, expanded significantly further after boosting. Likewise, after a relative delay during the priming phase, the splenic P1.7-2,4-specific memory B cells largely outnumbered those specific for P1.5-1,2-2, upon boosting. These trends were observed with different vaccine formulations of the porins. Our results show for the first time that B-cell subpopulations involved in a successfully maturated antibody response against a clinically relevant vaccine antigen are maintained at smaller population sizes than those associated with poor affinity maturation. This bears consequences for the interpretation of immunological memory data in clinical vaccine trials.

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