High-Throughput Ig Sequencing of Paired Blood and Spleen Samples Allows a Redefinition of Memory IgM Subsets in Humans

Abstract In humans, whether B cells with the IgM+IgD+CD27+ phenotype represent an independent lineage involved in T-independent responses, similar to mouse marginal zone B cells, or whether they are part of the germinal center-derived memory B-cell pool generated during responses to T-dependent antigens, is still a debated issue. To address this question, we performed high-throughput Ig sequencing of B-cell subsets from paired blood and spleen samples and analyzed the clonal relationships between them. We isolated and analyzed 3 different B cell subsets based on CD27 and IgD staining from both blood and spleen: IgD+CD27+ (MZ) - amplified with Cmu primers IgD-CD27+ (switched and IgM-only) with Cmu, Cgamma and Calpha primers IgD-CD27- (CD27- memory or double-negative DN) with the same three primers We obtained 95729 unique sequences that clustered in 49199 different clones: 1125 clones were shared between blood and spleen of the same B-cell subset, and 1681 clones were shared between different subsets, allowing us to trace their relationships. We analyzed these clones that share sequences from different subsets/tissues for their mutation frequency distribution, CDR3-length, and VH/JH family usage, and compared these different characteristics with the bulk of sequences from their respective subset of origin. The analysis of clones shared between blood and spleen for switched IgG/IgA and for MZ subsets suggests different recirculation dynamics. For switched cells, the blood appears to be a mixture of splenic and other lymphoid tissues B cells. For MZ B cells in contrast, the blood appear to be only composed of a subgroup of the splenic repertoire, in agreement with the observation that marginal zone B cells recirculate and are mainly generated in the spleen. Clonal relationships between the IgM clones (originating from the MZ, IgM-only and double negative compartments) show that the clones involved display the characteristics of IgM-only B cells whatever their subset of origin, even in the case of the paired MZ/double-negative sequences that were not supposed to include IgM-only sequences. We therefore conclude that the clones shared between the various IgM subsets do not represent b between them, but rather correspond to a heterogeneous phenotype of the IgM-only population that concerns both IgD and CD27 expression, leading to a partial overlap with the MZ and double-negative gates. Clones shared between the MZ and the switched IgG and IgA compartment also show, for their IgM part, the mutation and repertoire characteristics of IgM-only cells and not of MZ B cells, reinforcing the conclusion that IgM-only are true memory B cells, and constitute the only subset showing clonal relationships with switched memory B cells. In summary, we report that MZ B cells have different recirculation characteristics and do not show real clonal relationships with IgM-only and switched memory B cells, in agreement with the notion that they represent a distinct differentiation pathway. In contrast, the only precursor-product relationship between IgM memory and switched B cells appear to concern a B cell subset that has been described as "IgM-only", but appears to have a more heterogeneous expression of IgD than previously reported and therefore contribute to 3-15% of the MZ compartment. Searching for markers that would permit to discriminate between marginal zone and germinal center-derived IgM memory B cells is obviously required to further delineate their respective function. Disclosures No relevant conflicts of interest to declare.

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Telomere Elongation in B-Cells Is Independent of Class Switching.

Blood ◽

10.1182/blood.v112.11.1542.1542 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1542-1542

Author(s):

Alexander Roeth ◽

Dirk de Beer ◽

Marc Seifert ◽

Astrid Mueller ◽

Ulrich Duehrsen ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Telomere Length ◽

Germinal Center ◽

Cell Subset ◽

Memory B Cells ◽

Double Positive ◽

Telomere Elongation ◽

Cell Subpopulations ◽

B Cell Subsets

Abstract BACKGROUND: In contrast to human granulocytes and T-cells with a quite homogenous decrease in telomere lengths over age, telomere lengths of peripheral blood B-cell populations are highly heterogeneous with average telomere lengths of B-cells remaining relatively constant from middle age onward (Baerlocher et al, J. Leuk. Biol. 2003). So far, telomere elongation has been described in highly proliferating germinal center B-cells during the development from naive B-cells to memory B-cells (Norrback et al, Eur J Haematol 2001). During this same time period the process of class-switch recombination (CSR) occurs. Our aims were to analyze the telomere lengths in different B-cell subsets in order to elucidate telomere length dynamics in relationship to CSR. PATIENTS and METHODS: Buffy coats from 14 healthy donors were enriched for CD19+ B cells by magnetic cell separation. Naive B cells (IgM+/IgD+/CD27−), IgM-only B cells (IgM+/IgD−/low/CD27+), double-positive B cells (IgM+/IgD+/CD27+) and class-switched memory B cells (IgM−/IgD−/CD27+) were further separated by cell sorting. Telomere length of sorted B-cell subpopulations was measured by automated multicolour flow-FISH. RESULTS: Naive B-cells presented the shortest telomere length values (average telomere length, n=14: 7.2 kb ± 0.7 kb) compared to the other B-cell subsets. In comparison to the naive B-cell subset, the IgM only B-cell subset had 13% longer telomeres (average telomere length, n=14: 8.1 kb ± 1.3 kb), the double-positive B-cell subset had 10% longer telomeres (average telomere length, n=14: 7.9 kb ± 0.8 kb) and the memory B-cell subset had 22% longer telomeres (average telomere length, n=14: 8.7 kb ± 1.1 kb) (p < 0.0001). CONCLUSIONS: We are able to confirm longer telomeres in memory B cells than in naive B cells. For the first time, however, we can demonstrate that longer telomeres are also found in non-class switched B-cells. Based on these results telomere elongation does not coincide with the process of CSR. Additional studies are needed to assess whether telomere elongation can only take place in the germinal center or whether certain B-cell subsets are able to elongate their telomeres independent from the germinal center.

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Analysis of IgVH, BCL-6, PIM, RHO/TTF and PAX5 Mutational Status in Splenic and Nodal Marginal Zone B Cell Lymphoma Suggests a Particular B Cell Origin.

Blood ◽

10.1182/blood.v106.11.162.162 ◽

2005 ◽

Vol 106 (11) ◽

pp. 162-162 ◽

Cited By ~ 3

Author(s):

Alexandra Traverse-Glehen ◽

Aurelie Verney ◽

Lucille Baseggio ◽

Pascale Felman ◽

Evelyne Callet-Bauchu ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Somatic Mutations ◽

Marginal Zone ◽

Somatic Hypermutation ◽

B Cell Lymphoma ◽

Marginal Zone B Cells ◽

Mutational Status ◽

Frequent Absence

Abstract Background and Objectives Splenic and nodal marginal zone B cell lymphoma (SMZL and NMZL) have been recently identified as distinct clinicopathological entities in the WHO classification. These lymphomas entities may have a common origin in the marginal B-cell compartment of the lymphoid organs. However the precise cell of origin of marginal zone B cells, its status in the B cell differentiation pathway and the mechanisms involved in lymphomagenesis remain unclear. The most widely held view is that marginal zone B cells are mostly memory B cells. But the origin of these cells, especially the transit through germinal center pathway, remains contradictory. Somatically mutated variable-region of immunoglobulin genes and bcl-6 gene represent at this time faithful markers for exposure to the germinal center. In addition, aberrant somatic hypermutations have been suggested to contribute to the development of B-cell lymphomas, occurring in the 5′ sequence of several proto-oncogenes. Interestingly those mutation do not occur in normal germinal center B cells. Design and Methods: IgVH, BCL-6, PIM1, Rho/TTF and PAX 5 genes, highly mutated in DLBCL and other indolent lymphoma such as B-CLL, were analysed for the presence of somatic mutations from 50 marginal zone lymphoma tissue and blood samples (21 NMZL and 29 SMZL including 10 cases with numerous villous lymphoma cells in peripheral blood). According to the morphological and immunophenotypical analysis, the fraction of malignant cells in the specimen was 70% or more in all cases. Mutational analysis was restricted to the regions previously shown to contain more than 95% of mutations in DLBCL. PCR products were directly sequenced on both sides and perfomed in duplicate in two independent reactions. Results: Out of 18 NMZL cases analysed for IgVH mutational status (3 cases not analysed for IgVH) 15 cases were mutated and 21 out of 28 in SMZL cases. Mutation of BCL-6 was detected in only 1 NMZL patients (1/21) and 1 SMZL patients (1/29). For RhoH/TTF, PIM1, PAX5 the mutation average was also low with only 1 case mutated per group and per gene, with a different case mutated in each for each gene. Conclusion In summary, we demonstrate the low frequency of aberrant somatic mutations in SMZL and NMZL, suggesting that this process is probably not a major contributor to lymphomageneis. However the frequent absence of mutation in BCL6 suggest a particular differentiation pathway, as suggested before in normal marginal zone B cells, possibly without transit through the germinal center. Interestingly the relatively high frequency of VH mutated cases compared with the frequent absence of mutation of BCL6, considered as a specific germinal center tag, could suggest somatic hypermutation outside the germinal center. In addition the absence of hypermutation could be linked with the absence of recurrent translocation in SMZL and NMZL, the translocation process haveing been associated with somatic hypermutation dysfunction.

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B Cells with BCL2/IGH Translocation Compose a Distinctive Cell Population That May Serve as a Reservoir of Lymphoma of Germinal Center B-Cell Type.

Blood ◽

10.1182/blood.v112.11.2050.2050 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2050-2050

Author(s):

Tomomi Sakai ◽

Momoko Nishikori ◽

Masaharu Tashima ◽

Ryo Yamamoto ◽

Toshio Kitawaki ◽

...

Keyword(s):

Cell Cycle ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Marginal Zone ◽

Terminal Differentiation ◽

Follicular Dendritic Cells ◽

Marginal Zone B Cells ◽

Germinal Center B Cell ◽

Follicular B Cells

Abstract BCL2/IGH translocation is a hallmark of follicular lymphoma and diffuse large B-cell lymphoma of germinal center B-cell type. Although being a strong determinant of these histological subtypes, this translocation is considered to be insufficient by itself and further gene alterations are necessary for cellular transformation. In Eμ-BCL2 transgenic (Tg) mice, B-lineage cells are increased by several-fold compared to wild-type (WT) mice, but only 5–15 % of them develop disease in the first year of life. To clarify how the BCL2 translocation contributes to the development of specific lymphoma subtypes, we created two types of chimeric mouse models to characterize the biological features of BCL2-overexpressing B cells in normal individuals. First, we introduced CD19 promoter-driven BCL2 and its mutant genes to a minor population of murine bone marrow cells by using a lentiviral vector system and transplanted into irradiated mice. BCL2-overexpressing B cells showed increased follicular and reduced marginal zone populations. The same phenotypic shift was observed in B cells introducing BCL2-Y28F mutant that retained anti-apoptotic function, but a defective mutant BCL2-G142A and a mock vector did not affect B-cell phenotype. Additionally, BCL2-introduced B cells showed decreased cell size compared to those introduced BCL2-G142A and mock vectors. To assess the functional alteration of BCL2-overexpressing B cells, TNP-Ficoll binding experiment was performed. The result showed diminished T-cell independent response in parallel with decreased marginal zone B cells. The low transformation frequency of B cells in Eμ-BCL2 Tg mice has been partly explained by their propensity to reside in the G0 phase of the cell cycle (reviewed in Oncogene, 18:5268,1999). We hypothesized that the microenvironment of B cells in Eμ-BCL2 Tg mice might be altered by abnormal B cells themselves. To evaluate the influence of the different microenvironments on BCL2-overexpressing B cells, we next made Eμ-BCL2/CAG-GFP double Tg mice and transferred their bone marrow mononuclear cells into WT or Eμ-BCL2 Tg mice. Blastic cell population of BCL2+GFP+ B cells was larger in those transferred to WT mice compared to those transferred to Eμ-BCL2 Tg mice, regardless of the same phenotypic preference toward follicular B cells. BrdU uptake experiments demonstrated continuous cell cycle progression of the BCL2+GFP+ B cells in WT mice but repressed cell cycle of those in Eμ-BCL2 Tg mice. In immunohistochemical analysis, splenic follicles were disorganized with reduced follicular dendritic cells and inadequate T cell accumulation in Eμ-BCL2 Tg mice. Functional impairment of splenic follicles in Eμ-BCL2 Tg mice might be caused by decreased marginal zone B cell subset, as the antigen capture and delivery by marginal zone B cells was reported to play an important role in the development of follicular dendritic cells. To understand the fate of BCL2-overexpressing B cells after stimulation, we finally assessed their terminal differentiation capacity in vitro. Plasma cell differentiation was suppressed in B cells derived from Eμ-BCL2 Tg mice under either LPS or anti-IgM antibody stimulation. BCL2 is reported to impede the activity of transcription factor NF-AT (Proc Natl Acad Sci93:9545,1996; Nature386:728,1997), and we found that calcineurin inhibitor FK506 suppressed plasma cell differentiation of WT B cells. Gene regulation patterns of the Eμ-BCL2+ B cells were similar to B cells stimulated in the presence of FK506 as well, suggesting that repressed terminal differentiation in Eμ-BCL2+ B cells was partly caused by the suppressed activity of NF-AT. In summary, BCL2-deregulated B cells preferentially differentiate into follicular B cells, and as a result of decreased terminal differentiation in addition to their anti-apoptotic property, they may be obliged to survive and recirculate as memory B cells, and accumulate genetic abnormalities while they repeatedly pass through the germinal center. As the germinal center is the particular site where they can counterbalance the cell cycle-retarding effect of BCL2, it may be a specific place for generating lymphoma triggered by BCL2/IGH translocation. Our results emphasize the importance of the microenvironment of pre-malignant cells during transformation process, and suggest that a simple transgenic mouse model may not be always appropriate for the study of oncogenesis.

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Expression of a Dysfunctional Activation Induced Cytidine Deaminase Is Correlated with Disease Progression in Splenic Marginal Zone Lymphoma.

Blood ◽

10.1182/blood.v120.21.2397.2397 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2397-2397

Author(s):

Gabriel Brisou ◽

Laurent Jallades ◽

Alexandra Traverse-Glehen ◽

Francoise Berger ◽

Aurélie Verney ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Marginal Zone ◽

Cytidine Deaminase ◽

Marginal Zone Lymphoma ◽

Splenic Marginal Zone Lymphoma ◽

Marginal Zone B Cells ◽

Activation Induced Cytidine Deaminase ◽

Splicing Variants

Abstract Abstract 2397 B cells can undergo at least two differentiation pathways, dependent of T cells or not, starting from follicular or marginal zone B cells respectively. The T-independent response, less understood than the germinal center reaction, is triggered by specific antigens and arises from marginal zone B cells. During this development, some B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR), triggered by the same DNA editing enzyme called Activation Induced Cytidine Deaminase (AID). The splenic marginal zone lymphoma (SMZL) is a rare lymphoproliferative disorder characterized by a clonal expansion of B cells in the marginal zone of the spleen. These B-cells underwent SHM in roughly 60% of the cases but nearly none underwent CSR. These observations suggest that tumor clones originate from a particular activated B cell subset not transiting through the germinal center. In order to confirm this hypothesis, we focused our work on the status and impact of AID in this disease and worked on purified B cells extracted from spleen of well-characterized SMZL cases. We determined AID status by quantitative RT-PCR analysis on 27 SMZL samples and compared it with 5 controls. In the SMZL group the relative level of expression of AID is heterogeneous but two subgroups could be distinguished: one considered as expressing AID (14 cases out of the 27 analyzed), the remaining considered as not expressing AID. When we compared AID expression rate with occurrence of SHM and CSR, no clear correlation between AID expression and presence of SHM or CSR could be observed suggesting that AID, when expressed, is dysfunctional. To address this hypothesis, we first analyzed AID protein by immunohistochemistry and a good correlation between IHC signal and AID mRNA expression level has been observed. As AID gene was not mutated, we next focused our work on AID mRNA splicing variants as these variants exhibit different functions according to the domain of the protein they contain in a murine model. We found that SMZL B cells express various splicing variants of AID mRNA, some of those variants corresponding to the full length isoform (n = 6/17), and other variants corresponding to AID-ΔE4a (n = 2/17) or AID-ΔE4 (n = 7/17) isoforms known to be expressed in normal germinal center B cells as well as in Chronic Lymphocytic and Acute Lymphoblastic Leukemia. These findings indicate that although expressed at the mRNA and protein levels, AID may not be fully functional in SMZL cases. Finally we addressed the potential clinical significance of AID expression. We identified for that purpose a group of “progressive SMZL” patients that had received immuno-chemotherapy after splenectomy because of a significant risk of progression or transformation into aggressive large B cell lymphoma (n = 8/27) pre-empting outcome differences. We found a higher proportion of AID expressing patients in the defined “progressive SMZL” group (n = 7/8) as compared to the proportion found in the “indolent SMZL” group (n = 5/14, p = 0,03). Altogether, this data suggest that the B cell clone leading to SMZL originate from the marginal zone and support the hypothesis of a lymphoproliferative disorder affecting the T-independent response. AID expression in SMZL may reflect an advanced stage of the disease and could be correlated with the evolution of the lymphoma into a more clinically or pathologically aggressive form. Disclosures: No relevant conflicts of interest to declare.

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Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways

Blood ◽

10.1182/blood-2011-04-345579 ◽

2011 ◽

Vol 118 (8) ◽

pp. 2150-2158 ◽

Cited By ~ 217

Author(s):

Magdalena A. Berkowska ◽

Gertjan J. A. Driessen ◽

Vasilis Bikos ◽

Christina Grosserichter-Wagener ◽

Kostas Stamatopoulos ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Somatic Hypermutation ◽

Phenotypic Diversity ◽

Human Memory ◽

Memory B Cells ◽

Memory B Cell ◽

Effector Cells ◽

B Cell Subsets

Abstract Multiple distinct memory B-cell subsets have been identified in humans, but it remains unclear how their phenotypic diversity corresponds to the type of responses from which they originate. Especially, the contribution of germinal center-independent responses in humans remains controversial. We defined 6 memory B-cell subsets based on their antigen-experienced phenotype and differential expression of CD27 and IgH isotypes. Molecular characterization of their replication history, Ig somatic hypermutation, and class-switch profiles demonstrated their origin from 3 different pathways. CD27−IgG+ and CD27+IgM+ B cells are derived from primary germinal center reactions, and CD27+IgA+ and CD27+IgG+ B cells are from consecutive germinal center responses (pathway 1). In contrast, natural effector and CD27−IgA+ memory B cells have limited proliferation and are also present in CD40L-deficient patients, reflecting a germinal center-independent origin. Natural effector cells at least in part originate from systemic responses in the splenic marginal zone (pathway 2). CD27−IgA+ cells share low replication history and dominant Igλ and IgA2 use with gut lamina propria IgA+ B cells, suggesting their common origin from local germinal center-independent responses (pathway 3). Our findings shed light on human germinal center-dependent and -independent B-cell memory formation and provide new opportunities to study these processes in immunologic diseases.

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Optimisation of ex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses

Wellcome Open Research ◽

10.12688/wellcomeopenres.11386.2 ◽

2018 ◽

Vol 2 ◽

pp. 97 ◽

Cited By ~ 1

Author(s):

Luke Muir ◽

Paul F. McKay ◽

Velislava N. Petrova ◽

Oleksiy V. Klymenko ◽

Sven Kratochvil ◽

...

Keyword(s):

Cell Culture ◽

B Cells ◽

B Cell ◽

Cell Expansion ◽

Ex Vivo ◽

Cell Subset ◽

Human Memory ◽

Memory B Cells ◽

Memory B Cell ◽

B Cell Subsets

Background:Human memory B cells play a vital role in the long-term protection of the host from pathogenic re-challenge. In recent years the importance of a number of different memory B cell subsets that can be formed in response to vaccination or infection has started to become clear. To study memory B cell responses, cells can be culturedex vivo,allowing for an increase in cell number and activation of these quiescent cells, providing sufficient quantities of each memory subset to enable full investigation of functionality. However, despite numerous papers being published demonstrating bulk memory B cell culture, we could find no literature on optimised conditions for the study of memory B cell subsets, such as IgM+memory B cells.Methods:Following a literature review, we carried out a large screen of memory B cell expansion conditions to identify the combination that induced the highest levels of memory B cell expansion. We subsequently used a novel Design of Experiments approach to finely tune the optimal memory B cell expansion and differentiation conditions for human memory B cell subsets. Finally, we characterised the resultant memory B cell subpopulations by IgH sequencing and flow cytometry.Results:The application of specific optimised conditions induce multiple rounds of memory B cell proliferation equally across Ig isotypes, differentiation of memory B cells to antibody secreting cells, and importantly do not alter the Ig genotype of the stimulated cells. Conclusions:Overall, our data identify a memory B cell culture system that offers a robust platform for investigating the functionality of rare memory B cell subsets to infection and/or vaccination.

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Impact of Autologous Dendritic Cell Immunotherapy (Arcelis™) on B Cell Subsets in HIV-1-Infected Subjects Receiving Antiretroviral Therapy

Blood ◽

10.1182/blood.v112.11.80.80 ◽

2008 ◽

Vol 112 (11) ◽

pp. 80-80

Author(s):

Mohamed-Rachid Boulassel ◽

Bader Yassine-Diab ◽

Don Healey ◽

Charles Nicolette ◽

Rafick-Pierre Sékaly ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Immune Responses ◽

B Cell ◽

Plasma Cells ◽

Cell Subset ◽

Cd8 T Cell ◽

Memory B Cells ◽

B Cell Subsets ◽

Hiv 1

Abstract We demonstrated the enhancement of CD8-specific responses following the administration of an immune-based therapy consisting of dendritic cells (DC) electroporated with autologous amplified HIV-1 RNA and CD40 ligand (CD40 L) RNA manufactured by the Arcelis™ process in HIV patients receiving antiretroviral therapy (ART). We conducted a sub study on circulating B cell populations to further assess changes induced by this autologous DC therapy as CD40L is a major B cell co-stimulatory factor. To this end, we assessed B cell subset changes in relation to the proliferative capacity of CD4+ and CD8+ T cells response to DC targets containing the 4 HIV-1 antigens (Gag, Vpr, Rev, Nef). The co-expression of CD19, CD38, IgD, CD10, CD23, CD27, CD5, and CD138 were analyzed by multi-parametric flow cytometry to assess circulating B cell subsets such as naïve resting B-cells (Bm1), activated naïve B cells (Bm2), GC founder cells (Bm2’), centroblasts and centrocytes (Bm3 and Bm4), early memory B cells (eBm5), memory B cells (Bm5), IgD memory cells, plasma cells, and B-1 cells. Changes in B cells subsets were analyzed before and after the four intradermal injections of this immunotherapeutic product containing 1.2 × 107 DC. Ten ART treated subjects with undetectable viral load (< 50 copies/ml), median CD4+ count of 440 cells/μl (range: 316–1102), and with a CD4+ nadir > 200 cells/μl were studied. Throughout the study, no significant changes in CD4+ cell count, CD4/CD8 ratio, and no viral blips were noticed. The percentage of total B cells, Bm1, Bm2, Bm2′, eBm5, IgD memory, plasma cells, and B-1 cell subsets did not significantly change. However, a decrease in the percentage of Bm3 and Bm4 cells was found (0.36 [0.06–0.86] versus 0.11 [0.04–0.36]; P=0.05). Conversely, an important increase in the Bm5 cell subset was evidenced (10.4 [1.6–24.2] versus 18.1 [5.1–27.5]; P=0.005) suggesting a proliferation of B memory cells induced by DC immunization. In addition, the multifunctional and polyvalent CD8+ T cell proliferative responses to the 4 HIV genes used in this immunotherapy were noticed in 8 out of 9 subjects available for analysis and characterized by an effector memory phenotype. No CD4+ T cell immune responses were detected, consistent with the endogenous HLA class I loading of the antigens. Collectively, these results indicate that this immunotherapy induces an increase in the B memory cell population in the absence of inducing any clinically apparent autoimmunity along with strong HIV specific multifunctional CD8+ T cell specific immune responses.

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Single-cell transcriptomic analyses define distinct peripheral B cell subsets and discrete development pathways

10.1101/2020.09.03.281527 ◽

2020 ◽

Author(s):

Alexander Stewart ◽

Joseph Ng ◽

Gillian Wallis ◽

Vasiliki Tsioligka ◽

Franca Fraternali ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Single Cell ◽

Heterogeneous Population ◽

Memory B Cells ◽

Double Negative ◽

Memory Cells ◽

Relative Paucity ◽

Age Related ◽

B Cell Subsets

AbstractSeparation of B cells into different subsets has been useful to understand their different functions in various immune scenarios. In some instances, the subsets defined by phenotypic FACS separation are relatively homogeneous and so establishing the functions associated with them is straightforward. Other subsets, such as the “Double negative” (DN, CD19+CD27-IgD-) population, are more complex with reports of differing functionality which could indicate a heterogeneous population. Recent advances in single-cell techniques enable an alternative route to characterise cells based on their transcriptome. To maximise immunological insight, we need to match prior data from phenotype-based studies with the finer granularity of the single-cell transcriptomic signatures. We also need to be able to define meaningful B cell subsets from single cell analyses performed on PBMCs, where the relative paucity of a B cell signature means that defining B cell subsets within the whole is challenging. Here we provide a reference single-cell dataset based on phenotypically sorted B cells and an unbiased procedure to better classify functional B cell subsets in the peripheral blood, particularly useful in establishing a baseline cellular landscape and in extracting significant changes with respect to this baseline from single-cell datasets. We find 10 different clusters of B cells and applied a novel, geometry-inspired, method to RNA velocity estimates in order to evaluate the dynamical transitions between B cell clusters. This indicated the presence of two main developmental branches of memory B cells. One involves IgM memory cells and two DN subpopulations, culminating in a population thought to be associated with Age related B cells and the extrafollicular response. The other branch involves a third DN cluster which appears to be a precursor of classical memory cells. In addition, we identify a novel DN4 population, which is IgE rich and on its own developmental branch but with links to the classical memory branch.

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Optimisation of ex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses

Wellcome Open Research ◽

10.12688/wellcomeopenres.11386.1 ◽

2017 ◽

Vol 2 ◽

pp. 97 ◽

Cited By ~ 1

Author(s):

Luke Muir ◽

Paul F. McKay ◽

Velislava N. Petrova ◽

Oleksiy V. Klymenko ◽

Sven Kratochvil ◽

...

Keyword(s):

Cell Culture ◽

B Cells ◽

B Cell ◽

Cell Expansion ◽

Ex Vivo ◽

Cell Subset ◽

Human Memory ◽

Memory B Cells ◽

Memory B Cell ◽

B Cell Subsets

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NR4A1 Deletion in Marginal Zone B Cells Exacerbates Atherosclerosis in Mice—Brief Report

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314607 ◽

2020 ◽

Vol 40 (11) ◽

pp. 2598-2604

Author(s):

Meritxell Nus ◽

Gemma Basatemur ◽

Maria Galan ◽

Laia Cros-Brunsó ◽

Tian X. Zhao ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Marginal Zone ◽

Cell Function ◽

Cell Receptor ◽

Marginal Zone B Cells ◽

Follicular Helper ◽

Center Axis ◽

Cell Deletion

Objective: NR4A orphan receptors have been well studied in vascular and myeloid cells where they play important roles in the regulation of inflammation in atherosclerosis. NR4A1 (nerve growth factor IB) is among the most highly induced transcription factors in B cells following BCR (B-cell receptor) stimulation. Given that B cells substantially contribute to the development of atherosclerosis, we examined whether NR4A1 regulates B-cell function during atherogenesis. Approach and Results: We found that feeding Ldlr −/− mice a Western diet substantially increased Nr4a1 expression in marginal zone B (MZB) cells compared with follicular B cells. We then generated Ldlr −/− mice with complete B- or specific MZB-cell deletion of Nr4a1 . Complete B-cell deletion of Nr4a1 led to increased atherosclerosis, which was accompanied by increased T follicular helper cell–germinal center axis response, as well as increased serum total cholesterol and triglycerides levels. Interestingly, specific MZB-cell deletion of Nr4a1 increased atherosclerosis in association with an increased T follicular helper–germinal center response but without any impact on serum cholesterol or triglyceride levels. Nr4a1 −/− MZB cells showed decreased PDL1 (programmed death ligand-1) expression, which may have contributed to the enhanced T follicular helper response. Conclusions: Our findings reveal a previously unsuspected role for NR4A1 in the atheroprotective role of MZB cells.

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