Germinal center–dependent and –independent memory B cells produced throughout the immune response

Memory B cells comprise a heterogenous group of cells that differ in origin and phenotype. During the early phases of the immune response, activated B cells can differentiate into IgM-expressing memory cells, short-lived plasma cells, or seed germinal centers (GCs). The memory compartment is subsequently enriched by B cells that have been through several rounds of division and selection in the GC. Here, we report on the use of an unbiased lineage-tracking approach to explore the origins and properties of memory B cell subsets in mice with an intact immune system. We find that activated B cells continue to differentiate into memory B cells throughout the immune response. When defined on the basis of their origins, the memory B cells originating from activated B cells or GCs differ in isotype and overall gene expression, somatic hypermutation, and their affinity for antigen.

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Comparable Long-Term Persistence of Anti-FVIII Antibodies in Hemophilic E17 Mice on Different Genetic Backgrounds Despite Significant Differences in the Amplitude of the Immune Responses.

Blood ◽

10.1182/blood.v108.11.1027.1027 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1027-1027

Author(s):

Natalie Bauer ◽

Christina Hausl ◽

Rafi U. Ahmad ◽

Bernhard Baumgartner ◽

Hans Peter Schwarz ◽

...

Keyword(s):

Immune Response ◽

B Cells ◽

Genetic Background ◽

Neutralizing Antibodies ◽

Plasma Cells ◽

Memory B Cells ◽

Mouse Strains ◽

Igg Antibodies ◽

Specific Memory

Abstract About 30% of patients with severe hemophilia A develop neutralizing antibodies against FVIII (FVIII inhibitors) following replacement therapy. The type of FVIII gene mutation as well as other predisposing genetic factors contribute to the inhibitor phenotype. Based on these findings, we asked if the genetic background modulates the long-term persistence of anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells in the E17 murine hemophilia model. Furthermore, we asked if the recently described inhibition of memory-B-cell re-stimulation by high doses of FVIII is influenced by the genetic background of the murine model. E17 mice on two different genetic backgrounds (C57Bl/6J and Balb/c) were treated with four doses of 200 ng human FVIII at weekly intervals. Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were followed up to 12 months after the last dose of FVIII. Antibody titers and subclasses of antibodies (IgM, IgG1, IgG2a, IgG2b, IgG3) were measured by ELISA. Antibody secreting plasma cells in spleen and bone marrow were detected by ELISPOT as described (Hausl et al., Thromb Haemost 2002). The re-stimulation of FVIII-specific memory B cells was studied as described recently (Hausl et al., Blood 2005). Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were first detectable in E17 Balb/c mice. IgM antibodies in the circulation and IgM secreting plasma cells in the spleen were observed after the first dose of FVIII, IgG antibodies and IgG secreting plasma cells after the second dose. No anti-FVIII antibodies after the first dose of FVIII were observed in E17 C57BL/6J mice but both IgM and IgG antibodies as well as IgM and IgG producing plasma cells were detectable after the second dose of FVIII. The antibody response involved all IgG subclasses in both mouse strains. However, IgG1 was dominant in E17 Balb/c mice whereas IgG2a was dominant in E17 C57BL/6J mice. When the in vitro restimulation of FVIII-specific memory B cells was examined, similar patterns were observed for both mouse strains. Low concentrations of FVIII between 10 and 100 ng/ml FVIII restimulated memory B cells and induced their differentiation into antibody secreting plasma cells whereas high concentrations of FVIII between 1,000 and 20,000 ng/ml FVIII inhibited memory-B-cell-restimulation. These results indicate that the dose-dependent effect of FVIII on the restimulation of FVIII-specific memory B cells does not depend on the genetic background. The major difference between both hemophilic mouse strains was the amplitude of the anti-FVIII immune response. Peak titers of anti-FVIII antibodies and peak concentrations of anti-FVIII antibody secreting plasma cells in spleen and bone marrow were significantly higher in E17 C57BL/6J mice than in E17 Balb/c mice. Whether or not higher ELISA titers correlate with higher Bethesda titers of neutralizing antibodies is currently being investigated. Despite the substantial differences in the amplitude of the immune response, anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells persisted for the whole observation period of 12 months after the last dose of FVIII in both mouse strains. We conclude that the amplitude of the anti-FVIII immune response in hemophilic mice is significantly different between E17 C57BL/6J and E17 Balb/c mice. However, the persistence of the immune response is comparable.

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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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Polyreactivity and Somatic Hypermutation Analysis Reveals the Innate B Cell Origin of Human PF4/Heparin Reactive Antibodies

Blood ◽

10.1182/blood-2020-138781 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 34-35

Author(s):

Wen Zhu ◽

Lu Zhou ◽

Ting Zhao ◽

Yongwei Zheng ◽

Mei Yu ◽

...

Keyword(s):

Immune Response ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Healthy Subjects ◽

Somatic Hypermutation ◽

Memory B Cells ◽

Private Company ◽

Platelet Factor ◽

Polyreactive Antibodies

Heparin-induced thrombocytopenia (HIT) is a serious reaction to heparin treatment characterized by antibodies that recognize a complex formed between heparin and platelet factor 4 (PF4/H) and are capable of activating platelets and inducing a pro-thrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset of these antibodies are platelet-activating (pathogenic) and capable of causing HIT. We previously reported that we cloned B cells from six patients experiencing HIT and identified two types of PF4/H-binding antibodies: seven platelet-activating (PA) and 48 non-activating (NA). Comparison of the structural features in the PA, NA, and non PF4/H-binding (NB) clones showed that the length and the number of basic amino acid and tyrosine residue in the heavy chain complementarity determining region 3 (HCDR3) were significantly different, and was in the order of PA>NA>NB. Most significantly, the seven platelet-activating antibodies each have one of the two pathogenic motifs: RX1-2 R/KX1-2 R/H and YYYYY in an unusually long HCDR3 (≥ 20 residues). In the current study, we attempt to understand the origin of the B cells that produce the PA and NA antibodies and the nature of the immune response in HIT through analyzing somatic hypermutation and biological property of such antibodies. Longer HCDR3 and more basic Aas and Tyr residues in the HCDR3 are features of autoreactive and polyreactive antibodies. With this in mind, we tested PA and NA clones in a standard antinuclear antibody (ANA) assay and found that these clones were significantly more reactive than NB antibodies, and the plasma of HIT patients were significantly more reactive than normal plasma (Figure1). We then compared reactions of PA, NA and NB clones against a group of self and foreign antigens commonly used in polyreactivity assays: dsDNA, ssDNA, LPS, insulin, and keyhole limpet hemocyanin (KLH). About 90% of PA and NA clones were reactive to at least two antigens, this was true of only 20% of the NB clones, and the latter is consistent with the frequency of polyreactive clones in the IgG+ B cells (Figure2). Taken together, these data indicate that PA and NA antibodies are largely polyreactive. We then investigated the development of the PA and NA B cells through analyzing somatic hypermutation in the antibodies. Through analyzing the HCDR3 nucleotide insertion, trimming and VDJ segment usage, we found that longer HCDR3 typical of PF4/H-binding clones and the RKH and Y5 motifs identified in PA clones were the result of original recombination not somatic hypermutation. Consistently, the average number of nucleotide mutations in the VH genes of the binding clones was lower (PA and NA, 9.4 ± 9.5) compared to that of peripheral blood IgG+ memory B cells in healthy subjects (~18) (Figure3). Total mutation frequency in the VH and Vk CDRs of the PF4/H-binding PA and NA clones was comparable to that of the framework regions. This finding contrasts with findings made in peripheral blood IgG+ memory B cells of healthy subjects showing that the mutation frequencies are much higher in the CDRs than in the FRs of VH. Taken together, these findings suggest that affinity maturation plays a limited role in the evolution PF4/H-binding antibodies during the immune response that leads to HIT. In this study, we showed thay PF4/H-binding PA and NA IgGs are largely polyreactive antibodies and contain lower levels of mutations compared to IgG+ memory B cells. B1 and MZ B cells are innate B cells that are main producers of polyreactive natural antibodies and can respond to toll-like receptor signaling, quickly differentiate into antibody-secreting cells, and undergo IgG class switch extrafollicularly. Polyreactivity identified in the PF4/H-binding PA and NA IgGs supports the possibility that human B cells producing PF4/H-binding antibodies are innate B cells akin to MZ B cells shown to be a source of PF4/H antibodies in mice. A mutation rate lower than that of IgG+ memory cells in the PF4/H-binding IgGs is also consistent with an extrafollicular response typical of innate B cells. These observations would help to improve our understanding of the immunological responses and B cell origin in HIT patients. Disclosures Padmanabhan: Retham Technologies: Current equity holder in private company; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Versiti Blood Research Institute: Patents & Royalties.

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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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An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization

Blood ◽

10.1182/blood-2009-07-235960 ◽

2009 ◽

Vol 114 (25) ◽

pp. 5173-5181 ◽

Cited By ~ 144

Author(s):

Michel Jourdan ◽

Anouk Caraux ◽

John De Vos ◽

Geneviève Fiol ◽

Marion Larroque ◽

...

Keyword(s):

B Cells ◽

In Vitro Model ◽

Plasma Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Memory B Cells ◽

Vitro Model ◽

Intermediate Cells ◽

Activated B Cells

Abstract Human plasma cells (PCs) and their precursors play an essential role in humoral immune response but are rare and difficult to harvest. We report the generation of human syndecan-1+ and immunoglobulin secreting PCs starting from memory B cells in a 3-step and 10-day (D) culture, including a 6-fold cell amplification. We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells (activated B cells and plasmablasts) compared with memory B cells and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). We show this B cell–to-PC differentiation to involve IRF4 and AICDA expressions in D4 activated B cells, decrease of PAX5 and BCL6 expressions, and increase in PRDM1 and XBP1 expressions in D7 plasmablasts and D10 PCs. It involves down-regulation of genes controlled by Pax5 and induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). The detailed phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study PC dyscrasias, including multiple myeloma.

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Genome-Wide Histone Acetylation Profiling in Chronic Lymphocytic Leukemia Reveals a Distinctive Signature in Stereotyped Subset #8

Blood ◽

10.1182/blood-2019-127817 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1241-1241

Author(s):

Maria Tsagiopoulou ◽

Vicente Chapaprieta ◽

Nuria Russiñol ◽

Fotis Psomopoulos ◽

Nikos Papakonstantinou ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Research Funding ◽

Plasma Cells ◽

Somatic Hypermutation ◽

Memory B Cells ◽

Site Analysis ◽

Total Variability ◽

Cell Subpopulations ◽

Chromatin Acetylation

In CLL, subsets of patients carrying stereotyped B cell receptors (BcR) share similar biological and clinical features independently of IGHV gene somatic hypermutation status. Although the chromatin landscape of CLL as a whole has been recently characterized, it remains largely unexplored in stereotyped cases. Here, we analyzed the active chromatin regulatory landscape of 3 major CLL stereotyped subsets associated with clinical aggressiveness. We performed chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) with an antibody for the H3K27ac histone mark in sorted CLL cells from 19 cases, including clinically aggressive subsets #1 (clan I genes/IGKV(D)1-39, IG-unmutated CLL (U-CLL)(n=3)], #2 [IGHV3-21/IGLV3-21, IG-mutated CLL (M-CLL)(n=3)] and #8 [IGHV4-39/IGKV1(D)-39, U-CLL(n=3)] which we compared to non-stereotyped CLL cases [5 M-CLL|5 U-CLL]. In addition, a series of 15 normal B cell samples from different stages of B-cell differentiation were analyzed [naive B cells from peripheral blood (n=3), tonsillar naive B cells (n=3), germinal centre (GC) B cells (n=3), memory B cells (n=3), tonsillar plasma cells (n=3)]. Initial unsupervised principal component analysis (PCA) disclosed a distinct chromatin acetylation pattern in CLL, regardless of stereotypy status, versus normal B cells. CLL as a whole was found to be closer to naive and memory B cells rather than GC B cells and plasma cells. Detailed analysis of individual principal components (PC) revealed that PC4, which accounts for 5% of the total variability, segregated subset #8 cases and GC B cells from other CLLs and normal B cell subpopulations. Although PC4 accounts for only a small part of the total variability (5%), this suggests that subset #8 cases may share some chromatin features with proliferating GC B cells, in line with the fact that subset #8 BcR are IgG-switched. We also investigated whether stereotyped CLLs have different chromatin acetylation features compared to non-stereotyped CLLs matched by IGHV somatic hypermutation status and identified 878 Differential Regions (DR) in subset #8 vs. U-CLL, 84 DR in subset #1 vs. U-CLL and 66 DR in #2 compared vs. M-CLL. As subset #8 cases seemed to have the most distinct profile, we further characterized the detected regions. The 435 and 443 regions gaining and losing activation, respectively, mostly targeted promoters (29.5%) and regulatory elements located in introns (31%) and distal intergenic regions (21.8%). Hierarchical clustering based on the 878 DRs enabled the clear discrimination of subset #8 cases from U-CLL and normal B cells; however, it is worth noting that for several of these 878 DRs the acetylation patterns were shared between subset #8 and normal B cell subpopulations rather than subset #8 and U-CLL. Of note, 11/435 regions gaining activity on subset #8 were found within the gene encoding for the EBF1 transcription factor (TF); additional regions were associated with genes significant to CLL pathogenesis, e.g. TCF4 and E2F1. Moreover, 3 DRs losing activity in subset #8 were located within the CTLA4 gene and 2 DRs within the IL21R gene, which we have recently reported as hypermethylated and not expressed in subset #8. Next, we performed TF binding site analysis by MEME/AME suit, separately for regions gaining or losing activity, and identified significant enrichment (adj-p<0.001) on TFs such as AP-1, FOX, GATA, IRF. The regions losing activity in subset #8 showed a higher number of enriched TFs versus those gaining activity (165 vs 93 TFs), particularly displaying enrichment for many HOX family members . However, a cluster of TFs with enrichment on TF binding site analysis, such as FOXO1, FOXP1, MEF2D, PRDM1, RUNX1, RXRA, STAT6, were also located within the 878 DRs discriminating subset #8 from either U-CLL or normal B cell subpopulations. Taken together, subset #8 cases have a distinct chromatin acetylation signature which includes both loss and gain of active elements, shared features with proliferating GC B cells, and specific changes in chromatin activity of several genes and TFs relevant to B cell/CLL biology. These findings further underscore the concept that BcR stereotypy defines subsets of patients with consistent biological profile, while they may also be relevant to the particular clinical behavior of subset #8, known to be associated with the highest risk of Richter's transformation amongst all CLL. Disclosures Stamatopoulos: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

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EBV Can Induce Somatic Hypermutation in Naïve B Cells In Vitro but Ig Class Switching Requires T Cell Help.

Blood ◽

10.1182/blood.v108.11.2370.2370 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2370-2370

Author(s):

Sridhar Chaganti ◽

Noelia Begue Pastor ◽

Gouri Baldwin ◽

Claire Shannon-Lowe ◽

Regina Feederle ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germ Line ◽

Somatic Hypermutation ◽

Memory B Cells ◽

Class Switching ◽

B Cell Subsets ◽

T Cell Help

Abstract Following primary infection, Epstein-Barr virus (EBV) establishes life long persistence in the host IgD− CD27+ memory B cell compartment rather than the IgD+ CD27+ marginal zone (MZ)-like or the IgD+ CD27− naïve B cell compartments. One possible explanation for such exclusive persistence in memory B cells is that EBV preferentially infects memory B cells. Alternatively, the virus may infect all B cell subsets but then drive MZ and naïve B cells to acquire the Ig isotype-switched phenotype and hypermutated Ig genotype of memory cells. Here we ask whether there is any evidence for one or other hypothesis from in vitro experiments. B cells from healthy donor blood samples were FACS sorted on the basis of IgD/CD27 expression into naïve, MZ, and memory B cell subsets with purities of >99%, >97% and >98% respectively. Analysis of the IgVH sequence further confirmed purity of the FACS sorted B cell subsets. Accordingly, 102 of 105 IgVH sequences amplified from purified naïve B cells were germ-line where as the vast majority of sequences amplified from MZ and memory B cells were mutated. All three B cell subsets expressed equal amounts of CD21 (EBV receptor on B cells), bound similar amounts of virus, and transformed with equal efficiency to establish B lymphoblastoid cell lines (LCLs) in vitro. Naïve B cell transformants upregulated CD27 expression but retained the IgM+, IgD+ phenotype as determined by FACS analysis and RT-PCR; MZ-B derived LCLs likewise were IgM+, IgD+, CD27+; and memory-B derived LCLs were consistently CD27+, IgD− and expressed either IgG, IgA or in some cases IgM. Therefore, EBV infection per se did not induce class switching. However, both naïve and MZ-B derived LCLs could still be induced to switch to IgG in the presence of CD40 ligand and IL-4; signals that are normally provided by T cells in vivo. To assess if EBV infection might drive Ig hypermutation, we carried out IgVH sequence analysis on the naïve-B derived LCL clones. Interestingly, 42 of 114 clonal IgVH sequences amplified from naïve-B derived LCLs had 3 or more mutations and the patterns of mutation seen were consistent with that produced by somatic hypermutation (SHM). Furthermore, within some naïve-B cell derived LCL clones, there were both germ-line and mutated sequences all sharing the same VDJ rearrangement (CDR3 sequence), again implying sequence diversification following EBV transformation of a single naïve B cell. Some intraclonal variation of the already hypermutated IgVH sequence was also noted in memory and MZ-B derived LCLs further suggesting ongoing mutational activity. Consistent with this, activation-induced cytidine deaminase (AID) expression was upregulated in transformants as assessed by real time RT-PCR. Our in vitro data is therefore compatible with a model of EBV persistence where the virus infects all mature B cell subsets but then drives infected naïve B cells to acquire a memory genotype by inducing SHM. In addition, EBV infected naïve and MZ-B cells may undergo Ig class switching to acquire the IgD− CD27+ memory phenotype in the presence of T cell help in vivo. EBV’s ability to induce SHM may also contribute to the lymphomagenic potential of the virus in addition to its B cell transforming and growth promoting properties.

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IgM+ B-Cells Lacking CD27 Expression Display Somatically Mutated Ig VH Genes and Define a New Memory Population.

Blood ◽

10.1182/blood.v110.11.2630.2630 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2630-2630

Author(s):

Nicola J. Weston-Bell ◽

Mark R. Townsend ◽

Francesco Forconi ◽

Freda K. Stevenson ◽

Surinder S. Sahota

Keyword(s):

B Cells ◽

B Cell ◽

Plasma Cells ◽

Somatic Hypermutation ◽

Gene Mutations ◽

Memory B Cells ◽

Cell Leukaemia ◽

Germline Gene ◽

Cell Of Origin ◽

Class Switch

Abstract Somatic hypermutation (SHM) and isotype class switch recombination are critical mechanisms that diversify normal B-cells in response to antigen, and are generally confined to the germinal center (GC). Post-GC B-cells differentiate either into memory B-cells or antibody forming cells maturing as plasma cells. Memory B-cells which exit the GC have been regarded as invariably expressing CD27 and this population comprises both IgM+ and switched B-cells. It is, however, the presence of SHM that remains the defining feature of memory status in B-cells. Recently, it has been reported that dye extruding membrane ATP-binding transporters expressed by naive B-cells can distinguish them from memory B-cells which lack transporters and this, notably, led to the identification of IgG+ memory B-cells that did not express CD27. However, these observations were not correlated with SHM status. Here, we have examined these findings further and focused specifically on IgM+D+ B-cells in relation to our interest in mapping origins of B-cell tumors. Using dye-based transporter assays, we identified a substantial IgM+CD27− population in PBMNC from healthy individuals which lacked transporter activity. These cells were next isolated using a different strategy, employing IgD and CD27 expression, and a highly purified (>99%) IgD+CD27− fraction obtained by FACS sorting. There was virtually nil contamination by CD27+ B-cells. IgM transcripts were then specifically analyzed in this CD27− population for VH gene mutations by using Cmu downstream primers. We examined VH3 and VH5 gene transcripts from 2 separate sorts from a healthy donor following amplification by primary and nested RT-PCR and cloning products for sequence analysis. Of 104 VH3 clones from this CD27− fraction, 38 (37%) were germline and the remainder mutated (55% with 98–99.9% homology to germline, 9% <97.9%). Of these, 27% displayed 3–5 or more nucleotide mutations. Analysis of 124 VH5 clones from the same fraction showed 50% to be germline and the remainder mutated (45% with 98–99.9% homology, 5% <97.9%), of which 16% exhibited 3–5 or more mutations. Only 1 VH5 donor germline gene exists in the IgH repertoire, but 4 allelic variants which differ by 1 nucleotide are known. We mapped the VH5 germline gene in our donor using recombination sequence primers and only 1 allele was identified, identical to the donor gene mapped by database alignment, eliminating polymorphisms as a source of apparent base changes. Mutational load was also over and above a low level of PCR error which was quantified in the analysis. This confirmed that the levels of mutation detected in the rearranged VH5 transcripts in the IgM+D+CD27− population were generated by SHM. In contrast, between 74–90% of VH3 and VH5 transcripts from the IgM+D−CD27+ population were mutated, with a higher frequency of sequences (65–87%) displaying 3–5 or more mutations. Our data reveal the existence of a novel IgM+D+ population of normal B-cells which are somatically mutated but lack CD27. Unmutated IgM+D+ B-cells in this population point to heterogeneity in the CD27− B-cell pool. As yet, the clonal derivation of these mutated CD27− cells is undefined. Importantly, they have relevance for understanding the cell of origin in Hairy Cell leukaemia and Waldenstrom’s macroglobulinemia, where tumor cells are mutated yet lack CD27 expression.

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Recovery of Memory B-cell Subsets and Persistence of Antibodies in Convalescent COVID-19 Patients

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.21-0883 ◽

2021 ◽

Author(s):

Anuradha Rajamanickam ◽

Nathella Pavan Kumar ◽

Arul Nancy P ◽

Nandhini Selvaraj ◽

Saravanan Munisankar ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Neutralizing Antibodies ◽

Plasma Cells ◽

Ex Vivo ◽

Cell Subset ◽

Memory B Cells ◽

Memory B Cell ◽

Immature B Cells ◽

B Cell Subsets

It is essential to examine the longevity of the defensive immune response engendered by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. We examined the SARS-CoV-2-specific antibody responses and ex vivo memory B-cell subsets in seven groups of individuals with COVID-19 classified based on days since reverse-transcription polymerase chain reaction confirmation of SARS-CoV-2 infection. Our data showed that the levels of IgG and neutralizing antibodies started increasing from days 15 to 30 to days 61 to 90, and plateaued thereafter. The frequencies of naive B cells and atypical memory B cells decreased from days 15 to 30 to days 61 to 90, and plateaued thereafter. In contrast, the frequencies of immature B cells, classical memory B cells, activated memory B cells, and plasma cells increased from days 15 to 30 to days 61 to 90, and plateaued thereafter. Patients with severe COVID-19 exhibited increased frequencies of naive cells, atypical memory B cells, and activated memory B cells, and lower frequencies of immature B cells, central memory B cells, and plasma cells when compared with patients with mild COVID-19. Therefore, our data suggest modifications in memory B-cell subset frequencies and persistence of humoral immunity in convalescent individuals with COVID-19.

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Mirna Profiling Reveals Specific Patterns for Normal B Cell Subsets and B Cell Lymphomas with a Unique Burkitt Lymphoma Profile

Blood ◽

10.1182/blood.v112.11.3763.3763 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3763-3763

Author(s):

Jan-Lukas Robertus ◽

Geert Harms ◽

Rogier Reijmers ◽

Steven Pals ◽

Yolanthe Swart ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Hierarchical Clustering ◽

Burkitt Lymphoma ◽

Plasma Cells ◽

Memory B Cells ◽

Fold Change ◽

Differentially Expressed ◽

Differentially Expressed Mirnas ◽

B Cell Subsets

Abstract Abstract: MicroRNAs (miRNAs) are small (22–23nt) noncoding RNAs, which negatively regulate gene expression by inhibiting protein translation. MiRNAs play an important role in various cellular processes such as hematopoiesis. Deregulated expression is associated with development of B-cell lymphomas. The aim of this study was to define miRNA expression profiles to identify differentially expressed miRNAs in normal B cell subsets and malignancies derived from these subsets. Using Agilent Human miRNA microarrays the expression levels of 556 miRNAs were determined in 7 Mantle cell lymphoma (MCL), 7 Follicular lymphoma (FL), 7 paediatric Burkitt lymphoma (BL) and 13 Chronic lymphocytic lymphoma (CLL; 8 ZAP70 pos and 5 ZAP70 neg) cases, as well as in naïve, germinal center (GC), memory B cells and plasma cells obtained from 3 paediatric tonsils. Median normalisation was performed on all array data using GeneSpring GX 9.0.5. Differentially expressed miRNAs were defined by at least a four fold difference using ANOVA (p<0.05). Quantitive (q)RT-PCR was used for validation of the arrays. In the normal B-cell subsets 23 differentially expressed miRNAs were found with a four fold difference. Hierarchical clustering revealed a distinct pattern of these subsets. Specifically in GC B cells miR-15b, miR-17-5p and its seed family member miR- 106a were upregulated in comparison to the naïve, memory B cells and plasma cells. MiR-150, miR-221 and miR-222 were downregulated. Only miR-146a was significantly differentially expressed between naïve and memory B cell subsets. In the lymphoma cases a total of 59 miRNAs were differentially expressed. Hierarchical clustering of these miRNAs grouped all four lymphoma entities separately. ZAP70 positive and negative CLL cases did not cluster separately and none of the miRNAs were differentially expressed. Interestingly, 35 miRNAs were specifically deregulated in the Burkitt lymphomas as compared to MCL, FL and CLL, including miR-18a (up), miR-29a, miR-150, miR-155 and miR-222 (down). Almost all of these miRNAs were found not to be differentially expressed within the normal B cell subsets suggesting that these miRNAs might play a specific role in the biologic behaviour of BL. Comparison of BL to normal GC B cells revealed 15 miRNAs upregulated, including miR-126 and miR-145 (fold change >40), and 79 miRNAs downregulated, including miR-150, miR-155, miR-15, miR-16, miR- 17-5p, miR-21 and miR-222. In comparison with GC B cells FL showed 11 upregulated miRNAs, e.g. miR-143 and miR-145 (fold change >40) and 49 downregulated miRNAs. In comparison with memory B cells 21 miRNAs in the CLL group were upregulated, including miR-143 and miR-145 (fold change > 40) and 83 miRNAs were downregulated. Finally MCL was compared to the naïve B cells and showed 23 upregulated miRNAs with miR-126 showing the highest fold change (>40) and 70 downregulated miRNAs. In conclusion, we have identified specific miRNA profiles for MCL, BL, FL and CLL and also for normal B cell subsets. The differentially expressed miRNA profiles contain several miRNAs that have been shown to be directly or indirectly involved in B cell malignancies but also contain new potentially interesting miRNAs. We also have found several miRNAs that may play a tumor specific role in BL and it can be speculated that miRNAs contained in this profile may target genes related to the more aggressive clinical phenotype of BL.

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