scholarly journals Flice-Inhibitory Protein Is a Key Regulator of Germinal Center B Cell Apoptosis

2001 ◽  
Vol 193 (4) ◽  
pp. 447-458 ◽  
Author(s):  
Ana Hennino ◽  
Marion Bérard ◽  
Peter H. Krammer ◽  
Thierry Defrance
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Apoptosis ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Variable Region ◽  
Affinity Maturation ◽  
Death Domain ◽  
Cell Repertoire ◽  
Inhibitory Protein

Affinity maturation of the B cell response to antigen (Ag) takes place in the germinal centers (GCs) of secondary follicles. Two sequential molecular mechanisms underpin this process. First, the B cell repertoire is diversified through hypermutation of the immunoglobulin (Ig) variable region genes. Second, mutant B cell clones with improved affinity for Ag are positively selected by Ag and CD40 ligand (L). This selection step is contingent upon “priming” of GC B cells for apoptosis. The molecular means by which B cell apoptosis is initiated and controled in the GC remains unclear. Here, we show that GC B cell apoptosis is preceded by the rapid activation of caspase-8 at the level of CD95 death-inducing signaling complex (DISC). We found that GC B cells ex vivo display a preformed inactive DISC containing Fas-associated death domain–containing protein (FADD), procaspase-8, and the long isoform of cellular FADD-like IL-1β–converting enzyme-inhibitory protein (c-FLIPL) but not the CD95L. In culture, c-FLIPL is rapidly lost from the CD95 DISC unless GC B cells are exposed to the survival signal provided by CD40L. Our results suggest that (a) the death receptor signaling pathway is involved in the affinity maturation of antibodies, and (b) c-FLIPL plays an active role in positive selection of B cells in the GC.

scholarly journals Reprogramming the antigen specificity of B cells using genomeediting technologies

2018 ◽  
Author(s):  
James E. Voss ◽  
Alicia Gonzalez-Martin ◽  
Raiees Andrabi ◽  
Roberta P. Fuller ◽  
Ben Murrell ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cytidine Deaminase ◽  
Variable Region ◽  
Neutralizing Antibody ◽  
Surface Expression ◽  
Affinity Maturation ◽  
Cell Surface Expression ◽  
Human Antibody ◽  
Antigen Specificity

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody, PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved anti-HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses.

Bim establishes the B-cell repertoire from early to late in the immune response

2020 ◽  
Author(s):  
Akiko Sugimoto-Ishige ◽  
Michishige Harada ◽  
Miho Tanaka ◽  
Tommy Terooatea ◽  
Yu Adachi ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Affinity Maturation ◽  
Late Response ◽  
Cell Repertoire ◽  
High Affinity ◽  
B Cell Repertoire

Abstract In T cell-dependent antibody responses, some of the activated B cells differentiate along extrafollicular pathways into low-affinity memory and plasma cells, whereas others are involved in subsequent germinal center (GC) formation in follicular pathways, in which somatic hypermutation and affinity maturation occur. The present study demonstrated that Bim, a proapoptotic BH3-only member of the Bcl-2 family, contributes to the establishment of the B-cell repertoire from early to late stages of immune responses to T cell-dependent antigens. Extrafollicular plasma cells grew in the spleen during the early immune response, but their numbers rapidly declined with the appearance of GC-derived progeny in wild-type mice. By contrast, conditional Bim deficiency in B cells resulted in expansion of extrafollicular IgG1+ antibody-forming cells (AFCs) and this expansion was sustained during the late response, which hampered the formation of GC-derived high-affinity plasma cells in the spleen. Approximately 10% of AFCs in mutant mice contained mutated VH genes; thus, Bim deficiency appears not to impede the selection of high-affinity AFC precursor cells. These results suggest that Bim contributes to the replacement of low-affinity antibody by high-affinity antibody as the immune response progresses.

scholarly journals Excessive CD11c+Tbet+B cells promote aberrant TFHdifferentiation and affinity-based germinal center selection in lupus

2019 ◽  
Vol 116 (37) ◽  
pp. 18550-18560 ◽  
Author(s):  
Wenqian Zhang ◽  
Huihui Zhang ◽  
Shujun Liu ◽  
Fucan Xia ◽  
Zijian Kang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Molecular Mechanisms ◽  
Affinity Maturation ◽  
Antibody Affinity ◽  
Autoantibody Production ◽  
Cell Responses ◽  
Antigen Presenting

Excessive self-reactive and inadequate affinity-matured antigen-specific antibody responses have been reported to coexist in lupus, with elusive cellular and molecular mechanisms. Here, we report that the antigen-specific germinal center (GC) response―a process critical for antibody affinity maturation―is compromised in murine lupus models. Importantly, this defect can be triggered by excessive autoimmunity-relevant CD11c+Tbet+age-associated B cells (ABCs). In B cell-intrinsic Ship-deficient (ShipΔB) lupus mice, excessive CD11c+Tbet+ABCs induce deregulated follicular T-helper (TFH) cell differentiation through their potent antigen-presenting function and consequently compromise affinity-based GC selection. Excessive CD11c+Tbet+ABCs and deregulated TFHcell are also present in other lupus models and patients. Further, over-activated Toll-like receptor signaling in Ship-deficient B cells is critical for CD11c+Tbet+ABC differentiation, and blocking CD11c+Tbet+ABC differentiation in ShipΔB mice by ablating MyD88 normalizes TFHcell differentiation and rescues antigen-specific GC responses, as well as prevents autoantibody production. Our study suggests that excessive CD11c+Tbet+ABCs not only contribute significantly to autoantibody production but also compromise antigen-specific GC B-cell responses and antibody-affinity maturation, providing a cellular link between the coexisting autoantibodies and inadequate affinity-matured antigen-specific antibodies in lupus models and a potential target for treating lupus.

scholarly journals Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues

2004 ◽  
Vol 201 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Ki-Jong Rhee ◽  
Paul J. Jasper ◽  
Periannan Sethupathi ◽  
Malathy Shanmugam ◽  
Dennis Lanning ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Intestinal Microflora ◽  
Molecular Mechanisms ◽  
Amino Acid Sequences ◽  
Lymphoid Tissues ◽  
Antibody Repertoire ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
The Relationship

Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express VHn allotype immunoglobulin (Ig)M. Within weeks, the number of VHn B cells decreases, whereas VHa allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from VHn to VHa B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the VHn to VHa repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of VHa B cells. By comparing amino acid sequences of VHn and VHa Ig, we identified a putative VH ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with VHa B cells results in their selective expansion.

scholarly journals Ex vivo characterization and isolation of rare memory B cells with antigen tetramers

Blood ◽  
2011 ◽  
Vol 118 (2) ◽  
pp. 348-357 ◽  
Author(s):  
Bettina Franz ◽  
Kenneth F. May ◽  
Glenn Dranoff ◽  
Kai Wucherpfennig
Keyword(s):  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Memory B Cells ◽  
Cell Repertoire ◽  
Low Frequencies

Abstract Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B-cell repertoire can be missed when monomeric antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell sorted tetramer+ B cells with single-cell RT-PCR of the B-cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.

scholarly journals Highly sensitive and unbiased approach for elucidating antibody repertoires

2016 ◽  
Vol 113 (28) ◽  
pp. 7846-7851 ◽  
Author(s):  
Sherry G. Lin ◽  
Zhaoqing Ba ◽  
Zhou Du ◽  
Yu Zhang ◽  
Jiazhi Hu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germ Line ◽  
Variable Region ◽  
Cell Receptor ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
Variable Regions ◽  
Antibody Repertoires ◽  
B Lineage

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes.

scholarly journals A synthetic stroma-free germinal center niche using material-surface driven polyvalent signaling efficiently induces antigen-specific humoral immunity ex vivo

2017 ◽  
Author(s):  
Kyung-Ho Roh ◽  
Hannah K. Wilson ◽  
Pallab Pradhan ◽  
Kevin Bai ◽  
Caitlin D. Bohannon ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Antigen Presentation ◽  
Cell Population ◽  
Germinal Center ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Affinity Maturation ◽  
Material Surface ◽  
Peripheral Blood Mononuclear

AbstractB cells play a major role in the adaptive immune response by producing antigen-specific antibodies against pathogens and imparting immunological memory. Following infection or vaccination, antibody-secreting B cells and memory B cells are generated in specialized regions of lymph nodes and spleens, called germinal centers. Here, we report a fully synthetic ex-vivo system that recapitulates the generation of antigen-specific germinal-center (GC) like B cells using material-surface driven polyvalent signaling. This synthetic germinal center (sGC) reaction was effectively induced using biomaterial-based artificial “follicular T helper cells (TFH)” that provided both natural CD40-CD40L ligation as well as crosslinking of CD40; and by mimicking artificial “follicular dendritic cells (FDC)” to provide efficient, polyvalent antigen presentation. The artificial sGC reaction resulted in efficient B cell expansion, immunoglobulin (Ig) class switching, and expression of germinal center phenotypes. Antigen presentation during sGC reaction selectively enhanced the antigen-specific B cell population and induced somatic hyper-mutations for potential affinity maturation. The resulting B cell population consisted primarily of GC-like B cells (centrocytes) as well as some plasma-like B cells expressing CD138. With concurrent cell sorting, we successfully created highly enriched populations of antigen-specific B cells. Adoptive transfer of these GC-like B cells into non-irradiated isogeneic or non-lethally irradiated congenic recipient mice showed successful engraftment and survival of the donor cells for the 4 week test period. We show that this material-surface driven sGC reaction can be successfully applied to not only splenic B cells but also B cells isolated from more therapeutically relevant sources such as peripheral blood mononuclear cells (PBMCs), thus making our current work an exciting prospect in the new era of personalized medicine and custom-immunotherapy.

Expression of the AID protein in normal and neoplastic B cells

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3318-3325 ◽  
Author(s):  
Laura Pasqualucci ◽  
Roberta Guglielmino ◽  
Jane Houldsworth ◽  
Jessica Mohr ◽  
Said Aoufouchi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Variable Region ◽  
Affinity Maturation ◽  
Lymphocytic Leukemia ◽  
Antibody Affinity ◽  
Disease Subtype ◽  
Activation Induced Cytidine Deaminase

Abstract Somatic hypermutation (SHM) targets primarily the immunoglobulin variable region (IgV) genes in germinal center (GC) B cells, thereby allowing antibody affinity maturation. A malfunction of SHM, termed aberrant somatic hypermutation (ASHM), was found in about 50% of diffuse large B-cell lymphomas (DLBCLs), leading to mutations in the 5′ sequences of multiple genes, including oncogenes. Although the SHM mechanism is largely unknown, it was shown to require the activation-induced cytidine deaminase (AID) gene. AID mRNA is expressed in GC B cells and GC-derived lymphomas, but the pattern of expression of the AID protein is not known. Using 2 specific antibodies, here we show that the AID protein can be detected in GC centroblasts and their transformed counterpart (Burkitt lymphoma) but not in pre-GC B cells and post-GC neoplasms, including B-cell chronic lymphocytic leukemia and multiple myeloma. DLBCLs displayed variable levels of AID expression, which did not correlate with IgV ongoing hypermutation, ASHM, or disease subtype. Finally, both in normal and malignant B cells the AID protein appeared predominantly localized in the cytoplasm. These results indicate that the AID protein is specifically expressed in normal and transformed GC B cells; nonetheless, its predominantly cytoplasmic localization suggests that additional mechanisms may regulate its function and may be altered during lymphomagenesis. (Blood. 2004;104:3318-3325)

scholarly journals Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses

2019 ◽  
Vol 116 (19) ◽  
pp. 9511-9520 ◽  
Author(s):  
Hongsheng Wang ◽  
Shweta Jain ◽  
Peng Li ◽  
Jian-Xin Lin ◽  
Jangsuk Oh ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
Cell Development ◽  
Affinity Maturation

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.

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