scholarly journals Reprogramming the antigen specificity of B cells using genome-editing technologies

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
James E Voss ◽  
Alicia Gonzalez-Martin ◽  
Raiees Andrabi ◽  
Roberta P Fuller ◽  
Ben Murrell ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Genome Editing ◽  
Cytidine Deaminase ◽  
Variable Region ◽  
Neutralizing Antibody ◽  
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 (Ig) 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 (bnAb), 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 (LCs) 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 HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.

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.

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 Distinct clonal evolution of B-cells in HIV controllers with neutralizing antibody breadth

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Deniz Cizmeci ◽  
Giuseppe Lofano ◽  
Evan Rossignol ◽  
Anne-Sophie Dugast ◽  
Dongkyoon Kim ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Somatic Hypermutation ◽  
Clonal Evolution ◽  
Neutralizing Antibody ◽  
Affinity Maturation ◽  
Gene Repertoire ◽  
Immunoglobulin Gene ◽  
Hiv Controllers ◽  
Hiv 1

A minor subset of individuals infected with HIV-1 develop antibody neutralization breadth during the natural course of the infection, often linked to chronic, high-level viremia. Despite significant efforts, vaccination strategies have been unable to induce similar neutralization breadth and the mechanisms underlying neutralizing antibody induction remain largely elusive. Broadly neutralizing antibody responses can also be found in individuals who control HIV to low and even undetectable plasma levels in the absence of antiretroviral therapy, suggesting that high antigen exposure is not a strict requirement for neutralization breadth. We therefore performed an analysis of paired heavy and light chain B-cell receptor (BCR) repertoires in 12,591 HIV-1 envelope-specific single memory B-cells to determine alterations in the BCR immunoglobulin gene repertoire and B-cell clonal expansions that associate with neutralizing antibody breadth in 22 HIV controllers. We found that the frequency of genomic mutations in IGHV and IGLV was directly correlated with serum neutralization breadth. The repertoire of the most mutated antibodies was dominated by a small number of large clones with evolutionary signatures suggesting that these clones had reached peak affinity maturation. These data demonstrate that even in the setting of low plasma HIV antigenemia, similar to what a vaccine can potentially achieve, BCR selection for extended somatic hypermutation and clonal evolution can occur in some individuals suggesting that host-specific factors might be involved that could be targeted with future vaccine strategies.

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 Acetyltransferases GCN5 and PCAF are Required for B Cell Maturation in Mice

2021 ◽  
Author(s):  
Valentyn Oksenych
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
Cytidine Deaminase ◽  
Dna Recombination ◽  
Variable Region ◽  
Open Chromatin ◽  
Spleen And Lymph Nodes

B lymphocyte development includes two DNA recombination processes, the V(D)J recombination of immunoglobulin (Igh) gene variable region and class switching when the Igh constant regions are changed from IgM to IgG, IgA, or IgE. The V(D)J recombination is required for successful maturation of B cells from pro-B to pre-B to immature-B and then to mature B cells in the bone marrow. The CSR occurs outside the bone marrow when mature B cells migrate to peripheral lymphoid organs, such as spleen and lymph nodes. Both V(D)J recombination and CSR depend on an “open chromatin” state that makes DNA accessible to specific enzymes, recombination activating gene (RAG), and activation-induced cytidine deaminase (AID). Acetyltransferases GCN5 and PCAF possess redundant functions acetylating histone H3 lysine 9 (H3K9). Here, we generated by complex breeding a mouse model with B cells lacking both GCN5 and PCAF. We found that double-deficient mice possess low levels of mature B cells in the bone marrow and peripheral organs, accumulation of pro-B cells in bone marrow, and reduced CSR levels. We concluded that both GCN5 and PCAF are required for B cell development in vivo.

scholarly journals Acetyltransferases GCN5 and PCAF are required for B cell maturation

2021 ◽  
Author(s):  
Valentyn Oksenych
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
Cytidine Deaminase ◽  
Dna Recombination ◽  
Variable Region ◽  
Open Chromatin ◽  
Spleen And Lymph Nodes

B lymphocyte development includes two DNA recombination processes, the V(D)J recombina-tion of immunoglobulin (Igh) gene variable region and class switching when the Igh constant regions are changed from IgM to IgG, IgA, or IgE. The V(D)J recombination is required for suc-cessful maturation of B cells from pro-B to pre-B to immature-B and then to mature B cells in the bone marrow. The CSR occurs outside the bone marrow when mature B cells migrate to periph-eral lymphoid organs, such as spleen and lymph nodes. Both V(D)J recombination and CSR de-pend on an open chromatin state that makes DNA accessible to specific enzymes, recombina-tion activating gene (RAG), and activation-induced cytidine deaminase (AID). Acetyltransferases GCN5 and PCAF possess redundant functions acetylating histone H3 lysine 9 (H3K9). Here, we generated by complex breeding a mouse model with B cells lacking both GCN5 and PCAF. We found that double-deficient mice possess low levels of mature B cells in the bone marrow and peripheral organs, accumulation of pro-B cells in bone marrow, and reduced CSR levels. We concluded that both GCN5 and PCAF are required for B cell development in vivo.

scholarly journals In vitro affinity maturation of broader and more-potent variants of the HIV-1–neutralizing antibody CAP256-VRC26.25

2021 ◽  
Vol 118 (29) ◽  
pp. e2106203118
Author(s):  
Yiming Yin ◽  
Brian D. Quinlan ◽  
Tianling Ou ◽  
Yan Guo ◽  
Wenhui He ◽  
...  
Keyword(s):  
B Cell ◽  
Posttranslational Modifications ◽  
Neutralizing Antibodies ◽  
Cytidine Deaminase ◽  
Neutralizing Antibody ◽  
Affinity Maturation ◽  
Homology Directed Repair ◽  
Activation Induced Cytidine Deaminase ◽  
Hiv 1

Three variable 2 (V2) loops of HIV-1 envelope glycoprotein (Env) trimer converge at the Env apex to form the epitope of an important classes of HIV-1 broadly neutralizing antibodies (bNAbs). These V2-glycan/apex antibodies are exceptionally potent but less broad (∼60 to 75%) than many other bNAbs. Their CDRH3 regions are typically long, acidic, and tyrosine sulfated. Tyrosine sulfation complicates efforts to improve these antibodies through techniques such as phage or yeast display. To improve the breadth of CAP256-VRC26.25 (VRC26.25), a very potent apex antibody, we adapted and extended a B cell display approach. Specifically, we used CRISPR/Cas12a to introduce VRC26.25 heavy- and light-chain genes into their respective loci in a B cell line, ensuring that each cell expresses a single VRC26.25 variant. We then diversified these loci through activation-induced cytidine deaminase–mediated hypermutation and homology-directed repair using randomized CDRH3 sequences as templates. Iterative sorting with soluble Env trimers and further randomization selected VRC26.25 variants with successively improving affinities. Three mutations in the CDRH3 region largely accounted for this improved affinity, and VRC26.25 modified with these mutations exhibited greater breadth and potency than the original antibody. Our data describe a broader and more-potent form of VRC26.25 as well as an approach useful for improving the breadth and potency of antibodies with functionally important posttranslational modifications.

scholarly journals The AKT isoforms 1 and 2 drive B cell fate decisions during the germinal center response

2019 ◽  
Vol 2 (6) ◽  
pp. e201900506 ◽  
Author(s):  
Zilu Zhu ◽  
Ashima Shukla ◽  
Parham Ramezani-Rad ◽  
John R Apgar ◽  
Robert C Rickert
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Germinal Center ◽  
Cytidine Deaminase ◽  
Cell Receptor ◽  
Pi3k Pathway ◽  
Affinity Maturation ◽  
Cell Fate Decisions ◽  
Antibody Class

The PI3K pathway is integral for the germinal center (GC) response. However, the contribution of protein kinase B (AKT) as a PI3K effector in GC B cells remains unknown. Here, we show that mice lacking the AKT1 and AKT2 isoforms in B cells failed to form GCs, which undermined affinity maturation and antibody production in response to immunization. Upon B-cell receptor stimulation, AKT1/2–deficient B cells showed poor survival, reduced proliferation, and impaired mitochondrial and metabolic fitness, which collectively halted GC development. By comparison, Foxo1T24A mutant, which cannot be inactivated by AKT1/2 phosphorylation and is sequestered in the nucleus, significantly enhanced antibody class switch recombination via induction of activation-induced cytidine deaminase (AID) expression. By contrast, repression of FOXO1 activity by AKT1/2 promoted IRF4-driven plasma cell differentiation. Last, we show that T-cell help via CD40, but not enforced expression of Bcl2, rescued the defective GC response in AKT1/2–deficient animals by restoring proliferative expansion and energy production. Overall, our study provides mechanistic insights into the key role of AKT and downstream pathways on B cell fate decisions during the GC response.

scholarly journals Distinct clonal evolution of B-cells in HIV controllers with neutralizing antibody breadth

2020 ◽  
Author(s):  
Deniz Cizmeci ◽  
Giuseppe Lofano ◽  
Anne-Sophie Dugast ◽  
Dongkyoon Kim ◽  
Guy Cavet ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Somatic Hypermutation ◽  
Clonal Evolution ◽  
Neutralizing Antibody ◽  
Affinity Maturation ◽  
Gene Repertoire ◽  
Immunoglobulin Gene ◽  
Hiv Controllers ◽  
Hiv 1

AbstractA minor subset of individuals infected with HIV-1 develop antibody neutralization breadth during the natural course of the infection, often linked to chronic, high level viremia. Despite significant efforts, vaccination strategies have been unable to induce similar neutralization breadth and the mechanisms underlying neutralizing antibody induction remain largely elusive. Broadly neutralizing antibody responses can also be found in individuals who control HIV to low and even undetectable plasma levels in the absence of antiretroviral therapy, suggesting that high antigen exposure is not a strict requirement for neutralization breadth. We therefore performed an analysis of paired heavy and light chain B-cell receptor repertoires in 12,591 HIV-1 Envelope-specific single memory B-cells to determine alterations in the BCR immunoglobulin gene repertoire and B-cell clonal expansions that associate with neutralizing antibody breadth in 22 HIV controllers. We found that the frequency of genomic mutations in IGHV and IGLV was directly correlated with serum neutralization breadth. The repertoire of the most mutated antibodies was dominated by a small number of large clones with evolutionary signatures suggesting that these clones had reached peak affinity maturation. These data demonstrate that even in the setting of low plasma HIV antigenemia, similar to what a vaccine can potentially achieve, BCR selection for extended somatic hypermutation and clonal evolution can occur in some individuals suggesting that host-specific factors might be involved that could be targeted with future vaccine strategies.

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.

Sign in / Sign up

Export Citation Format

Share Document