scholarly journals The relationship between variable region determinants and antigen specificity on mitogen reactive B cell subsets.

1982 ◽  
Vol 156 (3) ◽  
pp. 924-929 ◽  
Author(s):  
D Primi ◽  
F Mami ◽  
C Le Guern ◽  
P A Cazenave
Keyword(s):  
B Cell ◽  
Variable Region ◽  
Antigen Specificity ◽  
Antigen Binding Site ◽  
V Region ◽  
Set Up ◽  
Cell Mitogen ◽  
B Cell Subsets ◽  
The Relationship ◽  
Beta Galactosidase

On the basis of previous frequency determinations we could set up large numbers of cultures, each containing less than one competent precursor B cell specific for beta-galactosidase or for each of three idiotopes previously found on a monoclonal anti-beta-galactosidase antibody. Cultures were polyclonally activated by either lipopolysaccharide or Nocardia-delipidated cell mitogen. Each culture supernatant was individually tested for hemagglutination activity against sheep erythrocytes coupled with beta-galactosidase or with each of the three purified monoclonal anti-idiotypic antibodies. The results showed that only a minority of those clones positive for only one or two idiotopes recognized antigen. However, all those clones simultaneously positive for the three V region determinants recognized beta-galactosidase. The implications of these results for our understanding of the relationship between the antigen-binding site and idiotope expression are discussed.

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.

scholarly journals Mitogen-reactive B cell subpopulations selectively express different sets of V regions.

1982 ◽  
Vol 156 (1) ◽  
pp. 181-190 ◽  
Author(s):  
D Primi ◽  
F Mami ◽  
C Le Guern ◽  
P A Cazenave
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Absolute Frequency ◽  
The Absolute ◽  
Cell Subpopulations ◽  
B Cell Subsets ◽  
Beta Galactosidase

The experiments presented here were designed to investigate whether the idiotypic repertoire is equally distributed among B cells subpopulations as defined by mitogen reactivity. To this end we used lipopolysaccharides (LPS) and Nocardia delipidated cell mitogens (NDCM), which are two mitogens that have been described to act on different B cell subsets. The repertoire can be defined in quantitative terms as the frequency of B cells that are precursors for clones secreting immunoglobulin with a given specificity or with a determinate idiotype. We determined, therefore, the absolute frequency of LPS- and NDCM-sensitive B lymphocytes secreting immunoglobulin molecules that bear three idiotopes originally found on a monoclonal anti-beta galactosidase antibody. Because the frequencies of B cells carrying one of these idiotypes are dramatically different in the LPS- and NDCM-sensitive B cells subsets, we conclude that the idiotypic repertoire is not randomly distributed among mitogen-reactive B cell subpopulations.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior

Blood ◽  
2021 ◽  
Author(s):  
Giorgia Chiodin ◽  
Joel D. Allen ◽  
Dean Bryant ◽  
Philip Rock ◽  
Enrica Antonia Martino ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Variable Region ◽  
Cell Receptor ◽  
Antigen Binding ◽  
Post Translational Modification ◽  
Antigen Binding Site ◽  
Lymphoma Cells ◽  
X Ray Crystallography ◽  
Significant Gene

Glycosylation of the surface immunoglobulin variable region is a remarkable follicular lymphoma-associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCL) which acquire N-glycosylation sites selectively in the immunoglobulin (Ig) complementary-determining-regions (CDR) of the antigen-binding sites. Mass-spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures due to burial in the CDR loops. Acquisition of sites occurs in ~50% of germinal center B-cell-like DLBCL, mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell-like DLBCL Ig, which rarely has sites in the CDR, and appears not to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites associate with more rapid disease progression and with significant gene-set enrichment of the B-cell receptor, PI3K/AKT/MTORC1, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin DC-SIGN, mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate-recognition-domain. Oligomannosylation of the tumor immunoglobulin is a post-translational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN-expressing M2-polarized macrophages.

Immunogenetic Profiling of Follicular Lymphoma Reveals Universal N-Glycosylation Sites Introduced into the B Cell Receptor by Somatic Mutation and Suggests Relevance to Lymphoma Pathogenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 606-606
Author(s):  
Christian H. Ottensmeier ◽  
Katy J. McCann ◽  
Peter Johnson ◽  
Freda K. Stevenoson
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Somatic Mutation ◽  
Essential Feature ◽  
Variable Region ◽  
Cell Receptor ◽  
Single Amino Acid ◽  
Cell Repertoire ◽  
Antigen Binding Site ◽  
Glycosylation Sites

Abstract Immunogenetic analysis of B-cell malignancies can provide important information that relates to the cellular origin and clonal history of these lymphomas and give clues as to possible pathogenic mechanisms. In follicular lymphoma (FL), immunoglobulin variable region (V) genes are commonly somatically mutated and display intraclonal heterogeneity consistent with location in the germinal centre (GC). In this analysis of 44 cases of FL we find that, with minor exceptions, both the VH and VL gene usage reflects that of the normal B cell repertoire, indicative of a common antigenic drive and in support of a final transforming event in the GC. We have previously reported a high incidence of potential N-glycosylation sites in the VH genes of FL, which have been introduced by the process of somatic mutation. Here we have assessed both the VH and VL genes and find that sites are universally present and further demonstrate that they are available for functional glycosylation. The majority of sites are found in VH (81%) and are located predominantly within CDR2 and CDR3, with few sites present in the FRs. Sites are also evident in VL (45%) where they are focused mainly in CDR3 and CDR1. A minor subset (10%) has sites in VL only. In total, 26 different N-glycosylation motifs were observed, with NIS being the most commonly used. The natural motif in the V4–34 germline gene appears unimportant, and can be lost. Scrutiny of the somatic mutations giving rise to these motifs reveals that the acquisition of sites was predominantly (73%) achieved by a single amino acid (aa) replacement to Asn at position 1 of the motif, either with or without an additional, non-essential aa replacement at another position. Common ‘hotspots’ were observed within the CDR2 for the VH gene segments V3–23, V3–48, V3–07 and V3–15. It appears likely that the acquisition of N-glycosylation sites in the antigen-binding site during somatic mutation in the GC and the subsequent addition of oligosaccharides is important to the lifestyle of FL and may provide a critical second tumorigenic event. In turn, it may be possible to exploit this seemingly essential feature to develop novel therapeutic approaches.

Is Autoantibody Production Related to Particular B-Cell Subsets and Variable Region Genes?

1987 ◽  
Vol 6 (5-6) ◽  
pp. 371-389 ◽  
Author(s):  
Guillaume Dighiero ◽  
Pascal Poncet ◽  
Thomas Matthes ◽  
Azad Kaushik
Keyword(s):  
B Cell ◽  
Variable Region ◽  
Autoantibody Production ◽  
B Cell Subsets ◽  
Variable Region Genes

scholarly journals Surprisingly Fast Interface and Elbow Angle Dynamics of Antigen-Binding Fragments

2020 ◽  
Vol 7 ◽  
Author(s):  
Monica L. Fernández-Quintero ◽  
Katharina B. Kroell ◽  
Martin C. Heiss ◽  
Johannes R. Loeffler ◽  
Patrick K. Quoika ◽  
...  
Keyword(s):  
Light Chain ◽  
Variable Region ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
Antigen Specificity ◽  
Antigen Binding Site ◽  
Elbow Angle ◽  
Distinct Distribution ◽  
Complementarity Determining Region

Fab consist of a heavy and light chain and can be subdivided into a variable (VH and VL) and a constant region (CH1 and CL). The variable region contains the complementarity-determining region (CDR), which is formed by six hypervariable loops, shaping the antigen binding site, the paratope. Apart from the CDR loops, both the elbow angle and the relative interdomain orientations of the VH–VL and the CH1–CL domains influence the shape of the paratope. Thus, characterization of the interface and elbow angle dynamics is essential to antigen specificity. We studied nine antigen-binding fragments (Fab) to investigate the influence of affinity maturation, antibody humanization, and different light-chain types on the interface and elbow angle dynamics. While the CDR loops reveal conformational transitions in the micro-to-millisecond timescale, both the interface and elbow angle dynamics occur on the low nanosecond timescale. Upon affinity maturation, we observe a substantial rigidification of the VH and VL interdomain and elbow-angle flexibility, reflected in a narrower and more distinct distribution. Antibody humanization describes the process of grafting non-human CDR loops onto a representative human framework. As the antibody framework changes upon humanization, we investigated if both the interface and the elbow angle distributions are changed or shifted. The results clearly showed a substantial shift in the relative VH–VL distributions upon antibody humanization, indicating that different frameworks favor distinct interface orientations. Additionally, the interface and elbow angle dynamics of five antibody fragments with different light-chain types are included, because of their strong differences in elbow angles. For these five examples, we clearly see a high variability and flexibility in both interface and elbow angle dynamics, highlighting the fact that Fab interface orientations and elbow angles interconvert between each other in the low nanosecond timescale. Understanding how the relative interdomain orientations and the elbow angle influence antigen specificity, affinity, and stability has broad implications in the field of antibody modeling and engineering.

scholarly journals Cellular origin and pathophysiology of chronic lymphocytic leukemia

2012 ◽  
Vol 209 (12) ◽  
pp. 2183-2198 ◽  
Author(s):  
Marc Seifert ◽  
Ludger Sellmann ◽  
Johannes Bloehdorn ◽  
Frederik Wein ◽  
Stephan Stilgenbauer ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Subset ◽  
Variable Region ◽  
Lymphocytic Leukemia ◽  
Independent Evidence ◽  
Cellular Origin ◽  
Germinal Center B Cell ◽  
B Cell Subsets

The cellular origin of chronic lymphocytic leukemia (CLL) is still debated, although this information is critical to understanding its pathogenesis. Transcriptome analyses of CLL and the main normal B cell subsets from human blood and spleen revealed that immunoglobulin variable region (IgV) gene unmutated CLL derives from unmutated mature CD5+ B cells and mutated CLL derives from a distinct, previously unrecognized CD5+CD27+ post–germinal center B cell subset. Stereotyped V gene rearrangements are enriched among CD5+ B cells, providing independent evidence for a CD5+ B cell derivation of CLL. Notably, these CD5+ B cell populations include oligoclonal expansions already found in young healthy adults, putatively representing an early phase in CLL development before the CLL precursor lesion monoclonal B cell lymphocytosis. Finally, we identified deregulated proteins, including EBF1 and KLF transcription factors, that were not detected in previous comparisons of CLL and conventional B cells.

scholarly journals Analysis of somatic mutation in five B cell subsets of human tonsil.

1994 ◽  
Vol 180 (1) ◽  
pp. 329-339 ◽  
Author(s):  
V Pascual ◽  
Y J Liu ◽  
A Magalski ◽  
O de Bouteiller ◽  
J Banchereau ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Somatic Mutation ◽  
Germinal Center ◽  
Variable Region ◽  
B Cell Differentiation ◽  
Phenotypic Markers ◽  
Vh Gene ◽  
B Cell Subsets

Using a series of phenotypic markers that include immunoglobulin (Ig)D, IgM, IgG, CD23, CD44, Bcl-2, CD38, CD10, CD77, and Ki67, human tonsillar B cells were separated into five fractions representing different stages of B cell differentiation that included sIgD+ (Bm1 and Bm2), germinal center (Bm3 and Bm4), and memory (Bm5) B cells. To establish whether the initiation of somatic mutation correlated with this phenotypic characterization, we performed polymerase chain reaction and subsequent sequence analysis of the Ig heavy chain variable region genes from each of the B cell subsets. We studied the genes from the smallest VH families (VH4, VH5, and VH6) in order to facilitate the mutational analysis. In agreement with previous reports, we found that the somatic mutation machinery is activated only after B cells reach the germinal center and become centroblasts (Bm3). Whereas 47 independently rearranged IgM transcripts from the Bm1 and Bm2 subsets were nearly germline encoded, 57 Bm3-, and Bm4-, and Bm5-derived IgM transcripts had accumulated an average of 5.7 point mutations within the VH gene segment. gamma transcripts corresponding to the same VH gene families were isolated from subsets Bm3, Bm4, and Bm5, and had accumulated an average of 9.5 somatic mutations. We conclude that the molecular events underlying the process of somatic mutation takes place during the transition from IgD+, CD23+ B cells (Bm2) to the IgD-, CD23-, germinal center centroblast (Bm3). Furthermore, the analysis of Ig variable region transcripts from the different subpopulations confirms that the pathway of B cell differentiation from virgin B cell throughout the germinal center up to the memory compartment can be traced with phenotypic markers. The availability of these subpopulations should permit the identification of the functional molecules relevant to each stage of B cell differentiation.

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.

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