Antibody repertoire deep sequencing reveals antigen-independent selection in maturing B cells

Kaplinsky, Joseph; Li, Anthony; Sun, Amy; Coffre, Maryaline; Koralov, Sergei B.; Arnaout, Ramy

doi:10.1073/pnas.1403278111

Cited by 46 publications

(56 citation statements)

References 39 publications

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“…These results are in accordance with, and extend, previous data (24) indicating that IgA can replace IgM to drive early B-cell development and instruct peripheral B-cell maturation. Whereas V H gene use may influence the FO versus MZ B-cell fate (27), our data reveal that BCR extrinsic factors critically contribute to this decision. Also, the development of FO and MZ B cells at the expense of B-1 B cells may reflect the expression by transnuclear B cells of a BCR specificity enabling selectively the development of a B2/MZ B-cell progenitor (28,29).…”

Section: Discussionmentioning

confidence: 74%

Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell

Kumar

Bach

Mainoldi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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In mammals, VDJ recombination is responsible for the establishment of a highly diversified preimmune antibody repertoire. Acquisition of a functional Ig heavy (H) chain variable (V) gene rearrangement is thought to prevent further recombination at the IgH locus. Here, we describe V H Q52 NT ; Vκgr32 NT Ig monoclonal mice reprogrammed from the nucleus of an intestinal IgA + plasma cell. In V H Q52 NT mice, IgA replaced IgM to drive early B-cell development and peripheral B-cell maturation. In V H Q52 NT animals, over 20% of mature B cells disrupted the single productive, nonautoimmune IgH rearrangement through VH replacement and exchanged it with a highly diversified pool of IgH specificities. VH replacement occurred in early pro-B cells, was independent of pre-B-cell receptor signaling, and involved predominantly one adjacent V H germ-line gene. VH replacement was also identified in 5% of peripheral B cells of mice inheriting a different productive V H rearrangement expressed in the form of an IgM H chain. In summary, editing of a productive IgH rearrangement through VH replacement can account for up to 20% of the IgH repertoire expressed by mature B cells. A functional immune system relies on the ability of B-lymphocytes to recognize foreign antigens in a highly specific fashion through the Ig receptor (also called B-cell receptor, or BCR). Each BCR consists of two identical Ig heavy (H) and light (L) chains, which are expressed on the surface of the B cell together with an Igα/Igβ heterodimer to form a signaling unit. In most vertebrates, the ability of the immune system to generate a highly diversified repertoire of antibody specificities relies on the stochastic assembly of variable (V), diversity (D), and joining (J) gene segments that encode for the antigen-binding domain of IgH and IgL chains of the BCR. This process, called V(D)J recombination, is mediated by the ordered recruitment at Ig loci of the RAG-1/2 endonucleases, followed by nonhomologous endjoining factors that catalyze the joining of the cleaved DNA segments (1). The latter processes are often accompanied by trimming and insertion of n-nucleotides at junctional ends. All together these mechanisms contribute to generate a highly diversified pool of Ig specificities.Ig receptor editing provides B cells with the opportunity to exchange BCR specificity through secondary VDJ recombination. Whereas IgL chain editing was shown to play a central role in the neutralization of autoimmune BCR specificities expressed by newly generated immature B cells (2), the contribution of IgH receptor editing to antibody repertoire diversification has remained largely unappreciated. Two mechanisms promote secondary IgH rearrangements. In V H -to-J H direct joining, RAG proteins cleave at Recombination Signal Sequences (RSSs) of V H and J H elements lying, respectively, upstream and downstream of the original V H rearrangement, followed by microhomology-driven joining of the Ig gene segments. This mechanism has been mainly observed in IgH knock-in mice carrying nonpr...

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Section: Discussionmentioning

confidence: 74%

Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell

Kumar

Bach

Mainoldi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Furthermore, B-cell signaling is sensitive to small conformational changes in the B-cell receptor (50). Modulation of the Fab elbow angle dynamics may have a favorable effect on B-cell receptor signaling (51).…”

Section: Discussionmentioning

confidence: 99%

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

Koenig¹,

Lee²,

Walters³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. Deciphering the molecular mechanism of SHMs in improving antibodies is critical for understanding the evolution of the immune repertoire. It is well recognized that mutations at the CDRs or FWR structurally adjacent to the CDRs can modulate and optimize the antigen-binding interface (4-7), and several studies have used saturated mutagenesis of CDR position to explore the effect of various mutations on affinity and specificity (8-11).The role of SHM in antigen-distal FWR is less well understood, however. Previous studies have suggested that the antigen-distal FWR contributes primarily to the variable domain structural integrity; thus, mutations at these positions would be either detrimental to or neutral for antigen-binding function (12)(13)(14). Other authors have characterized the potential of antigen-distal framework mutation as beneficial for antigen-binding function. For example, framework mutations can compensate for the destabilizing effect of mutations at CDRs needed for antigen binding (15). In other cases, non-CDR SHMs have been shown to be required for the neutralization activity of broadly neutralizing anti-HIV antibodies (16).Thus far, the roles of SHM have been studied primarily by examining the functional consequences of reverting the somatic mutation of selected antibodies back to the germline sequences (15-21). Although these studies have demonstrated that antibodies depend on somatic mutations to achieve high-affinity antigen binding, the approach is limited to the small set of mutations present in a given antibody.Here we took a systematic approach to assessing the mutability of the whole variable domain for maintaining or improving folding stability and antigen binding. We used a welloptimized anti-vascular endothelial cell growth factor (VEGF), G6.31, with high affinity (K d = 0.4 nM) and excellent thermostability [fragment antigen-binding (Fab) melting temperature (T m ) = 84°C]. G6.31 derives its HC and LC variable domains ...

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“…However, diverse B‐cell subsets may be involved in a response, including naive, marginal zone (MZ) and memory (immunoglobulin M (IgM) and IgG) B cells, depending on the type of antigen, previous exposure, and route of immunization. Analysis of different B‐cell subsets revealed differences in their sequence, and VDJ gene segment composition, 6 , 7 and thus interrogation of the BCR repertoire on a subset‐by‐subset basis could potentially be used for fine delineation of which B cells are involved in vaccine responses.…”

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confidence: 99%

BCR repertoire sequencing: different patterns of B‐cell activation after two Meningococcal vaccines

et al. 2015

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Next-generation sequencing was used to investigate the B cell receptor heavy chain transcript repertoire of different B cell subsets (naïve, marginal zone, IgM memory and IgG memory) at baseline, and of plasma cells 7 days following administration of serogroup ACWY meningococcal polysaccharide and protein-polysaccharide conjugate vaccines. Baseline B cell subsets could be distinguished from each other using a small number of repertoire properties (clonality, mutation from germ-line and complementarity-determining region 3 length) that were conserved between individuals. However, analyzing the complementarity-determining region 3 amino acid sequence (which is particularly important for antigen binding) of the baseline subsets showed few sequences shared between individuals. In contrast, day 7 plasma cells demonstrated nearly tenfold greater sequence sharing between individuals than the baseline subsets, consistent with the plasma cells being induced by the vaccine antigen, and sharing specificity for a more limited range of epitopes. By annotating plasma cell sequences based on IgG subclass usage and mutation, and also comparing them to the sequences of the baseline cell subsets, we were able to identify different signatures after the polysaccharide and conjugate vaccines. Plasma cells produced after conjugate vaccination were predominantly IgG1, and most related to IgG memory cells. In contrast, after polysaccharide vaccination, the plasma cells were predominantly IgG2, less mutated, and were equally likely to be related to marginal zone, IgM memory or IgG memory cells. High-throughput B cell repertoire sequencing thus provides a unique insight into patterns of B cell activation not possible from more conventional measures of immunogenicity.

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Antibody repertoire deep sequencing reveals antigen-independent selection in maturing B cells

Cited by 46 publications

References 39 publications

Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell

Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

BCR repertoire sequencing: different patterns of B‐cell activation after two Meningococcal vaccines

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