High-Throughput Sequencing of the Zebrafish Antibody Repertoire

Weinstein, Joshua A.; Jiang, Ning; White, Richard Allen; Fisher, Daniel S.; Quake, Stephen R.

doi:10.1126/science.1170020

Cited by 418 publications

(458 citation statements)

References 13 publications

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“…Full-length V regions for HC and LC were assembled with human HC and LC constant regions and transfected into HEK293 cells. A sampling of the stably selected library by highthroughput sequencing (HTP) provided a lower estimate of 6 × 10 7 total diversity of combinatorially expressed antibody sequences (25). The library was designed to provide multiple initial candidates with germline V-gene segments for further maturation by SHM, and is termed ABELmAb (AnaptysBio Evolving Library of monoclonal Antibodies).…”

Section: Resultsmentioning

confidence: 99%

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies

Bowers

Horlick

Neben

et al. 2011

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

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A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human D J regions was used to isolate low-affinity antibodies to human β nerve growth factor (hβNGF). These antibodies, initially naïve to SHM, were subjected to AID-directed SHM in vitro and selected using the same mammalian cell display system, as illustrated by the maturation of one of the antibodies to low pM K D . This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs. affinity maturation | mammalian display

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Section: Resultsmentioning

confidence: 99%

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies

Bowers

Horlick

Neben

et al. 2011

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

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“…This conundrum was elegantly answered many years ago by an expansion of the primary Ab repertoire through genetic recombination of VDJ gene segments and by resultant combinatorial diversity arising from rearranged heterodimers of light and heavy chains (1). Recent high-throughput sequencing data in zebra fish and humans have provided further insights into the extent of the expressed Ab repertoire, as envisaged by Tonegawa et al (2,3) However, quantitative assessment, even after considering other possible mechanisms, proves this diversity to be inadequate for recognition of infinite Ags (4).…”

mentioning

confidence: 94%

Structural Elucidation of the Mechanistic Basis of Degeneracy in the Primary Humoral Response

Khan

Salunke

2012

The Journal of Immunology

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The mechanistic basis for efficient combating of the infinite range of foreign Ags by the limited repertoire of naive Abs expressed on primary B cell surfaces during their first encounter was addressed through elegantly designed crystallographic analyses. Resolution of the discrepancy arising from the limited number of possible germline Ab receptors on primary B cells for recognizing the unlimited pool of possible Ags has been attempted by invoking the degenerate recognition potential of the germline Abs. Structural analyses of germline mAb BBE6.12H3 in an Ag-free state, as well as bound to four different peptide Ags, established the correlation of its degenerate specificity with conformational versatility of the paratope. Six distinct paratope topologies observed for a single germline mAb provided a quantitative description of the primary Ag recognition repertoire at the tertiary structural level. Each of the four different peptide Ags was bound specifically to a distinct conformation of the paratope, which was also different from that of the Ag-free states of the same germline mAb. A minimal conserved motif in the pristine Ag-combining site essential for multispecificity and Ag binding-mediated change in the elbow angle of Fab was also discernible. It is proposed that the generation of a primary Ab repertoire involves large, yet finite, germline Ab clones, each capable of adopting discrete conformations, which in turn exhibit diverse binding modes.

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“…The available data suggest that convergent recombination [37][38][39][40] and biases during recombination [33,37,41] are the major contributors of TCR sharing in TCR repertoires among individuals. Convergent recombination is the process whereby multiple recombination events 'converge' to produce the same nucleotide sequence and multiple nucleotide sequences "converge" to encode the same amino-acid sequence (Figure 1), which results in different TCR sequences to be generated with differential frequencies during recombination [37][38][39][40].…”

Section: How Does Initial V(d)j Recombination Determine Tcr Sharing?mentioning

confidence: 99%

“…Convergent recombination is the process whereby multiple recombination events 'converge' to produce the same nucleotide sequence and multiple nucleotide sequences "converge" to encode the same amino-acid sequence (Figure 1), which results in different TCR sequences to be generated with differential frequencies during recombination [37][38][39][40]. Recombinatorial biases include biased V/D/J gene usage and combination, bias in the number of nucleotide deletions at the coding ends of V/D/J gene segments, bias in the number of nucleotide additions and bias in base usage at the V-D/ D-J junctions [33,37,[41][42][43]. How those two determinants generate the substantial sharing of TCRs among individuals during initial recombination is discussed below.…”

Section: How Does Initial V(d)j Recombination Determine Tcr Sharing?mentioning

confidence: 99%

Determinants of public T cell responses

et al. 2012

Cell Res

118

112

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Historically, sharing T cell receptors (TCRs) between individuals has been speculated to be impossible, considering the dramatic discrepancy between the potential enormity of the TCR repertoire and the limited number of T cells generated in each individual. However, public T cell response, in which multiple individuals share identical TCRs in responding to a same antigenic epitope, has been extensively observed in a variety of immune responses across many species. Public T cell responses enable individuals within a population to generate similar antigen-specific TCRs against certain ubiquitous pathogens, leading to favorable biological outcomes. However, the relatively concentrated feature of TCR repertoire may limit T cell response in a population to some other pathogens. It could be a great benefit for human health if public T cell responses can be manipulated. Therefore, the mechanistic insight of public TCR generation is important to know. Recently, high-throughput DNA sequencing has revolutionized the study of immune receptor repertoires, which allows a much better understanding of the factors that determine the overlap of TCR repertoire among individuals. Here, we summarize the current knowledge on public T-cell response and discuss future challenges in this field.

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High-Throughput Sequencing of the Zebrafish Antibody Repertoire

Cited by 418 publications

References 13 publications

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies

Structural Elucidation of the Mechanistic Basis of Degeneracy in the Primary Humoral Response

Determinants of public T cell responses

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