Single-Cell and Deep Sequencing of IgG-Switched Macaque B Cells Reveal a Diverse Ig Repertoire following Immunization

Sundling, Christopher; Zhang, Zhenhai; Phad, Ganesh E.; Sheng, Zizhang; Wang, Yimeng; Mascola, John R.; Li, Yuxing; Wyatt, Richard T.; Shapiro, Lawrence; Hedestam, Gunilla B. Karlsson

doi:10.4049/jimmunol.1303334

Cited by 54 publications

(90 citation statements)

References 49 publications

(67 reference statements)

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“…Being DNA-based, HTGTS-Rep-seq also bypasses a major limitation of RNA-based methods for certain applications by quantitatively capturing the frequency of Ig rearrangements in a population regardless of their expression level or whether they are productive or nonproductive. Current means to address biases due to multiplex PCR or varying expression levels between cells include the use of universal identifiers (25,36,37) or single cell methods (38), but HTGTS-Rep-seq can accurately identify a population repertoire profile without the additional cost or steps of synthesizing primers with random barcodes, or sorting for single cells.…”

Section: Resultsmentioning

confidence: 99%

Highly sensitive and unbiased approach for elucidating antibody repertoires

Lin

et al. 2016

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

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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-Repseq), 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 DJ H 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. 1). In this process, the V, D, and J coding ends are generated as covalent hairpins that must be opened and that are often further processed, before being joined by classical nonhomologous end joining (2). Processing of V, D, J coding ends can involve generation of deletions or insertions of nucleotides at the junction regions (2), including the frequent de novo addition of nucleotides by the terminal deoxynucleotidyl transferase component of the V(D)J recombination process (3). Notably the V(D)J junctional region encodes a major antigen contact region of the antibody variable region, known as complementarity determining region 3 (CDR3), and thus these junctional diversification processes make a huge contribution to antibody diversity.The mouse IgH locus spans 2.7 megabases (Mb). There are 100s of V H s in the several megabase distal portion of the IgH, with the number varying substantially in certain mouse strains (4). The V H s lie ∼100 kb upstream from a 50-kb region containing 13 D H s, which is followed several kilobases downstream by a 2-kb region containing four J H s. The IgH constant region (C H ) exons lie downstream of the J H s. After assembly of a V H DJ H exon, transcription initiates upstream of the V H and terminates downstream of the C H exons, with V(D)J and C H portions being fused...

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Section: Resultsmentioning

confidence: 99%

Highly sensitive and unbiased approach for elucidating antibody repertoires

Lin

et al. 2016

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

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“…There was a large expansion of V H 4 family BCRs within all RMs ( fig. S9), a finding that may partially reflect the preferential usage of V H 4 in RM class-switched IgG B cells (20). Overall, SHM accounted for between 0 and 18% of the nucleotides in the V H genes (Fig.…”

Section: Development Of Cross-reactive Neutralization Activity and Afmentioning

confidence: 95%

Quality and quantity of T _FH cells are critical for broad antibody development in SHIV _AD8 infection

Lynch²,

et al. 2015

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Broadly neutralizing antibodies (bNAbs) protect against HIV-1 infection, yet how they are generated during chronic infection remains unclear. It is known that T follicular helper (TFH) cells are needed to promote affinity maturation of B cells during an immune response; however, the role of TFH during HIV-1 infection is undefined within lymph node germinal centers (GCs). We use nonhuman primates to investigate the relationship in the early stage of chronic SHIVAD8 (simian-human immunodeficiency virus AD8) infection between envelope (Env)-specific TFH cells, Env-specific B cells, virus, and the generation of bNAbs during later infection. We found that both the frequency and quality of Env-specific TFH cells were associated with an expansion of Env-specific immunoglobulin G-positive GC B cells and broader neutralization across HIV clades. We also found a correlation between breadth of neutralization and the degree of somatic hypermutation in Env-specific memory B cells. Finally, we observed high viral loads and greater diversity of Env sequences in rhesus macaques that developed cross-reactive neutralization as compared to those that did not. These studies highlight the importance of boosting high-quality TFH populations as part of a robust vaccine regimen aimed at eliciting bNabs.

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“…Finally, the relatively poor annotation of the macaque Ig locus, as compared to the human counterpart, has also been a major obstacle. However, recent progress has been made in annotating the macaque Ig locus and designing primers for amplification of macaque single cell Ig rearrangements (Sundling et al, 2012b) leading to an efficient isolation of CD4 binding site specific antibodies from macaques vaccinated with an HIV antigen (Sundling et al, 2014). …”

Section: Introductionmentioning

confidence: 99%

Vaccine-induced plasmablast responses in rhesus macaques: Phenotypic characterization and a source for generating antigen-specific monoclonal antibodies

Silveira

Kasturi

Kovalenkov

et al. 2015

Journal of Immunological Methods

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Over 100 broadly neutralizing antibodies have been isolated from a minority of HIV infected patients, but the steps leading to the selection of plasmacells producing such antibodies remain incompletely understood, hampering the development of vaccines able to elicit them. Rhesus macaques have become a preferred animal model system used to study SIV/HIV, for the characterization and development of novel therapeutics and vaccines as well as to understand pathogenesis. However, most of our knowledge about the dynamics of antibody responses is limited to the analysis of serum antibodies or monoclonal antibodies generated from memory B cells. In a vaccine setting, relatively little is known about the early cellular responses that elicit long-lived plasma cells and memory B cells and the tools to dissect plasmablast responses are not available in macaques. In the current study, we show that the majority (>80%) of the vaccine-induced plasmablast response are antigen-specific by functional ELISPOT assays. While plasmablasts are easily defined and isolated in humans, those same phenotypic markers have not been useful for identifying macaque plasmablasts. Here we describe an approach that allows for the isolation and single cell sorting of vaccine-induced plasmablasts. Finally, we show that isolated plasmablasts can be used to efficiently recover antigen-specific monoclonal antibodies through single cell expression cloning. This will allow detailed studies of the early plasmablast responses in rhesus macaques, enabling the characterization of both their repertoire breadth as well as the epitope specificity and functional qualities of the antibodies they produce, not only in the context of SIV/HIV vaccines but for many other pathogens/vaccines as well.

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Single-Cell and Deep Sequencing of IgG-Switched Macaque B Cells Reveal a Diverse Ig Repertoire following Immunization

Cited by 54 publications

References 49 publications

Highly sensitive and unbiased approach for elucidating antibody repertoires

Highly sensitive and unbiased approach for elucidating antibody repertoires

Quality and quantity of T _FH cells are critical for broad antibody development in SHIV _AD8 infection

Vaccine-induced plasmablast responses in rhesus macaques: Phenotypic characterization and a source for generating antigen-specific monoclonal antibodies

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Single-Cell and Deep Sequencing of IgG-Switched Macaque B Cells Reveal a Diverse Ig Repertoire following Immunization

Cited by 54 publications

References 49 publications

Highly sensitive and unbiased approach for elucidating antibody repertoires

Highly sensitive and unbiased approach for elucidating antibody repertoires

Quality and quantity of T FH cells are critical for broad antibody development in SHIV AD8 infection

Vaccine-induced plasmablast responses in rhesus macaques: Phenotypic characterization and a source for generating antigen-specific monoclonal antibodies

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Quality and quantity of T _FH cells are critical for broad antibody development in SHIV _AD8 infection