Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching

Horns, Felix; Vollmers, Christopher; Croote, Derek; Mackey, Sally; Swan, Gary E.; Dekker, Cornelia L.; Davis, Mark M.; Quake, Stephen R.

doi:10.7554/elife.16578

Cited by 122 publications

(161 citation statements)

References 57 publications

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“…Genetic remnants of secondary recombination in humans have previously been detected in IG switch regions of IGHG1 and IGHG2 6 . In addition, transcripts from clonally‐related cells utilizing distinct IGHG subclasses have been identified in large‐scale Ig repertoire studies 13 , 23 . Interestingly, these B cells did not acquire more SHM, which is again suggestive of uncoupling between SHM and CSR.…”

Section: Discussionmentioning

confidence: 95%

Human IgG2‐ and IgG4‐expressing memory B cells display enhanced molecular and phenotypic signs of maturity and accumulate with age

et al. 2017

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The mechanisms involved in sequential immunoglobulin G (IgG) class switching are still largely unknown. Sequential IG class switching is linked to higher levels of somatic hypermutation (SHM) in vivo, but it remains unclear if these are generated temporally during an immune response or upon activation in a secondary response. We here aimed to uncouple these processes and to distinguish memory B cells from primary and secondary immune responses. SHM levels and IgG subclasses were studied with 454 pyrosequencing on blood mononuclear cells from young children and adults as models for primary and secondary immunological memory. Additional sequencing and detailed immunophenotyping with IgG subclass-specific antibodies was performed on purified IgG+ memory B-cell subsets. In both children and adults, SHM levels were higher in transcripts involving more downstream-located IGHG genes (esp. IGHG2 and IGHG4). In adults, SHM levels were significantly higher than in children, and downstream IGHG genes were more frequently utilized. This was associated with increased frequencies of CD27+IgG+ memory B cells, which contained higher levels of SHM, more IGHG2 usage, and higher expression levels of activation markers than CD27−IgG+ memory B cells. We conclude that secondary immunological memory accumulates with age and these memory B cells express CD27, high levels of activation markers, and carry high SHM levels and frequent usage of IGHG2. These new insights contribute to our understanding of sequential IgG subclass switching and show a potential relevance of using serum IgG2 levels or numbers of IgG2-expressing B cells as markers for efficient generation of memory responses.

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Section: Discussionmentioning

confidence: 95%

Human IgG2‐ and IgG4‐expressing memory B cells display enhanced molecular and phenotypic signs of maturity and accumulate with age

et al. 2017

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“…(33), p. 3058]. They have described sequential CSR from IgM to proximal classes (IgG3, IgG1, or IgA1) and then to distal classes (IgG2, IgG4, or IgA2) in healthy twin samples.…”

Section: Discussionmentioning

confidence: 99%

Different Somatic Hypermutation Levels among Antibody Subclasses Disclosed by a New Next-Generation Sequencing-Based Antibody Repertoire Analysis

Kitaura¹,

Yamashita²,

Ayabe³

et al. 2017

Front. Immunol.

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A diverse antibody repertoire is primarily generated by the rearrangement of V, D, and J genes and subsequent somatic hypermutation (SHM). Class-switch recombination (CSR) produces various isotypes and subclasses with different functional properties. Although antibody isotypes and subclasses are considered to be produced by both direct and sequential CSR, it is still not fully understood how SHMs accumulate during the process in which antibody subclasses are generated. Here, we developed a new next-generation sequencing (NGS)-based antibody repertoire analysis capable of identifying all antibody isotype and subclass genes and used it to examine the peripheral blood mononuclear cells of 12 healthy individuals. Using a total of 5,480,040 sequences, we compared percentage frequency of variable (V), junctional (J) sequence, and a combination of V and J, diversity, length, and amino acid compositions of CDR3, SHM, and shared clones in the IgM, IgD, IgG3, IgG1, IgG2, IgG4, IgA1, IgE, and IgA2 genes. The usage and diversity were similar among the immunoglobulin (Ig) subclasses. Clonally related sequences sharing identical V, D, J, and CDR3 amino acid sequences were frequently found within multiple Ig subclasses, especially between IgG1 and IgG2 or IgA1 and IgA2. SHM occurred most frequently in IgG4, while IgG3 genes were the least mutated among all IgG subclasses. The shared clones had almost the same SHM levels among Ig subclasses, while subclass-specific clones had different levels of SHM dependent on the genomic location. Given the sequential CSR, these results suggest that CSR occurs sequentially over multiple subclasses in the order corresponding to the genomic location of IGHCs, but CSR is likely to occur more quickly than SHMs accumulate within Ig genes under physiological conditions. NGS-based antibody repertoire analysis should provide critical information on how various antibodies are generated in the immune system.

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“…IGH repertoires were obtained using previously published protocols (39, 40) with small modifications, via the following steps: reverse transcription (RT) with IGH constant domain–specific primers containing random barcodes (table S7) on 250 ng of total RNA extracted from PBMCs; second-strand synthesis with IGH variable domain–specific primers containing random barcodes (table S8); AMPure purification (Beckman Coulter); polymerase chain reaction (PCR) amplification incorporating Illumina adapters and sample multiplexing indices; 150–base pair paired-end NextSeq sequencing (Illumina); and analysis using pRESTO (41), IMGT/HighV-QUEST (42), Change-O (43), and R scripts deposited at https://github.com/cdebourcy/scleroderma. During analysis, the random barcodes contained in the primers were used as UIDs, allowing RNA transcript counting and amplification bias correction: We collapsed reads that had identical UIDs so as to count each RNA molecule of the starting material only once, regardless of the degree of PCR amplification the molecule underwent.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of the human antibody repertoire after B cell depletion in systemic sclerosis

et al. 2017

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Systemic sclerosis with pulmonary arterial hypertension (SSc-PAH) is a debilitating and frequently lethal disease of unknown cause lacking effective treatment options. Lymphocyte anomalies and autoantibodies observed in systemic sclerosis have suggested an autoimmune character. We study the clonal structure of the B cell repertoire in SSc-PAH using immunoglobulin heavy chain (IGH) sequencing before and after B cell depletion. We found SSc-PAH to be associated with anomalies in B cell development, namely, altered VDJ rearrangement frequencies (reduced IGHV2-5 segment usage) and an increased somatic mutation–fixation probability in expanded B cell lineages. SSc-PAH was also characterized by anomalies in B cell homeostasis, namely, an expanded immunoglobulin D–positive (IgD+) proportion with reduced mutation loads and an expanded proportion of highly antibody-secreting cells. Disease signatures pertaining to IGHV2-5 segment usage, IgD proportions, and mutation loads were temporarily reversed after B cell depletion. Analyzing the time course of B cell depletion, we find that the kinetics of naïve replenishment are predictable from baseline measurements alone, that release of plasma cells into the periphery can precede naïve replenishment, and that modes of B cell receptor diversity are highly elastic. Our findings reveal humoral immune signatures of SSc-PAH and uncover determinism in the effects of B cell depletion on the antibody repertoire.

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Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching

Cited by 122 publications

References 57 publications

Human IgG2‐ and IgG4‐expressing memory B cells display enhanced molecular and phenotypic signs of maturity and accumulate with age

Human IgG2‐ and IgG4‐expressing memory B cells display enhanced molecular and phenotypic signs of maturity and accumulate with age

Different Somatic Hypermutation Levels among Antibody Subclasses Disclosed by a New Next-Generation Sequencing-Based Antibody Repertoire Analysis

Dynamics of the human antibody repertoire after B cell depletion in systemic sclerosis

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