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Molecular footprints of a germinal center derivation of human IgM+(IgD+)CD27+ B cells and the dynamics of memory B cell generation.

Seifert M, Küppers R - J. Exp. Med. (2009)

Bottom Line: These findings provide genetic evidence for a GC derivation of somatically mutated IgM(+) B cells and indicate that adult humans harbor a large population of IgM(+)IgD(+) post-GC memory B cells.Furthermore, the analysis revealed that a highly diverse and often very large population of memory B cells is generated from a given GC B cell clone, and that (preferentially IgM) memory B cells are generated already early in the GC reaction.This provides novel insights into the dynamics of GC reactions and the generation of a memory B cell repertoire.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, D-45122 Essen, Germany.

ABSTRACT
The origin of IgM(+)CD27(+) B lymphocytes with mutated IgV genes, which account for approximately 20% of human peripheral blood (PB) B cells, is controversially discussed. A generation in a primary diversification pathway, in T cell-independent immune responses, or in T cell-dependent germinal center (GC) reactions has been proposed. We show here that IgM(+)IgD(+)CD27(+) and IgM(+)IgD(-/low)CD27(+) B cell subsets carry, like class-switched memory B cells, mutations in the Bcl6 gene as a genetic trait of a GC experience. Moreover, the identification of PB IgM(+)IgD(+)CD27(+) B cells clonally related to GC-derived IgG(+) memory B cells with shared and distinct IgV gene mutations demonstrates the GC origin also of the former subset. These findings provide genetic evidence for a GC derivation of somatically mutated IgM(+) B cells and indicate that adult humans harbor a large population of IgM(+)IgD(+) post-GC memory B cells. Furthermore, the analysis revealed that a highly diverse and often very large population of memory B cells is generated from a given GC B cell clone, and that (preferentially IgM) memory B cells are generated already early in the GC reaction. This provides novel insights into the dynamics of GC reactions and the generation of a memory B cell repertoire.

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Flow charts of the experimental approaches used to identify clonally related sequences in PB IgM+ and IgG+ B cells. (A) PAGE–based approach. (B) CDRIII-specific primer−based approach.
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fig1: Flow charts of the experimental approaches used to identify clonally related sequences in PB IgM+ and IgG+ B cells. (A) PAGE–based approach. (B) CDRIII-specific primer−based approach.

Mentions: Our first approach to identify such clonally related cells was based on a PAGE strategy and aimed at very large clones. Considering the number of cells that could be used per PCR reaction (30,000) and the total number of IgG+CD27+ and IgM+IgD+CD27+ B cells in the PB (∼6 × 108 per subset), we calculated that a putative clone should have at least 20,000 members in each of the two subsets to be detectable by this approach. Several aliquots of 30,000 IgG+CD27+ and IgM+IgD+CD27+ B cells each were sorted from PB of 10 healthy donors. Extracted RNA was separately reverse transcribed with gene-specific primers for Cγ and Cμ/Cδ and amplified with a VH1 family-specific primer and the same Cγ and Cμ/Cδ primers (Fig. 1 A). The Cγ primer used in this and the experiment described further below is specific for the Cγ1 and Cγ3 genes, which are classically used by post-GC memory B cells, but does not amplify Cγ2 transcripts, which mainly derive from TI responses (Shackelford et al., 1988). All CH primers were complementary to positions in equal distance from the first coding nucleotide of the corresponding CH gene. Thus, an identical CDRIII length of putative clone members in IgM+IgD+CD27+ and IgG+CD27+ B cells would result in equal-size products in a CDRIII-spectratyping analysis, performed on neighboring lanes of a PAGE gel. Such bands of interest were cut out from the gel, reamplified, and sequenced. Overall, 120 pairs of identical length were analyzed from sorted cells of the 10 donors. Most of these products (85%) were oligo- to polyclonal and not further followed up. From the monoclonal products in one instance, clonally related VH1-18–expressing IgM and IgG transcripts were obtained, and after cloning of the amplificates 7 distinct IgG+ and 2 distinct IgM+ members of the clone were identified (Fig. 2 A). The clone members contained between 3 and 9 VH gene mutations per sequence, of which 1 to 7 were shared by at least two clone members. Importantly, the genealogical tree depicting the most likely clonal evolution revealed that several IgG+ clone members descended from a proposed intermediate with only a single point mutation, whereas the two IgM sequences shared 5 additional point mutations with the other IgG+ sequences. We conclude that the IgM+ B cells acquired most if not all of their mutations in the clonal evolution of the GC B cell clone that gave rise to these memory B cells.


Molecular footprints of a germinal center derivation of human IgM+(IgD+)CD27+ B cells and the dynamics of memory B cell generation.

Seifert M, Küppers R - J. Exp. Med. (2009)

Flow charts of the experimental approaches used to identify clonally related sequences in PB IgM+ and IgG+ B cells. (A) PAGE–based approach. (B) CDRIII-specific primer−based approach.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806629&req=5

fig1: Flow charts of the experimental approaches used to identify clonally related sequences in PB IgM+ and IgG+ B cells. (A) PAGE–based approach. (B) CDRIII-specific primer−based approach.
Mentions: Our first approach to identify such clonally related cells was based on a PAGE strategy and aimed at very large clones. Considering the number of cells that could be used per PCR reaction (30,000) and the total number of IgG+CD27+ and IgM+IgD+CD27+ B cells in the PB (∼6 × 108 per subset), we calculated that a putative clone should have at least 20,000 members in each of the two subsets to be detectable by this approach. Several aliquots of 30,000 IgG+CD27+ and IgM+IgD+CD27+ B cells each were sorted from PB of 10 healthy donors. Extracted RNA was separately reverse transcribed with gene-specific primers for Cγ and Cμ/Cδ and amplified with a VH1 family-specific primer and the same Cγ and Cμ/Cδ primers (Fig. 1 A). The Cγ primer used in this and the experiment described further below is specific for the Cγ1 and Cγ3 genes, which are classically used by post-GC memory B cells, but does not amplify Cγ2 transcripts, which mainly derive from TI responses (Shackelford et al., 1988). All CH primers were complementary to positions in equal distance from the first coding nucleotide of the corresponding CH gene. Thus, an identical CDRIII length of putative clone members in IgM+IgD+CD27+ and IgG+CD27+ B cells would result in equal-size products in a CDRIII-spectratyping analysis, performed on neighboring lanes of a PAGE gel. Such bands of interest were cut out from the gel, reamplified, and sequenced. Overall, 120 pairs of identical length were analyzed from sorted cells of the 10 donors. Most of these products (85%) were oligo- to polyclonal and not further followed up. From the monoclonal products in one instance, clonally related VH1-18–expressing IgM and IgG transcripts were obtained, and after cloning of the amplificates 7 distinct IgG+ and 2 distinct IgM+ members of the clone were identified (Fig. 2 A). The clone members contained between 3 and 9 VH gene mutations per sequence, of which 1 to 7 were shared by at least two clone members. Importantly, the genealogical tree depicting the most likely clonal evolution revealed that several IgG+ clone members descended from a proposed intermediate with only a single point mutation, whereas the two IgM sequences shared 5 additional point mutations with the other IgG+ sequences. We conclude that the IgM+ B cells acquired most if not all of their mutations in the clonal evolution of the GC B cell clone that gave rise to these memory B cells.

Bottom Line: These findings provide genetic evidence for a GC derivation of somatically mutated IgM(+) B cells and indicate that adult humans harbor a large population of IgM(+)IgD(+) post-GC memory B cells.Furthermore, the analysis revealed that a highly diverse and often very large population of memory B cells is generated from a given GC B cell clone, and that (preferentially IgM) memory B cells are generated already early in the GC reaction.This provides novel insights into the dynamics of GC reactions and the generation of a memory B cell repertoire.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, D-45122 Essen, Germany.

ABSTRACT
The origin of IgM(+)CD27(+) B lymphocytes with mutated IgV genes, which account for approximately 20% of human peripheral blood (PB) B cells, is controversially discussed. A generation in a primary diversification pathway, in T cell-independent immune responses, or in T cell-dependent germinal center (GC) reactions has been proposed. We show here that IgM(+)IgD(+)CD27(+) and IgM(+)IgD(-/low)CD27(+) B cell subsets carry, like class-switched memory B cells, mutations in the Bcl6 gene as a genetic trait of a GC experience. Moreover, the identification of PB IgM(+)IgD(+)CD27(+) B cells clonally related to GC-derived IgG(+) memory B cells with shared and distinct IgV gene mutations demonstrates the GC origin also of the former subset. These findings provide genetic evidence for a GC derivation of somatically mutated IgM(+) B cells and indicate that adult humans harbor a large population of IgM(+)IgD(+) post-GC memory B cells. Furthermore, the analysis revealed that a highly diverse and often very large population of memory B cells is generated from a given GC B cell clone, and that (preferentially IgM) memory B cells are generated already early in the GC reaction. This provides novel insights into the dynamics of GC reactions and the generation of a memory B cell repertoire.

Show MeSH