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Germinal center founder cells display propensity for apoptosis before onset of somatic mutation.

Lebecque S, de Bouteiller O, Arpin C, Banchereau J, Liu YJ - J. Exp. Med. (1997)

Bottom Line: They express low levels of Bcl-2, high levels of Fas, and undergo rapid apoptosis in culture.The smaller nonproliferating sIgM+IgD+CD38+ B cells displayed a lower level of somatic mutation in their immunoglobulin variable region genes compared with the large proliferating ones.Unmutated sIgM+IgD-CD38+ tonsillar B cells may thus represent germinal center founder cells in which the program for apoptotic cell death is triggered before the onset of somatic mutation, allowing the selection of the germline antibody repertoire at an early stage.

View Article: PubMed Central - PubMed

Affiliation: Schering-Plough, Laboratory for Immunological Research, Dardilly, France.

ABSTRACT
B lymphocytes undergo affinity maturation of their antigen receptors within germinal centers. These anatomical structures develop in secondary lymphoid organs from the clonal expansion of a few antigen-specific founder B cells, whose isolation and characterization are reported here. Human germinal center founder cells express the naive B cell markers surface IgM and IgD as well as the germinal center B cell markers CD10 and CD38. They express low levels of Bcl-2, high levels of Fas, and undergo rapid apoptosis in culture. The smaller nonproliferating sIgM+IgD+CD38+ B cells displayed a lower level of somatic mutation in their immunoglobulin variable region genes compared with the large proliferating ones. Unmutated sIgM+IgD-CD38+ tonsillar B cells may thus represent germinal center founder cells in which the program for apoptotic cell death is triggered before the onset of somatic mutation, allowing the selection of the germline antibody repertoire at an early stage.

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Schematic representation of mutations in rearranged VH5-μ sequences. Each line represents one VH5 sequence. VH5-2 sequences are underlined. Leader region, CDR1, and CDR2 are indicated. Mutations present in the DJH regions are not shown. Mutations are represented by different  symbols. •, replacement mutation; ‖, silent mutation; ○, stop codon. (A and B) VH5-μ sequences from slgM+IgD+CD38− B cells and  sIgM+IgD+CD38+ B cells, respectively. The upper, middle, and lower groups of sequences represent three different tonsil samples. (C and D) VH5-μ  sequences from medium-sized and large sIgM+IgD+CD38+ B cells, respectively, that were isolated from the fourth tonsil.
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Figure 3: Schematic representation of mutations in rearranged VH5-μ sequences. Each line represents one VH5 sequence. VH5-2 sequences are underlined. Leader region, CDR1, and CDR2 are indicated. Mutations present in the DJH regions are not shown. Mutations are represented by different symbols. •, replacement mutation; ‖, silent mutation; ○, stop codon. (A and B) VH5-μ sequences from slgM+IgD+CD38− B cells and sIgM+IgD+CD38+ B cells, respectively. The upper, middle, and lower groups of sequences represent three different tonsil samples. (C and D) VH5-μ sequences from medium-sized and large sIgM+IgD+CD38+ B cells, respectively, that were isolated from the fourth tonsil.

Mentions: VH5-μ sequence analysis has been successfully used to trace the progression of human tonsillar B cell subsets from the virgin to the memory compartment (11). Therefore, 32 VH5-μ sequences from sIgM+IgD+ CD38+ B cells were compared to 30 VH5-μ sequences from sIgM+IgD+CD38− naive B cells of three tonsil samples (Fig. 3, A and B). In agreement with our previous reports, all 30 sequences from naive B cells contain 0–2 mutations per sequence, with an overall mutation frequency of 2 × 10−3 bp (20 mutations/9,600 bp) that is barely distinguishable from the PCR Taq-error rate (1 × 10−3 bp). Within 32 sequences from sIgM+IgD+CD38+ B cells, while 17 might be considered germline (0–2 mutations/sequence), 15 have accumulated from 3–13 mutations, with an average mutation frequency of 20 × 10−3 bp (88 mutations/4,480 bp). The replacement mutations in 12 less mutated sequences (3.2, 3.4, 3.10, 3.14, 3b.5, 3b.6, 3b.9, 3b.13, 3c.5, 3c.7, 3c.9, and 3c.12) are randomly distributed within the CDRs and FWs, an indication of the absence of antigen-driven selection. In contrast, sequences 3.11, 3b.7, and 3c.6, which have accumulated 10–13 mutations, showed replacement mutations mainly focused in the CDRs, suggesting that these cells have undergone antigen-driven selection. Unlike the VH5-γ sequences from sIgD−CD38+ GC B cells and VH5-δ sequences from sIgM−IgD+CD38+ reported previously (11, 28), no clonal relatedness could be found among the VH5-μ transcripts from tonsillar sIgM+ IgD+CD38+ B cells. To establish whether the mediumsized nonproliferating cells from this population are those with unmutated V regions, sIgM+IgD+CD38+ B cells were isolated from a fourth tonsil and fractionated according to their size. The subset of medium-sized sIgM+IgD+CD38+ B cells was enriched for unmutated B cells (Fig. 3, C and D) as five out of nine sequences from the medium-sized B cells displayed less than two mutations and were therefore considered as nonmutated. In contrast, only one out of nine sequences from the large B cells was unmutated. In those two subsets, the most mutated sequences show features of antigen-driven selection.


Germinal center founder cells display propensity for apoptosis before onset of somatic mutation.

Lebecque S, de Bouteiller O, Arpin C, Banchereau J, Liu YJ - J. Exp. Med. (1997)

Schematic representation of mutations in rearranged VH5-μ sequences. Each line represents one VH5 sequence. VH5-2 sequences are underlined. Leader region, CDR1, and CDR2 are indicated. Mutations present in the DJH regions are not shown. Mutations are represented by different  symbols. •, replacement mutation; ‖, silent mutation; ○, stop codon. (A and B) VH5-μ sequences from slgM+IgD+CD38− B cells and  sIgM+IgD+CD38+ B cells, respectively. The upper, middle, and lower groups of sequences represent three different tonsil samples. (C and D) VH5-μ  sequences from medium-sized and large sIgM+IgD+CD38+ B cells, respectively, that were isolated from the fourth tonsil.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196025&req=5

Figure 3: Schematic representation of mutations in rearranged VH5-μ sequences. Each line represents one VH5 sequence. VH5-2 sequences are underlined. Leader region, CDR1, and CDR2 are indicated. Mutations present in the DJH regions are not shown. Mutations are represented by different symbols. •, replacement mutation; ‖, silent mutation; ○, stop codon. (A and B) VH5-μ sequences from slgM+IgD+CD38− B cells and sIgM+IgD+CD38+ B cells, respectively. The upper, middle, and lower groups of sequences represent three different tonsil samples. (C and D) VH5-μ sequences from medium-sized and large sIgM+IgD+CD38+ B cells, respectively, that were isolated from the fourth tonsil.
Mentions: VH5-μ sequence analysis has been successfully used to trace the progression of human tonsillar B cell subsets from the virgin to the memory compartment (11). Therefore, 32 VH5-μ sequences from sIgM+IgD+ CD38+ B cells were compared to 30 VH5-μ sequences from sIgM+IgD+CD38− naive B cells of three tonsil samples (Fig. 3, A and B). In agreement with our previous reports, all 30 sequences from naive B cells contain 0–2 mutations per sequence, with an overall mutation frequency of 2 × 10−3 bp (20 mutations/9,600 bp) that is barely distinguishable from the PCR Taq-error rate (1 × 10−3 bp). Within 32 sequences from sIgM+IgD+CD38+ B cells, while 17 might be considered germline (0–2 mutations/sequence), 15 have accumulated from 3–13 mutations, with an average mutation frequency of 20 × 10−3 bp (88 mutations/4,480 bp). The replacement mutations in 12 less mutated sequences (3.2, 3.4, 3.10, 3.14, 3b.5, 3b.6, 3b.9, 3b.13, 3c.5, 3c.7, 3c.9, and 3c.12) are randomly distributed within the CDRs and FWs, an indication of the absence of antigen-driven selection. In contrast, sequences 3.11, 3b.7, and 3c.6, which have accumulated 10–13 mutations, showed replacement mutations mainly focused in the CDRs, suggesting that these cells have undergone antigen-driven selection. Unlike the VH5-γ sequences from sIgD−CD38+ GC B cells and VH5-δ sequences from sIgM−IgD+CD38+ reported previously (11, 28), no clonal relatedness could be found among the VH5-μ transcripts from tonsillar sIgM+ IgD+CD38+ B cells. To establish whether the mediumsized nonproliferating cells from this population are those with unmutated V regions, sIgM+IgD+CD38+ B cells were isolated from a fourth tonsil and fractionated according to their size. The subset of medium-sized sIgM+IgD+CD38+ B cells was enriched for unmutated B cells (Fig. 3, C and D) as five out of nine sequences from the medium-sized B cells displayed less than two mutations and were therefore considered as nonmutated. In contrast, only one out of nine sequences from the large B cells was unmutated. In those two subsets, the most mutated sequences show features of antigen-driven selection.

Bottom Line: They express low levels of Bcl-2, high levels of Fas, and undergo rapid apoptosis in culture.The smaller nonproliferating sIgM+IgD+CD38+ B cells displayed a lower level of somatic mutation in their immunoglobulin variable region genes compared with the large proliferating ones.Unmutated sIgM+IgD-CD38+ tonsillar B cells may thus represent germinal center founder cells in which the program for apoptotic cell death is triggered before the onset of somatic mutation, allowing the selection of the germline antibody repertoire at an early stage.

View Article: PubMed Central - PubMed

Affiliation: Schering-Plough, Laboratory for Immunological Research, Dardilly, France.

ABSTRACT
B lymphocytes undergo affinity maturation of their antigen receptors within germinal centers. These anatomical structures develop in secondary lymphoid organs from the clonal expansion of a few antigen-specific founder B cells, whose isolation and characterization are reported here. Human germinal center founder cells express the naive B cell markers surface IgM and IgD as well as the germinal center B cell markers CD10 and CD38. They express low levels of Bcl-2, high levels of Fas, and undergo rapid apoptosis in culture. The smaller nonproliferating sIgM+IgD+CD38+ B cells displayed a lower level of somatic mutation in their immunoglobulin variable region genes compared with the large proliferating ones. Unmutated sIgM+IgD-CD38+ tonsillar B cells may thus represent germinal center founder cells in which the program for apoptotic cell death is triggered before the onset of somatic mutation, allowing the selection of the germline antibody repertoire at an early stage.

Show MeSH
Related in: MedlinePlus