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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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Subpopulations of tonsillar B cells. (A) Double staining of total tonsil B cells with anti–IgD-PE and anti–CD38-FITC, showing four  B cell subsets. The sIgD+CD38+ B cells represent 5–15% of total tonsil  B cells from three representative samples. (B) Phenotypic analysis by threecolor flow cytometry. Anti–IgD-tricolor and anti–CD38-PE are used together with other third antibodies directly conjugated with FITC. Anti– IgD-PE and anti–CD38-FITC were used together with Hoechst 33342.  For Bcl-2 and Ki67 staining, the cells were permeabilized by saponin  (0.33 g/100 ml in PBS, 1% BSA) for 15 min on ice. (C) Cell sorting of  sIgD+CD38+ B cells into sIgM+ and sIgM− subsets.
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Figure 1: Subpopulations of tonsillar B cells. (A) Double staining of total tonsil B cells with anti–IgD-PE and anti–CD38-FITC, showing four B cell subsets. The sIgD+CD38+ B cells represent 5–15% of total tonsil B cells from three representative samples. (B) Phenotypic analysis by threecolor flow cytometry. Anti–IgD-tricolor and anti–CD38-PE are used together with other third antibodies directly conjugated with FITC. Anti– IgD-PE and anti–CD38-FITC were used together with Hoechst 33342. For Bcl-2 and Ki67 staining, the cells were permeabilized by saponin (0.33 g/100 ml in PBS, 1% BSA) for 15 min on ice. (C) Cell sorting of sIgD+CD38+ B cells into sIgM+ and sIgM− subsets.

Mentions: Human tonsillar B cells that coexpress sIgD and CD38 have been identified, which represent 5–15% of total human tonsillar B cells (Fig. 1 A). Three-color flow cytometry shows that sIgD+CD38+ B cells express low levels of the naive B cell markers CD23, CD44, and IgM, while they express high levels of the germinal center markers CD71, CD10, and CD77 (Fig. 1 B). Unlike the sIgD+CD38− naive B cells, they express low levels of Bcl-2, high levels of Fas, and many of them express the proliferation associated nuclear antigen Ki67 (Fig. 1 B). The sIgD+CD38+ B cells were sorted into a sIgM+ subset (3–9% of total tonsillar B cells) and a sIgM− subset (2–6% of total tonsillar B cells) (Fig. 1 C). Our recent study has shown that sIgM−IgD+CD38+ B cells represent a peculiar population of highly mutated GC centroblasts (28). We have now further characterized the sIgM+IgD+CD38+ B cell subset which displays a phenotype intermediate between follicular mantle naive B cells and GC B cells.


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)

Subpopulations of tonsillar B cells. (A) Double staining of total tonsil B cells with anti–IgD-PE and anti–CD38-FITC, showing four  B cell subsets. The sIgD+CD38+ B cells represent 5–15% of total tonsil  B cells from three representative samples. (B) Phenotypic analysis by threecolor flow cytometry. Anti–IgD-tricolor and anti–CD38-PE are used together with other third antibodies directly conjugated with FITC. Anti– IgD-PE and anti–CD38-FITC were used together with Hoechst 33342.  For Bcl-2 and Ki67 staining, the cells were permeabilized by saponin  (0.33 g/100 ml in PBS, 1% BSA) for 15 min on ice. (C) Cell sorting of  sIgD+CD38+ B cells into sIgM+ and sIgM− subsets.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196025&req=5

Figure 1: Subpopulations of tonsillar B cells. (A) Double staining of total tonsil B cells with anti–IgD-PE and anti–CD38-FITC, showing four B cell subsets. The sIgD+CD38+ B cells represent 5–15% of total tonsil B cells from three representative samples. (B) Phenotypic analysis by threecolor flow cytometry. Anti–IgD-tricolor and anti–CD38-PE are used together with other third antibodies directly conjugated with FITC. Anti– IgD-PE and anti–CD38-FITC were used together with Hoechst 33342. For Bcl-2 and Ki67 staining, the cells were permeabilized by saponin (0.33 g/100 ml in PBS, 1% BSA) for 15 min on ice. (C) Cell sorting of sIgD+CD38+ B cells into sIgM+ and sIgM− subsets.
Mentions: Human tonsillar B cells that coexpress sIgD and CD38 have been identified, which represent 5–15% of total human tonsillar B cells (Fig. 1 A). Three-color flow cytometry shows that sIgD+CD38+ B cells express low levels of the naive B cell markers CD23, CD44, and IgM, while they express high levels of the germinal center markers CD71, CD10, and CD77 (Fig. 1 B). Unlike the sIgD+CD38− naive B cells, they express low levels of Bcl-2, high levels of Fas, and many of them express the proliferation associated nuclear antigen Ki67 (Fig. 1 B). The sIgD+CD38+ B cells were sorted into a sIgM+ subset (3–9% of total tonsillar B cells) and a sIgM− subset (2–6% of total tonsillar B cells) (Fig. 1 C). Our recent study has shown that sIgM−IgD+CD38+ B cells represent a peculiar population of highly mutated GC centroblasts (28). We have now further characterized the sIgM+IgD+CD38+ B cell subset which displays a phenotype intermediate between follicular mantle naive B cells and GC B cells.

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