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FLICE-inhibitory protein is a key regulator of germinal center B cell apoptosis.

Hennino A, Bérard M, Krammer PH, Defrance T - J. Exp. Med. (2001)

Bottom Line: We found that GC B cells ex vivo display a preformed inactive DISC containing Fas-associated death domain-containing protein (FADD), procaspase-8, and the long isoform of cellular FADD-like IL-1beta-converting enzyme-inhibitory protein (c-FLIP(L)) but not the CD95L.In culture, c-FLIP(L) is rapidly lost from the CD95 DISC unless GC B cells are exposed to the survival signal provided by CD40L.Our results suggest that (a) the death receptor signaling pathway is involved in the affinity maturation of antibodies, and (b) c-FLIP(L) plays an active role in positive selection of B cells in the GC.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), U404 Immunité et Vaccination, Lyon, Cedex 07, France.

ABSTRACT
Affinity maturation of the B cell response to antigen (Ag) takes place in the germinal centers (GCs) of secondary follicles. Two sequential molecular mechanisms underpin this process. First, the B cell repertoire is diversified through hypermutation of the immunoglobulin (Ig) variable region genes. Second, mutant B cell clones with improved affinity for Ag are positively selected by Ag and CD40 ligand (L). This selection step is contingent upon "priming" of GC B cells for apoptosis. The molecular means by which B cell apoptosis is initiated and controlled in the GC remains unclear. Here, we show that GC B cell apoptosis is preceded by the rapid activation of caspase-8 at the level of CD95 death-inducing signaling complex (DISC). We found that GC B cells ex vivo display a preformed inactive DISC containing Fas-associated death domain-containing protein (FADD), procaspase-8, and the long isoform of cellular FADD-like IL-1beta-converting enzyme-inhibitory protein (c-FLIP(L)) but not the CD95L. In culture, c-FLIP(L) is rapidly lost from the CD95 DISC unless GC B cells are exposed to the survival signal provided by CD40L. Our results suggest that (a) the death receptor signaling pathway is involved in the affinity maturation of antibodies, and (b) c-FLIP(L) plays an active role in positive selection of B cells in the GC.

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GC B cells do not produce CD95L. (A) RT and PCR amplification of the CD95L and β-actin transcripts were performed on RNA extracts from freshly isolated GC B cells and from PBLs which had been successively stimulated for 72 h with PHA (5 μg/ml) and 6 h with PMA (10 ng/ml) and ionomycin (0.5 μg/ml) (act. PBL). (B–D) Freshly isolated GC B cells were cultured in complete medium (CM) in the presence or absence of the antagonistic anti-CD95 mAb ZB4 (1 μg/ml). GC B cells were assessed after 1, 2, 3, and 4 h of culture for their mitochondrial transmembrane potential (B), externalization of PS (C), and activation of caspase-3 (D) using a PE-conjugated anti-active caspase-3 Ab. The results are expressed as means of the (B) percent viable cells, (C) annexin V+ cells, and (D) cells with activated caspase-3, calculated from duplicate determinations. The difference between duplicate measurements never exceeded 10% of the mean values. Representative of three distinct experiments.
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Figure 6: GC B cells do not produce CD95L. (A) RT and PCR amplification of the CD95L and β-actin transcripts were performed on RNA extracts from freshly isolated GC B cells and from PBLs which had been successively stimulated for 72 h with PHA (5 μg/ml) and 6 h with PMA (10 ng/ml) and ionomycin (0.5 μg/ml) (act. PBL). (B–D) Freshly isolated GC B cells were cultured in complete medium (CM) in the presence or absence of the antagonistic anti-CD95 mAb ZB4 (1 μg/ml). GC B cells were assessed after 1, 2, 3, and 4 h of culture for their mitochondrial transmembrane potential (B), externalization of PS (C), and activation of caspase-3 (D) using a PE-conjugated anti-active caspase-3 Ab. The results are expressed as means of the (B) percent viable cells, (C) annexin V+ cells, and (D) cells with activated caspase-3, calculated from duplicate determinations. The difference between duplicate measurements never exceeded 10% of the mean values. Representative of three distinct experiments.

Mentions: Next we examined whether clustering of CD95 and the subsequent formation of a DISC in GC B cells was consecutive to the binding of CD95L. It has been demonstrated that upon prolonged stimulation through their Ag receptor, T cells can express and release CD95L that promotes their autocrine suicide 26. Two types of experiments were undertaken to test whether the aggregation of CD95 on isolated GC B cells was consecutive to their endogenous production of CD95L. First, we examined by RT-PCR the expression of the CD95L transcript in freshly isolated GC B cells. As shown in Fig. 6 A, the CD95L message was not detectable in GC B cells, whereas it was expressed by PHA-activated PBLs which had been restimulated for 6 h with PMA and ionomycin. Second, we estimated whether blockade of the CD95–CD95L interaction by the antagonistic anti-CD95 mAb ZB4 could prevent apoptosis of GC B cells cultured for 1, 2, 3, and 4 h in the absence of exogenous stimuli. Data shown in Fig. 6bFig. cFig. d, indicate that mAb ZB4 failed to prevent the Δψm drop, PS exposure, and activation of caspase-3 in cultured GC B cells. This suggests that aggregation of CD95 on GC B cells is not because of an autocrine production loop of CD95L. To test the possible involvement of exogenous CD95L in multimerization of CD95, we next examined whether CD95 was physically bound to its ligand in GC B cells. For this purpose, we determined whether CD95L was part of the CD95 DISC in GC B cells. The proteins associated with FADD were thus immunoprecipitated in freshly isolated GC B cells and in the human leukemic T cells H9 which had been treated for 6 h with an anti-CD3 mAb. The H9 cell line was chosen as a positive control since ligation of the TCR–CD3 complex on these cells promotes their apoptosis via the endogenous release of CD95L and its subsequent binding to CD95 27. As shown in Fig. 7, both CD95 and CD95L are coprecipitated with FADD in CD3-activated H9 cells. Two bands migrating respectively as a 40/42-kD species and as a 26-kD species are revealed by the anti-CD95L mAb in the H9 immunoprecipitates. These two bands are likely to represent the membrane-bound and the soluble form of CD95L, respectively. They were also found in OKT3-treated Jurkat T cells (data not shown). In contrast, despite the fact that CD95 was efficiently coprecipitated with FADD in GC B cells, no signal was obtained with the anti-CD95L mAb in these immunoprecipitates. In agreement with our observation that GC B cells lack the CD95L transcript, whole cell lysates prepared from GC B cells were also negative for the expression of the CD95L protein. Altogether, these data suggest that CD95L is not responsible for oligomerization of CD95 and formation of a DISC in GC B cells.


FLICE-inhibitory protein is a key regulator of germinal center B cell apoptosis.

Hennino A, Bérard M, Krammer PH, Defrance T - J. Exp. Med. (2001)

GC B cells do not produce CD95L. (A) RT and PCR amplification of the CD95L and β-actin transcripts were performed on RNA extracts from freshly isolated GC B cells and from PBLs which had been successively stimulated for 72 h with PHA (5 μg/ml) and 6 h with PMA (10 ng/ml) and ionomycin (0.5 μg/ml) (act. PBL). (B–D) Freshly isolated GC B cells were cultured in complete medium (CM) in the presence or absence of the antagonistic anti-CD95 mAb ZB4 (1 μg/ml). GC B cells were assessed after 1, 2, 3, and 4 h of culture for their mitochondrial transmembrane potential (B), externalization of PS (C), and activation of caspase-3 (D) using a PE-conjugated anti-active caspase-3 Ab. The results are expressed as means of the (B) percent viable cells, (C) annexin V+ cells, and (D) cells with activated caspase-3, calculated from duplicate determinations. The difference between duplicate measurements never exceeded 10% of the mean values. Representative of three distinct experiments.
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Related In: Results  -  Collection

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Figure 6: GC B cells do not produce CD95L. (A) RT and PCR amplification of the CD95L and β-actin transcripts were performed on RNA extracts from freshly isolated GC B cells and from PBLs which had been successively stimulated for 72 h with PHA (5 μg/ml) and 6 h with PMA (10 ng/ml) and ionomycin (0.5 μg/ml) (act. PBL). (B–D) Freshly isolated GC B cells were cultured in complete medium (CM) in the presence or absence of the antagonistic anti-CD95 mAb ZB4 (1 μg/ml). GC B cells were assessed after 1, 2, 3, and 4 h of culture for their mitochondrial transmembrane potential (B), externalization of PS (C), and activation of caspase-3 (D) using a PE-conjugated anti-active caspase-3 Ab. The results are expressed as means of the (B) percent viable cells, (C) annexin V+ cells, and (D) cells with activated caspase-3, calculated from duplicate determinations. The difference between duplicate measurements never exceeded 10% of the mean values. Representative of three distinct experiments.
Mentions: Next we examined whether clustering of CD95 and the subsequent formation of a DISC in GC B cells was consecutive to the binding of CD95L. It has been demonstrated that upon prolonged stimulation through their Ag receptor, T cells can express and release CD95L that promotes their autocrine suicide 26. Two types of experiments were undertaken to test whether the aggregation of CD95 on isolated GC B cells was consecutive to their endogenous production of CD95L. First, we examined by RT-PCR the expression of the CD95L transcript in freshly isolated GC B cells. As shown in Fig. 6 A, the CD95L message was not detectable in GC B cells, whereas it was expressed by PHA-activated PBLs which had been restimulated for 6 h with PMA and ionomycin. Second, we estimated whether blockade of the CD95–CD95L interaction by the antagonistic anti-CD95 mAb ZB4 could prevent apoptosis of GC B cells cultured for 1, 2, 3, and 4 h in the absence of exogenous stimuli. Data shown in Fig. 6bFig. cFig. d, indicate that mAb ZB4 failed to prevent the Δψm drop, PS exposure, and activation of caspase-3 in cultured GC B cells. This suggests that aggregation of CD95 on GC B cells is not because of an autocrine production loop of CD95L. To test the possible involvement of exogenous CD95L in multimerization of CD95, we next examined whether CD95 was physically bound to its ligand in GC B cells. For this purpose, we determined whether CD95L was part of the CD95 DISC in GC B cells. The proteins associated with FADD were thus immunoprecipitated in freshly isolated GC B cells and in the human leukemic T cells H9 which had been treated for 6 h with an anti-CD3 mAb. The H9 cell line was chosen as a positive control since ligation of the TCR–CD3 complex on these cells promotes their apoptosis via the endogenous release of CD95L and its subsequent binding to CD95 27. As shown in Fig. 7, both CD95 and CD95L are coprecipitated with FADD in CD3-activated H9 cells. Two bands migrating respectively as a 40/42-kD species and as a 26-kD species are revealed by the anti-CD95L mAb in the H9 immunoprecipitates. These two bands are likely to represent the membrane-bound and the soluble form of CD95L, respectively. They were also found in OKT3-treated Jurkat T cells (data not shown). In contrast, despite the fact that CD95 was efficiently coprecipitated with FADD in GC B cells, no signal was obtained with the anti-CD95L mAb in these immunoprecipitates. In agreement with our observation that GC B cells lack the CD95L transcript, whole cell lysates prepared from GC B cells were also negative for the expression of the CD95L protein. Altogether, these data suggest that CD95L is not responsible for oligomerization of CD95 and formation of a DISC in GC B cells.

Bottom Line: We found that GC B cells ex vivo display a preformed inactive DISC containing Fas-associated death domain-containing protein (FADD), procaspase-8, and the long isoform of cellular FADD-like IL-1beta-converting enzyme-inhibitory protein (c-FLIP(L)) but not the CD95L.In culture, c-FLIP(L) is rapidly lost from the CD95 DISC unless GC B cells are exposed to the survival signal provided by CD40L.Our results suggest that (a) the death receptor signaling pathway is involved in the affinity maturation of antibodies, and (b) c-FLIP(L) plays an active role in positive selection of B cells in the GC.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), U404 Immunité et Vaccination, Lyon, Cedex 07, France.

ABSTRACT
Affinity maturation of the B cell response to antigen (Ag) takes place in the germinal centers (GCs) of secondary follicles. Two sequential molecular mechanisms underpin this process. First, the B cell repertoire is diversified through hypermutation of the immunoglobulin (Ig) variable region genes. Second, mutant B cell clones with improved affinity for Ag are positively selected by Ag and CD40 ligand (L). This selection step is contingent upon "priming" of GC B cells for apoptosis. The molecular means by which B cell apoptosis is initiated and controlled in the GC remains unclear. Here, we show that GC B cell apoptosis is preceded by the rapid activation of caspase-8 at the level of CD95 death-inducing signaling complex (DISC). We found that GC B cells ex vivo display a preformed inactive DISC containing Fas-associated death domain-containing protein (FADD), procaspase-8, and the long isoform of cellular FADD-like IL-1beta-converting enzyme-inhibitory protein (c-FLIP(L)) but not the CD95L. In culture, c-FLIP(L) is rapidly lost from the CD95 DISC unless GC B cells are exposed to the survival signal provided by CD40L. Our results suggest that (a) the death receptor signaling pathway is involved in the affinity maturation of antibodies, and (b) c-FLIP(L) plays an active role in positive selection of B cells in the GC.

Show MeSH
Related in: MedlinePlus