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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: Second, mutant B cell clones with improved affinity for Ag are positively selected by Ag and CD40 ligand (L).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.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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Composition of the CD95 DISC in freshly isolated and cultured GC B cells. (A) CD95 or (B) FADD was immunoprecipitated (IP) from 107 freshly isolated GC B cells or at different times after setting the same cells in culture in complete medium as described in Materials and Methods. The immunoprecipitates were washed, subjected to 10% SDS PAGE, and successively probed with the anti–caspase-8, FADD, CD95, and c-FLIP Abs. Representative of three experiments.
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Figure 5: Composition of the CD95 DISC in freshly isolated and cultured GC B cells. (A) CD95 or (B) FADD was immunoprecipitated (IP) from 107 freshly isolated GC B cells or at different times after setting the same cells in culture in complete medium as described in Materials and Methods. The immunoprecipitates were washed, subjected to 10% SDS PAGE, and successively probed with the anti–caspase-8, FADD, CD95, and c-FLIP Abs. Representative of three experiments.

Mentions: Two cell types, each preferentially using one of two different CD95 signaling pathways, have been identified 25. In type I cells, cleavage of caspase-8 occurs at the level of the DISC within minutes of CD95 ligation, before the loss of Δψm. In type II cells, DISC formation is strongly reduced, and activation of caspase-8 is delayed and mainly occurs downstream of the mitochondria, consecutive to Δψm disruption. Although mitochondria are perturbed in both cell types, only type II cells depend on the proapoptotic function of the mitochondria to execute the death program initiated by ligation of CD95. Two observations suggested that caspase-8 could be activated through its association with a death receptor signaling complex in GC B cells. First, the p16/p18 cleavage products of caspase-8 are detectable very early after the onset of GC B cell cultures. Second, the caspase-8 inhibitory peptide z-IETD prevented not only PS exposure but also the mitochondrial alterations in cultured GC B cells (data not shown). These findings led us to test the hypothesis that caspase-8 could be activated by association with the CD95 DISC. For this purpose, the proteins associated with either CD95 or FADD were immunoprecipitated in freshly isolated GC B cells or in GC B cells cultured in complete medium without exogenous stimuli for different lengths of time. Both CD95 and FADD immunoprecipitates were analyzed by Western blotting using monoclonal or polyclonal Abs directed against the components of the DISC (CD95, FADD, and caspase-8) or against the natural antagonist of the death receptor signaling pathway c-FLIP. As illustrated by Fig. 5 A, FADD, the proform of caspase-8, and c-FLIPL are found in the CD95 immunoprecipitates obtained from freshly isolated GC B cells. c-FLIPS was below the threshold of detection in all experiments performed. Upon culturing in complete medium, CD95 remains associated with FADD and caspase-8 but there is already a strong reduction of c-FLIPL expression in the CD95 immunoprecipitates at the earliest time point (10 min). From 30 min of culture onwards, c-FLIPL is no longer associated with the CD95 DISC. Expression of c-FLIP in the FADD immunoprecipitates could not be assessed because both the anti-FADD and anti–c-FLIP mAbs are of the same isotype. However, examination of the FADD immunoprecipitates (Fig. 5 B) confirmed that FADD, CD95, and procaspase-8 are readily associated in GC B cells ex vivo. Altogether, these results provide several pieces of information. First, GC B cells contain a preformed CD95 DISC which includes FADD, the zymogen form of caspase-8, and the natural CD95 antagonist c-FLIPL. Second, caspase-8 is activated at the level of the CD95 DISC during spontaneous GC B cell apoptosis. Third, c-FLIPL disappears from the multimolecular CD95 signaling complex within minutes of in vitro culturing.


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)

Composition of the CD95 DISC in freshly isolated and cultured GC B cells. (A) CD95 or (B) FADD was immunoprecipitated (IP) from 107 freshly isolated GC B cells or at different times after setting the same cells in culture in complete medium as described in Materials and Methods. The immunoprecipitates were washed, subjected to 10% SDS PAGE, and successively probed with the anti–caspase-8, FADD, CD95, and c-FLIP Abs. Representative of three experiments.
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Related In: Results  -  Collection

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Figure 5: Composition of the CD95 DISC in freshly isolated and cultured GC B cells. (A) CD95 or (B) FADD was immunoprecipitated (IP) from 107 freshly isolated GC B cells or at different times after setting the same cells in culture in complete medium as described in Materials and Methods. The immunoprecipitates were washed, subjected to 10% SDS PAGE, and successively probed with the anti–caspase-8, FADD, CD95, and c-FLIP Abs. Representative of three experiments.
Mentions: Two cell types, each preferentially using one of two different CD95 signaling pathways, have been identified 25. In type I cells, cleavage of caspase-8 occurs at the level of the DISC within minutes of CD95 ligation, before the loss of Δψm. In type II cells, DISC formation is strongly reduced, and activation of caspase-8 is delayed and mainly occurs downstream of the mitochondria, consecutive to Δψm disruption. Although mitochondria are perturbed in both cell types, only type II cells depend on the proapoptotic function of the mitochondria to execute the death program initiated by ligation of CD95. Two observations suggested that caspase-8 could be activated through its association with a death receptor signaling complex in GC B cells. First, the p16/p18 cleavage products of caspase-8 are detectable very early after the onset of GC B cell cultures. Second, the caspase-8 inhibitory peptide z-IETD prevented not only PS exposure but also the mitochondrial alterations in cultured GC B cells (data not shown). These findings led us to test the hypothesis that caspase-8 could be activated by association with the CD95 DISC. For this purpose, the proteins associated with either CD95 or FADD were immunoprecipitated in freshly isolated GC B cells or in GC B cells cultured in complete medium without exogenous stimuli for different lengths of time. Both CD95 and FADD immunoprecipitates were analyzed by Western blotting using monoclonal or polyclonal Abs directed against the components of the DISC (CD95, FADD, and caspase-8) or against the natural antagonist of the death receptor signaling pathway c-FLIP. As illustrated by Fig. 5 A, FADD, the proform of caspase-8, and c-FLIPL are found in the CD95 immunoprecipitates obtained from freshly isolated GC B cells. c-FLIPS was below the threshold of detection in all experiments performed. Upon culturing in complete medium, CD95 remains associated with FADD and caspase-8 but there is already a strong reduction of c-FLIPL expression in the CD95 immunoprecipitates at the earliest time point (10 min). From 30 min of culture onwards, c-FLIPL is no longer associated with the CD95 DISC. Expression of c-FLIP in the FADD immunoprecipitates could not be assessed because both the anti-FADD and anti–c-FLIP mAbs are of the same isotype. However, examination of the FADD immunoprecipitates (Fig. 5 B) confirmed that FADD, CD95, and procaspase-8 are readily associated in GC B cells ex vivo. Altogether, these results provide several pieces of information. First, GC B cells contain a preformed CD95 DISC which includes FADD, the zymogen form of caspase-8, and the natural CD95 antagonist c-FLIPL. Second, caspase-8 is activated at the level of the CD95 DISC during spontaneous GC B cell apoptosis. Third, c-FLIPL disappears from the multimolecular CD95 signaling complex within minutes of in vitro culturing.

Bottom Line: Second, mutant B cell clones with improved affinity for Ag are positively selected by Ag and CD40 ligand (L).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.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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