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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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c-FLIPL is upregulated in CD40L- or anti-Ig–stimulated GC B cells. (A) Freshly isolated GC B cells (0) were cultured for 12 h in complete medium with (+) or without (−) soluble trimeric CD40L or immobilized anti-Ig Abs. Cell lysates were separated on 10% SDS-PAGE and analyzed sequentially by Western blot for expression of FADD, caspase-8, and c-FLIP. (B and C) GC B cells were cultured for the indicated periods of time with or without soluble trimeric CD40L and processed for analysis of PS exposure (B) or for analysis of c-FLIP expression by Western blot. CM, complete medium. (C) In B, results are expressed as means of the percent apoptotic cells (annexin V+) calculated from duplicate determinations. The difference between duplicate measurements never exceeded 10% of the mean values. The blot shown in C was subjected to densitometry scanning analysis. The relative intensity of c-FLIPL expression at each time point (indicated below the c-FLIP blot) was estimated by the ratio between the intensity of the c-FLIPL and β-actin bands. Representative of three separate experiments.
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Figure 8: c-FLIPL is upregulated in CD40L- or anti-Ig–stimulated GC B cells. (A) Freshly isolated GC B cells (0) were cultured for 12 h in complete medium with (+) or without (−) soluble trimeric CD40L or immobilized anti-Ig Abs. Cell lysates were separated on 10% SDS-PAGE and analyzed sequentially by Western blot for expression of FADD, caspase-8, and c-FLIP. (B and C) GC B cells were cultured for the indicated periods of time with or without soluble trimeric CD40L and processed for analysis of PS exposure (B) or for analysis of c-FLIP expression by Western blot. CM, complete medium. (C) In B, results are expressed as means of the percent apoptotic cells (annexin V+) calculated from duplicate determinations. The difference between duplicate measurements never exceeded 10% of the mean values. The blot shown in C was subjected to densitometry scanning analysis. The relative intensity of c-FLIPL expression at each time point (indicated below the c-FLIP blot) was estimated by the ratio between the intensity of the c-FLIPL and β-actin bands. Representative of three separate experiments.

Mentions: It has been shown previously that engagement of CD40 or BCR protects GC B cells from spontaneous apoptosis in vitro and promotes expression of the bcl-2 protein 28. This finding supported the concept that reinduction of antiapoptotic members of the bcl-2 family was instrumental in positive selection of mutated B cell clones which express a high affinity BCR for the relevant Ag. Since we had found that entry of a GC B cell into apoptosis is correlated with downregulation of c-FLIPL, we next investigated whether the antiapoptotic signals provided by surrogate Ag or CD40L could prevent the loss of c-FLIPL in cultured GC B cells. For this purpose, Western blot analysis of c-FLIPL, FADD, and caspase-8 was carried out in lysates of GC B cells cultured for 12 h in the presence or absence of either trimeric CD40L or immobilized anti-Ig Abs. As expected, CD40L and anti-Ig Abs inhibit both PS exposure and the activation of caspase-3 in GC B cell cultures (data not shown). As shown in Fig. 8 A, the p18 cleavage product of caspase-8 was clearly detectable in cultures of GC B cells carried out in complete medium, whereas expression of c-FLIPL was lost. In contrast, the p18 form of active caspase-8 was undetectable in both CD40L- and anti-Ig–treated cells. Furthermore, inhibition of caspase-8 processing promoted by ligation of CD40 or BCR was tightly correlated with the upregulation of c-FLIPL. To further document that c-FLIPL is instrumental in promoting survival in CD40L-treated cells, time course experiments were carried out in which both PS exposure and expression of c-FLIPL were simultaneously monitored in cultured GC B cells stimulated or not with CD40L. As illustrated by Fig. 8 B, the antiapoptotic effect of CD40L on GC B cells, as estimated by the reduction of the proportion of cells binding annexin V, was not detectable before 4 h of culture. In contrast, reexpression of c-FLIPL revealed by Western blot analysis (Fig. 8 C) was already found within 2 h of CD40L stimulation and thus preceded the inhibition of PS exposure promoted by CD40L. Altogether, these findings suggest that c-FLIPL contributes to the survival signal provided by surrogate Ag or CD40L.


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)

c-FLIPL is upregulated in CD40L- or anti-Ig–stimulated GC B cells. (A) Freshly isolated GC B cells (0) were cultured for 12 h in complete medium with (+) or without (−) soluble trimeric CD40L or immobilized anti-Ig Abs. Cell lysates were separated on 10% SDS-PAGE and analyzed sequentially by Western blot for expression of FADD, caspase-8, and c-FLIP. (B and C) GC B cells were cultured for the indicated periods of time with or without soluble trimeric CD40L and processed for analysis of PS exposure (B) or for analysis of c-FLIP expression by Western blot. CM, complete medium. (C) In B, results are expressed as means of the percent apoptotic cells (annexin V+) calculated from duplicate determinations. The difference between duplicate measurements never exceeded 10% of the mean values. The blot shown in C was subjected to densitometry scanning analysis. The relative intensity of c-FLIPL expression at each time point (indicated below the c-FLIP blot) was estimated by the ratio between the intensity of the c-FLIPL and β-actin bands. Representative of three separate experiments.
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Figure 8: c-FLIPL is upregulated in CD40L- or anti-Ig–stimulated GC B cells. (A) Freshly isolated GC B cells (0) were cultured for 12 h in complete medium with (+) or without (−) soluble trimeric CD40L or immobilized anti-Ig Abs. Cell lysates were separated on 10% SDS-PAGE and analyzed sequentially by Western blot for expression of FADD, caspase-8, and c-FLIP. (B and C) GC B cells were cultured for the indicated periods of time with or without soluble trimeric CD40L and processed for analysis of PS exposure (B) or for analysis of c-FLIP expression by Western blot. CM, complete medium. (C) In B, results are expressed as means of the percent apoptotic cells (annexin V+) calculated from duplicate determinations. The difference between duplicate measurements never exceeded 10% of the mean values. The blot shown in C was subjected to densitometry scanning analysis. The relative intensity of c-FLIPL expression at each time point (indicated below the c-FLIP blot) was estimated by the ratio between the intensity of the c-FLIPL and β-actin bands. Representative of three separate experiments.
Mentions: It has been shown previously that engagement of CD40 or BCR protects GC B cells from spontaneous apoptosis in vitro and promotes expression of the bcl-2 protein 28. This finding supported the concept that reinduction of antiapoptotic members of the bcl-2 family was instrumental in positive selection of mutated B cell clones which express a high affinity BCR for the relevant Ag. Since we had found that entry of a GC B cell into apoptosis is correlated with downregulation of c-FLIPL, we next investigated whether the antiapoptotic signals provided by surrogate Ag or CD40L could prevent the loss of c-FLIPL in cultured GC B cells. For this purpose, Western blot analysis of c-FLIPL, FADD, and caspase-8 was carried out in lysates of GC B cells cultured for 12 h in the presence or absence of either trimeric CD40L or immobilized anti-Ig Abs. As expected, CD40L and anti-Ig Abs inhibit both PS exposure and the activation of caspase-3 in GC B cell cultures (data not shown). As shown in Fig. 8 A, the p18 cleavage product of caspase-8 was clearly detectable in cultures of GC B cells carried out in complete medium, whereas expression of c-FLIPL was lost. In contrast, the p18 form of active caspase-8 was undetectable in both CD40L- and anti-Ig–treated cells. Furthermore, inhibition of caspase-8 processing promoted by ligation of CD40 or BCR was tightly correlated with the upregulation of c-FLIPL. To further document that c-FLIPL is instrumental in promoting survival in CD40L-treated cells, time course experiments were carried out in which both PS exposure and expression of c-FLIPL were simultaneously monitored in cultured GC B cells stimulated or not with CD40L. As illustrated by Fig. 8 B, the antiapoptotic effect of CD40L on GC B cells, as estimated by the reduction of the proportion of cells binding annexin V, was not detectable before 4 h of culture. In contrast, reexpression of c-FLIPL revealed by Western blot analysis (Fig. 8 C) was already found within 2 h of CD40L stimulation and thus preceded the inhibition of PS exposure promoted by CD40L. Altogether, these findings suggest that c-FLIPL contributes to the survival signal provided by surrogate Ag or CD40L.

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.

Show MeSH
Related in: MedlinePlus