Limits...
Differential requirement of MALT1 for BAFF-induced outcomes in B cell subsets.

Tusche MW, Ward LA, Vu F, McCarthy D, Quintela-Fandino M, Ruland J, Gommerman JL, Mak TW - J. Exp. Med. (2009)

Bottom Line: MALT1(-/-) MZ B cells also express higher amounts of TRAF3, a known negative regulator of BAFF receptor-mediated signaling, and TRAF3 was found to interact with MALT1.Furthermore, phenotypes associated with overexpression of BAFF, including increased MZ B cell numbers, elevated serum immunoglobulin titers, and spontaneous germinal center formation, were found to be dependent on B cell-intrinsic MALT1 expression.Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

ABSTRACT
B cell activation factor of the TNF family (BAFF) activates noncanonical nuclear factor kappaB (NF-kappaB) heterodimers that promote B cell survival. We show that although MALT1 is largely dispensable for canonical NF-kappaB signaling downstream of the B cell receptor, the absence of MALT1 results in impaired BAFF-induced phosphorylation of NF-kappaB2 (p100), p100 degradation, and RelB nuclear translocation in B220(+) B cells. This corresponds with impaired survival of MALT1(-/-) marginal zone (MZ) but not follicular B cells in response to BAFF stimulation in vitro. MALT1(-/-) MZ B cells also express higher amounts of TRAF3, a known negative regulator of BAFF receptor-mediated signaling, and TRAF3 was found to interact with MALT1. Furthermore, phenotypes associated with overexpression of BAFF, including increased MZ B cell numbers, elevated serum immunoglobulin titers, and spontaneous germinal center formation, were found to be dependent on B cell-intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction.

Show MeSH

Related in: MedlinePlus

MALT1 is dispensable for B cell proliferation and canonical NF-κB activation induced by BCR engagement. (A) B220+ B cells were isolated from spleens of WT (white bars), Bcl10−/− (black bars), and MALT1−/− (gray bars) mice and stimulated for 24 or 48 h with 5 µg/ml anti-IgM (i), or for 24 h with the indicated doses of anti-IgM (ii) or anti-CD40 (iii). Proliferation was assessed by [3H]thymidine incorporation. Results shown are means ± SD of three replicates per genotype and are representative of three independent analyses. (B) Purified splenic B cells from WT, Bcl10−/−, and MALT1−/− mice were stimulated with 5 µg/ml anti-IgM for the indicated times, and lysates were Western blotted to detect IκBα. Results shown are representative of three independent analyses. (C) Nuclear lysates were prepared from unstimulated B cells or from B cells that had been treated for 8 h with anti-IgM from the indicated genotypes. Lysates were subjected to EMSA to detect up-regulation of binding to NF-κB DNA consensus sequences (p65). Results shown are representative of at least three independent analyses. (D) MZ and FO B cells were sorted from the indicated genotypes. Cells were treated with 5 µg/ml anti-IgM for the indicated times, and viability was assessed using annexin V/PI staining as described. Experiments shown are representative of three independent analyses. (E) Splenocytes from the indicated genotypes were cultured for 12 h with 5 µg/ml anti-IgM, and B7 up-regulation was assessed in the MZ and FO compartments as described in Materials and methods. Anti-IgM–treated samples (blue) were treated with 5 µg/ml for 12 h. Numbers to the left of gels represent kilodaltons.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2806610&req=5

fig1: MALT1 is dispensable for B cell proliferation and canonical NF-κB activation induced by BCR engagement. (A) B220+ B cells were isolated from spleens of WT (white bars), Bcl10−/− (black bars), and MALT1−/− (gray bars) mice and stimulated for 24 or 48 h with 5 µg/ml anti-IgM (i), or for 24 h with the indicated doses of anti-IgM (ii) or anti-CD40 (iii). Proliferation was assessed by [3H]thymidine incorporation. Results shown are means ± SD of three replicates per genotype and are representative of three independent analyses. (B) Purified splenic B cells from WT, Bcl10−/−, and MALT1−/− mice were stimulated with 5 µg/ml anti-IgM for the indicated times, and lysates were Western blotted to detect IκBα. Results shown are representative of three independent analyses. (C) Nuclear lysates were prepared from unstimulated B cells or from B cells that had been treated for 8 h with anti-IgM from the indicated genotypes. Lysates were subjected to EMSA to detect up-regulation of binding to NF-κB DNA consensus sequences (p65). Results shown are representative of at least three independent analyses. (D) MZ and FO B cells were sorted from the indicated genotypes. Cells were treated with 5 µg/ml anti-IgM for the indicated times, and viability was assessed using annexin V/PI staining as described. Experiments shown are representative of three independent analyses. (E) Splenocytes from the indicated genotypes were cultured for 12 h with 5 µg/ml anti-IgM, and B7 up-regulation was assessed in the MZ and FO compartments as described in Materials and methods. Anti-IgM–treated samples (blue) were treated with 5 µg/ml for 12 h. Numbers to the left of gels represent kilodaltons.

Mentions: Several groups have shown that MALT1 is necessary for MZ and B1 B cell development, whereas Bcl10 leads to a more profound developmental defect affecting FO, MZ, and B1 B cells (Ruefli-Brasse et al., 2003; Ruland et al., 2003; Xue et al., 2003). Though Bcl10 has been shown to impinge on the activation of NF-κB downstream of both TCR and BCR and is essential for B cell survival, MALT1 appears to play a more subtle role in B cell survival after BCR engagement, and its role in the survival of FO versus MZ B cells has not been determined (Ruefli-Brasse et al., 2003; Ruland et al., 2003). We first examined whether MALT1 is required for B cell proliferation triggered by BCR engagement by stimulating purified, resting B220+ B cells from WT, MALT1−/−, and Bcl10−/− mice with anti-IgM and monitoring [3H]thymidine incorporation. As expected, the proliferation of Bcl10−/− B cells was significantly reduced, but no statistically significant difference in proliferation was noted between WT and MALT1−/− B cells (Fig. 1 A, i). MALT1+/+ and MALT1−/− B cells were also treated with increasing doses of anti-IgM, and proliferation was assessed at 24 h (Fig. 1 A, ii). No significant differences were noted between genotypes. Consistent with this result, anti-IgM treatment stimulated IκBα degradation in WT and MALT1−/− B cells but not in Bcl10−/− B cells (Fig. 1 B). Furthermore, anti-IgM induced the up-regulation of p65-dependent DNA binding in WT and MALT1−/− B cells but not in Bcl10−/− B cells (Fig. 1 C). The CD40 pathway is also important in B cell activation and proliferation. We therefore also examined the role of MALT1 in anti-CD40–mediated B cell proliferation. We found no differences in B cell proliferation in response to anti-CD40 treatment of WT versus MALT1−/− B cells (Fig. 1 A, iii). Furthermore, CD40L stimulation also induced IκBα degradation and p100 processing in WT and MALT1−/− B cells (Fig. S1, A and B). Therefore, our data imply that CD40-mediated signaling pathways are intact in MALT1−/− B cells.


Differential requirement of MALT1 for BAFF-induced outcomes in B cell subsets.

Tusche MW, Ward LA, Vu F, McCarthy D, Quintela-Fandino M, Ruland J, Gommerman JL, Mak TW - J. Exp. Med. (2009)

MALT1 is dispensable for B cell proliferation and canonical NF-κB activation induced by BCR engagement. (A) B220+ B cells were isolated from spleens of WT (white bars), Bcl10−/− (black bars), and MALT1−/− (gray bars) mice and stimulated for 24 or 48 h with 5 µg/ml anti-IgM (i), or for 24 h with the indicated doses of anti-IgM (ii) or anti-CD40 (iii). Proliferation was assessed by [3H]thymidine incorporation. Results shown are means ± SD of three replicates per genotype and are representative of three independent analyses. (B) Purified splenic B cells from WT, Bcl10−/−, and MALT1−/− mice were stimulated with 5 µg/ml anti-IgM for the indicated times, and lysates were Western blotted to detect IκBα. Results shown are representative of three independent analyses. (C) Nuclear lysates were prepared from unstimulated B cells or from B cells that had been treated for 8 h with anti-IgM from the indicated genotypes. Lysates were subjected to EMSA to detect up-regulation of binding to NF-κB DNA consensus sequences (p65). Results shown are representative of at least three independent analyses. (D) MZ and FO B cells were sorted from the indicated genotypes. Cells were treated with 5 µg/ml anti-IgM for the indicated times, and viability was assessed using annexin V/PI staining as described. Experiments shown are representative of three independent analyses. (E) Splenocytes from the indicated genotypes were cultured for 12 h with 5 µg/ml anti-IgM, and B7 up-regulation was assessed in the MZ and FO compartments as described in Materials and methods. Anti-IgM–treated samples (blue) were treated with 5 µg/ml for 12 h. Numbers to the left of gels represent kilodaltons.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806610&req=5

fig1: MALT1 is dispensable for B cell proliferation and canonical NF-κB activation induced by BCR engagement. (A) B220+ B cells were isolated from spleens of WT (white bars), Bcl10−/− (black bars), and MALT1−/− (gray bars) mice and stimulated for 24 or 48 h with 5 µg/ml anti-IgM (i), or for 24 h with the indicated doses of anti-IgM (ii) or anti-CD40 (iii). Proliferation was assessed by [3H]thymidine incorporation. Results shown are means ± SD of three replicates per genotype and are representative of three independent analyses. (B) Purified splenic B cells from WT, Bcl10−/−, and MALT1−/− mice were stimulated with 5 µg/ml anti-IgM for the indicated times, and lysates were Western blotted to detect IκBα. Results shown are representative of three independent analyses. (C) Nuclear lysates were prepared from unstimulated B cells or from B cells that had been treated for 8 h with anti-IgM from the indicated genotypes. Lysates were subjected to EMSA to detect up-regulation of binding to NF-κB DNA consensus sequences (p65). Results shown are representative of at least three independent analyses. (D) MZ and FO B cells were sorted from the indicated genotypes. Cells were treated with 5 µg/ml anti-IgM for the indicated times, and viability was assessed using annexin V/PI staining as described. Experiments shown are representative of three independent analyses. (E) Splenocytes from the indicated genotypes were cultured for 12 h with 5 µg/ml anti-IgM, and B7 up-regulation was assessed in the MZ and FO compartments as described in Materials and methods. Anti-IgM–treated samples (blue) were treated with 5 µg/ml for 12 h. Numbers to the left of gels represent kilodaltons.
Mentions: Several groups have shown that MALT1 is necessary for MZ and B1 B cell development, whereas Bcl10 leads to a more profound developmental defect affecting FO, MZ, and B1 B cells (Ruefli-Brasse et al., 2003; Ruland et al., 2003; Xue et al., 2003). Though Bcl10 has been shown to impinge on the activation of NF-κB downstream of both TCR and BCR and is essential for B cell survival, MALT1 appears to play a more subtle role in B cell survival after BCR engagement, and its role in the survival of FO versus MZ B cells has not been determined (Ruefli-Brasse et al., 2003; Ruland et al., 2003). We first examined whether MALT1 is required for B cell proliferation triggered by BCR engagement by stimulating purified, resting B220+ B cells from WT, MALT1−/−, and Bcl10−/− mice with anti-IgM and monitoring [3H]thymidine incorporation. As expected, the proliferation of Bcl10−/− B cells was significantly reduced, but no statistically significant difference in proliferation was noted between WT and MALT1−/− B cells (Fig. 1 A, i). MALT1+/+ and MALT1−/− B cells were also treated with increasing doses of anti-IgM, and proliferation was assessed at 24 h (Fig. 1 A, ii). No significant differences were noted between genotypes. Consistent with this result, anti-IgM treatment stimulated IκBα degradation in WT and MALT1−/− B cells but not in Bcl10−/− B cells (Fig. 1 B). Furthermore, anti-IgM induced the up-regulation of p65-dependent DNA binding in WT and MALT1−/− B cells but not in Bcl10−/− B cells (Fig. 1 C). The CD40 pathway is also important in B cell activation and proliferation. We therefore also examined the role of MALT1 in anti-CD40–mediated B cell proliferation. We found no differences in B cell proliferation in response to anti-CD40 treatment of WT versus MALT1−/− B cells (Fig. 1 A, iii). Furthermore, CD40L stimulation also induced IκBα degradation and p100 processing in WT and MALT1−/− B cells (Fig. S1, A and B). Therefore, our data imply that CD40-mediated signaling pathways are intact in MALT1−/− B cells.

Bottom Line: MALT1(-/-) MZ B cells also express higher amounts of TRAF3, a known negative regulator of BAFF receptor-mediated signaling, and TRAF3 was found to interact with MALT1.Furthermore, phenotypes associated with overexpression of BAFF, including increased MZ B cell numbers, elevated serum immunoglobulin titers, and spontaneous germinal center formation, were found to be dependent on B cell-intrinsic MALT1 expression.Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

ABSTRACT
B cell activation factor of the TNF family (BAFF) activates noncanonical nuclear factor kappaB (NF-kappaB) heterodimers that promote B cell survival. We show that although MALT1 is largely dispensable for canonical NF-kappaB signaling downstream of the B cell receptor, the absence of MALT1 results in impaired BAFF-induced phosphorylation of NF-kappaB2 (p100), p100 degradation, and RelB nuclear translocation in B220(+) B cells. This corresponds with impaired survival of MALT1(-/-) marginal zone (MZ) but not follicular B cells in response to BAFF stimulation in vitro. MALT1(-/-) MZ B cells also express higher amounts of TRAF3, a known negative regulator of BAFF receptor-mediated signaling, and TRAF3 was found to interact with MALT1. Furthermore, phenotypes associated with overexpression of BAFF, including increased MZ B cell numbers, elevated serum immunoglobulin titers, and spontaneous germinal center formation, were found to be dependent on B cell-intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction.

Show MeSH
Related in: MedlinePlus