. 2013 Nov 27;5(4):1022-35.
Epub 2013 Nov 14.
Context-specific BAFF-R Signaling by the NF-κB and PI3K Pathways
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Context-specific BAFF-R Signaling by the NF-κB and PI3K Pathways
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BAFF is a soluble factor required for B cell maturation and survival. BAFF-R signals via the noncanonical NF-κB pathway regulated by the TRAF3/NIK/IKK1 axis. We show that deletion of Ikk1 during early B cell development causes a partial impairment in B cell maturation and BAFF-dependent survival, but inactivation of Ikk1 in mature B cells does not affect survival. We further show that BAFF-R employs CD19 to promote survival via phosphatidylinositol 3-kinase (PI3K), and that coinactivation of Cd19 and Ikk1 causes a profound block in B cell maturation at the transitional stage. Consistent with a role for PI3K in BAFF-R function, inactivation of PTEN mediates a partial rescue of B cell maturation and function in Baff(-/-) animals. Elevated PI3K signaling also circumvents BAFF-dependent survival in a spontaneous B cell lymphoma model. These findings indicate that the combined activities of PI3K and IKK1 drive peripheral B cell differentiation and survival in a context-dependent manner.
Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.
Figure 1. IKK1-deficient mature B cells show normal
in vivo survival and BAFF-mediated survival in vitro
Ikk1 deletion was induced in mature B cells by tamoxifen injection of Ikk1 mice on 3 consecutive days. L/LhCD20Tam Cre+ Ikk1 L/LhCD20Tam Cre − or Ikk1 mice were used as controls (ctrl). Mice were sacrificed 1 week or 2 weeks after the last tamoxifen injection and the percentage of B cells in the spleen was determined by flow cytometry. Graphs show means +SD from 3 independent experiments. (B) The percentage of YFP+ B cells 7 days after tamoxifen injection was comparable between CD21 +/+ hCD20Tam Cre+ intCD23 hi follicular B cells and CD21 hiCD23 int/low MZ B cells. YFP expression was not detected in non-B cells (B220 −). Shown is a representative of n=2 experiments. (C) To study BAFF-mediated survival in vitro, mice were sacrificed after the last tamoxifen injection, and B cells were purified and stimulated with 10 ng/mL BAFF. The percentage of viable B cells 3 days or 5 days after culture was determined by flow cytometry. Graphs show mean +SD from 3 independent experiments. (D) Splenic B cells from tamoxifen-treated Ikk1 mice were stimulated over night with 25 ng/mL BAFF or incubated in medium alone. p100 cleavage and p52 generation were visualized by western blotting. Absence of IKK1 in Cre+ cells (YFP+) was confirmed by western blot analysis. Actin was used as loading control. Shown is a representative of n=2 experiments. L/LhCD20Tam Cre+
Figure 2. IKK1 deletion early in B cell development results in an incomplete block in B cell maturation
(A) Graphs show total cell numbers of B cells (left panel) and B cell subsets (middle and right panel) in spleens obtained from
Ikk1 and control mice. L/LCD19 Cre+ Ikk1 or L/LCD19 Cre− Ikk1 mice were used as controls (ctrl). B cell subsets were identified by cell surface markers: B220 +/+ CD19 Cre+ +=total B cells, B220 +CD21 intIgM low=mature B cells, B220 +CD21 lowIgM hi=T1 B cells, B220 +CD21 hiCD23 hiIgM hi=T2 B cells, B220 +CD21 hiCD23 int/low=MZ B cells. (B) B cell maturation in the spleen was analyzed by flow cytometry. Plots are representative of >11 mice analyzed. (C) Mice were continuously provided BrdU in the drinking water and euthanized after 7d, 14d or 21d of treatment. Cells were harvested from the spleen and the bone marrow and stained with an anti-BrdU antibody and for surface markers as follows: (left) splenic follicular (B220 +, IgM +, CD23 hi, CD21 lo) B cells; (center) bone marrow B cell progenitors (B220 +, IgM −, IgD −); (right) recirculating mature B cells (B220 +, IgD +, IgM lo) in the bone marrow. Four experimental and CD19Cre control mice (10–15w old) were used per time point and rates of turnover calculated by linear regression analysis. (D) B cells from spleens enriched for mature B cells (CD23 +CD43 −), or from LNs (B220 +CD43 −) were stimulated with 10 ng/mL BAFF and the percentage of viable cells was determined by flow cytometry after 3 days and/or 5 days in culture. The graph summarizes n=7 samples for each genotype and time point. (E) Protein lysates from freshly-isolated splenic B cells were assayed for p100 cleavage by western blotting (left panel). p100 processing to p52 in LN B cells stimulated overnight with 25 ng/mL BAFF versus unstimulated cells (right panel).
Figure 3. Constitutively-active PI3K restores B cell development in
Baff mice −/−
(A) Flow cytometry of B220+ splenic cells from
Pten, +/+Baff +/+Cd19 Cre Pten, L/LBaff +/+Cd19 Cre Pten, and +/+Baff −/−Cd19 Cre Pten mice. Data are representative of n>8 mice per group. (B) Absolute numbers of splenocytes and splenic B220 L/LBaff −/−Cd19 Cre + B cells (top panel), and splenic B cell subsets (bottom panel). Shown is n=7 mice per group from n=5 experiments; small horizontal lines indicate mean. (C) Expression of CD21/35 on B220 +-gated IgM loIgD hi splenic B cells from Pten, +/+Baff +/+Cd19 Cre Pten, L/LBaff +/+Cd19 Cre Pten, and +/+Baff −/−Cd19 Cre Pten mice. MFI=mean fluorescence intensity. (D) ELISA of nitrophenol-specific IgM (top) or IgG (bottom) in the sera of L/LBaff −/−Cd19 Cre Pten, +/+Baff +/+Cd19 Cre Pten, L/LBaff +/+Cd19 Cre Pten, and +/+Baff −/−Cd19 Cre Pten mice prior to immunization (day 0), and 7 or 14 days post-immunization with 100 μg NP-KLH in alum. (E) Flow cytometric analysis of splenic GC B cells (B220 L/LBaff −/−Cd19 Cre + gated) from immunized mice (top). Graph summarizes the percent of B220 +GL7 +Fas + B cells 14 days post-immunization (bottom).
Figure 4. Upregulation of activation markers and proliferation are restored in
Baff B cells lacking −/− Pten
(A) Flow cytometric analysis of CD69 expression on
Pten, +/+Baff +/+Cd19 Cre Pten, L/LBaff +/+Cd19 Cre Pten, and +/+Baff −/−Cd19 Cre Pten B cells following stimulation with indicated mitogens. (B) As in (A), expression of CD86. (C) Purified splenic B cells from L/LBaff −/−Cd19 Cre Pten, +/+Baff +/+Cd19 Cre Pten, L/LBaff +/+Cd19 Cre Pten, and +/+Baff −/−Cd19 Cre Pten mice were stimulated as indicated. Proliferation was determined at 48 hours by L/LBaff −/−Cd19 Cre 3H-thymidine incorporation. All assays were conducted in triplicate and SDs are shown as error bars. Data are representative of 3 independent experiments with n=2 mice per group per experiment. (D) Pten or +/+Cd19 Cre Pten mature lymph node B cells were left untreated or were cultured in the presence of BAFF, and cell viability was assessed by Annexin V (AnnV) and propidium iodide (PI) staining. Graph shows the percent of live (AnnV L/LCd19 Cre −PI −) cells at each time point. Data are representative of n=3 experiments with 5 mice/group total.
Figure 5. Lymphoma development in
Pten mice occurs in a BAFF-independent manner L/LShip L/LCd19 Cre
(A) Flow cytometric analysis of B220
+-gated splenic cells from Pten, Pten+/+Ship+/+ +/+Ship +/+Baff +/+Cd19 Cre Baff, −/−Cd19 Cre Pten, and L/LShip L/LBaff +/+Cd19 Cre Pten mice. Data are representative of 2 independent experiments with n≥2–3 mice per group. (B) Absolute numbers of splenocytes and splenic B cells (n=3 mice per group; small horizontal lines indicate mean). (C) Kaplan-Meier survival curve of L/LShip L/LBaff −/−Cd19 Cre Pten (n=7), +/+Ship +/+Baff +/+Cd19 Cre Pten (n=6), and L/LShip L/LBaff +/+Cd19 Cre Pten (n=9) mice. (D) Expansion of B220 L/LShip L/LBaff −/−Cd19 Cre −/lowCD19 + lymphoma B cells in peripheral blood of Pten, +/+Ship +/+Baff +/+Cd19 Cre Pten, and L/LShip L/LBaff +/+Cd19 Cre Pten mice determined by flow cytometry at indicated time points. Data shown are from two representative animals for each group. The L/LShip L/LBaff −/−Cd19 Cre Pten animal shown in bottom row died before 9 months of age. L/LShip L/LBaff +/+Cd19 Cre
Figure 6. Augmented PI3K signaling by BAFF-R does not affect the non-canonical NF-κB pathway and promotes Mcl-1 function
Pten and +/+Baff +/+Cd19 Cre Pten B cells were left untreated, or were cultured in the presence of BAFF or anti-IgM F(ab′) L/LBaff +/+Cd19 Cre 2 fragments. Activation of non-canonical NF-κB was determined by western blotting with antibodies against p100/p52. Membranes were stripped and reprobed for actin as a loading control. (B) Western blots of protein lysates from freshly-isolated Pten, +/+Baff +/+Cd19 Cre Pten, L/LBaff +/+Cd19 Cre Pten, or +/+Baff −/−Cd19 Cre Pten splenic B cells probed with anti-pAkt1 (S473), anti-GSK-3β (S9), anti-Mcl-1, or anti-Akt1 antibodies. (C) L/LBaff −/−Cd19 Cre Pten and +/+Cd19 Cre Pten B cells were left untreated or were treated with BAFF. Lysates were generated and Mcl-1 was immunoprecipitated. Immunoprecipitates were resolved by SDS-PAGE and membranes probed with antibodies against Bim and Mcl-1. L/LCd19 Cre
Figure 7. BAFF-induced signaling is attenuated in B cells lacking expression of CD19
(A) Western blots of protein lysates from
Traf3 or +/+Cd19 Cre Traf3 splenic B cells treated for indicated time points with BAFF were probed with anti-pAkt1 (S473) or anti-tAkt1 antibodies. (B) Western blots of protein lysates from L/LCd19 Cre Cd19 or +/+ Cd19 splenic B cells treated for indicated time points with 25 ng/mL BAFF were probed with anti-pCD19 (Y513), anti-CD19, anti-pAkt1 (S473), or anti-tAkt1 antibodies. (C) −/− Cd19 or +/+ Cd19 LN B cells were left untreated or were cultured in the presence of 25ng/mL BAFF and the percentage of viable cells was determined by flow cytometry after 3 days and/or 5 days in culture (left panel). Graphs summarize data from 3 individual mice in technical triplicates per genotype. LN B cells from −/− Ikk1 (IKK1 and CD19 double deficient) and control mice were treated with 10ng/mL BAFF or were cultured in medium alone and cell viability was assessed 3 days later (right panel). Graphs summarize results from 3 independent experiments with 7 control samples and 3 L/LCD19 Cre/Cre Ikk1 samples in total. These measurements were part of the experiments described in Figure 2C, therefore, results shown for L/LCD19 Cre/Cre Ikk1 samples can be directly compared with L/LCD19 Cre/Cre Ikk1 samples shown in Figure 2C. (D) Total splenic B cells numbers and cell numbers of indicated B cell subsets from L/LCD19 Cre/+ CD19 and control mice are shown in the top panel. B cell subsets were defined as in Figure 2A. Analysis of total cell numbers for −/− Ikk1 and control mice is shown in the bottom panel. These mice were analyzed in parallel with mice presented in Figure 2A, therefore, results shown for L/LCD19 Cre/Cre Ikk1 mice can be directly compared with data from L/LCD19 Cre/Cre Ikk1 mice shown in Figure 2A. L/LCD19 Cre/+
All figures (7)
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Antigens, CD19 / immunology
B-Cell Activating Factor / genetics
B-Cell Activating Factor / immunology*
B-Cell Activation Factor Receptor / immunology
B-Lymphocytes / immunology
Cell Survival / immunology
I-kappa B Kinase / genetics
I-kappa B Kinase / immunology*
Lymphocyte Activation / immunology
Lymphoma, B-Cell / genetics
Lymphopoiesis / immunology*
NF-kappa B p52 Subunit / immunology
PTEN Phosphohydrolase / genetics
PTEN Phosphohydrolase / immunology
Phosphatidylinositol 3-Kinases / immunology*
Signal Transduction / immunology
TNF Receptor-Associated Factor 3 / genetics
TNF Receptor-Associated Factor 3 / immunology
B-Cell Activation Factor Receptor
TNF Receptor-Associated Factor 3
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