Context-specific BAFF-R signaling by the NF-κB and PI3K pathways

Abstract

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.

Figure 1. IKK1-deficient mature B cells show normal in vivo survival and BAFF-mediated survival in vitro

(A) Ikk1 deletion was induced in mature B cells by tamoxifen injection of Ikk1^L/LhCD20Tam^Cre+ mice on 3 consecutive days. Ikk1^L/LhCD20Tam^Cre− or Ikk1^+/+ hCD20Tam^Cre+ 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^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^L/LhCD20Tam^Cre+ 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.

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^L/LCD19^Cre+ and control mice. Ikk1^L/LCD19^Cre− or Ikk1^+/+ CD19^Cre+ mice were used as controls (ctrl). B cell subsets were identified by cell surface markers: B220⁺=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^+/+Baff^−/−Cd19^Cre, and Pten^L/LBaff^−/−Cd19^Cre mice. Data are representative of n>8 mice per group. (B) Absolute numbers of splenocytes and splenic B220⁺ 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^+/+Baff^−/−Cd19^Cre, and Pten^L/LBaff^−/−Cd19^Cre mice. MFI=mean fluorescence intensity. (D) ELISA of nitrophenol-specific IgM (top) or IgG (bottom) in the sera of Pten^+/+Baff^+/+Cd19^Cre, Pten^L/LBaff^+/+Cd19^Cre, Pten^+/+Baff^−/−Cd19^Cre, and Pten^L/LBaff^−/−Cd19^Cre 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⁺ 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^+/+Baff^−/−Cd19^Cre, and Pten^L/LBaff^−/−Cd19^Cre B cells following stimulation with indicated mitogens. (B) As in (A), expression of CD86. (C) Purified splenic B cells from Pten^+/+Baff^+/+Cd19^Cre, Pten^L/LBaff^+/+Cd19^Cre, Pten^+/+Baff^−/−Cd19^Cre, and Pten^L/LBaff^−/−Cd19^Cre mice were stimulated as indicated. Proliferation was determined at 48 hours by 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^+/+Cd19^Cre or Pten^L/LCd19^Cre 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⁻PI⁻) cells at each time point. Data are representative of n=3 experiments with 5 mice/group total.

Figure 5. Lymphoma development in Pten^L/LShip^L/LCd19^Cre mice occurs in a BAFF-independent manner

(A) Flow cytometric analysis of B220⁺-gated splenic cells from Pten^+/+Ship^+/+Baff^+/+Cd19^Cre, Pten+/+Ship+/+Baff^−/−Cd19^Cre, Pten^L/LShip^L/LBaff^+/+Cd19^Cre, and Pten^L/LShip^L/LBaff^−/−Cd19^Cre 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 Pten^+/+Ship^+/+Baff^+/+Cd19^Cre (n=7), Pten^L/LShip^L/LBaff^+/+Cd19^Cre (n=6), and Pten^L/LShip^L/LBaff^−/−Cd19^Cre (n=9) mice. (D) Expansion of B220^−/lowCD19⁺ lymphoma B cells in peripheral blood of Pten^+/+Ship^+/+Baff^+/+Cd19^Cre, Pten^L/LShip^L/LBaff^+/+Cd19^Cre, and Pten^L/LShip^L/LBaff^−/−Cd19^Cre mice determined by flow cytometry at indicated time points. Data shown are from two representative animals for each group. The Pten^L/LShip^L/LBaff^+/+Cd19^Cre animal shown in bottom row died before 9 months of age.

Figure 6. Augmented PI3K signaling by BAFF-R does not affect the non-canonical NF-κB pathway and promotes Mcl-1 function

(A) Pten^+/+Baff^+/+Cd19^Cre and Pten^L/LBaff^+/+Cd19^Cre B cells were left untreated, or were cultured in the presence of BAFF or anti-IgM F(ab′)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^+/+Baff^−/−Cd19^Cre, or Pten^L/LBaff^−/−Cd19^Cre splenic B cells probed with anti-pAkt1 (S473), anti-GSK-3β (S9), anti-Mcl-1, or anti-Akt1 antibodies. (C) Pten^+/+Cd19^Cre and Pten^L/LCd19^Cre 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.

Figure 7. BAFF-induced signaling is attenuated in B cells lacking expression of CD19

(A) Western blots of protein lysates from Traf3^+/+Cd19^Cre or Traf3^L/LCd19^Cre 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 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^L/LCD19^Cre/Cre (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 Ikk1^L/LCD19^Cre/Cre samples in total. These measurements were part of the experiments described in Figure 2C, therefore, results shown for Ikk1^L/LCD19^Cre/Cre samples can be directly compared with Ikk1^L/LCD19^Cre/+ samples shown in Figure 2C. (D) Total splenic B cells numbers and cell numbers of indicated B cell subsets from 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^L/LCD19^Cre/Cre 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 Ikk1^L/LCD19^Cre/Cre mice can be directly compared with data from Ikk1^L/LCD19^Cre/+ mice shown in Figure 2A.