BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway

The B cell activating factor belonging to the tumor necrosis factor family (BAFF) is required for B cell survival and maturation. The mechanisms by which BAFF mediates B cell survival are less understood. We found that BAFF and a proliferation-inducing ligand (APRIL), which are related, block B cell antigen receptor (BCR)–induced apoptosis upstream of mitochondrial damage, which is consistent with a role for Bcl-2 family proteins. BCR ligation strongly increased expression of the proapoptotic Bcl-2 homology 3–only Bcl-2 protein Bim in both WEHI-231 and splenic B cells, and increases in Bim were reversed by BAFF or APRIL. Small interfering RNA vector–mediated suppression of Bim blocked BCR-induced apoptosis. BAFF also induced Bim phosphorylation and inhibited BCR-induced association of Bim with Bcl-2. BAFF induced delayed but sustained stimulation of extracellular signal–regulated kinase (ERK) and its activators, mitogen-activated protein kinase/ERK activating kinase (MEK) and c-Raf, and MEK inhibitors promoted accumulation and dephosphorylation of Bim. These results suggest that BAFF inhibits BCR-induced death by down-regulating Bim via sustained ERK activation, demonstrating that BAFF directly regulates Bim function. Although transitional immature type 1 (T1) B cell numbers are normal in Bim−/− mice, T2 and follicular mature B cells are elevated and marginal zone B cells are reduced. Our results suggest that mature B cell homeostasis is maintained by BAFF-mediated regulation of Bim

B cell-activating factor (BAFF) from dendritic cells, monocytes and neutrophils is required for B cell maturation and autoantibody production in SLE-like autoimmune disease

Purpose and methodsB cell-activating factor (BAFF) contributes to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Although several anti-BAFF Abs and derivatives have been developed for the treatment of SLE, the specific sources of BAFF that sustain autoantibody (auto-Ab) producing cells have not been definitively identified. Using BAFF-RFP reporter mice, we identified major changes in BAFF-producing cells in two mouse spontaneous lupus models (Tlr7 Tg mice and Sle1), and in a pristane-induced lupus (PIL) model.ResultsFirst, we confirmed that similar to their wildtype Tlr7 Tg and Sle1 mice counterparts, BAFF-RFP Tlr7 Tg mice and BAFF-RFP Sle1 mice had increased BAFF serum levels, which correlated with increases in plasma cells and auto-Ab production. Next, using the RFP reporter, we defined which cells had dysregulated BAFF production. BAFF-producing neutrophils (Nphs), monocytes (MOs), cDCs, T cells and B cells were all expanded in the spleens of BAFF-RFP Tlr7 Tg mice and BAFF-RFP Sle1 mice compared to controls. Furthermore, Ly6Chi inflammatory MOs and T cells had significantly increased BAFF expression per cell in both spontaneous lupus models, while CD8- DCs up-regulated BAFF expression only in the Tlr7 Tg mice. Similarly, pristane injection of BAFF-RFP mice induced increases in serum BAFF levels, auto-Abs, and the expansion of BAFF-producing Nphs, MOs, and DCs in both the spleen and peritoneal cavity. BAFF expression in MOs and DCs, in contrast to BAFF from Nphs, was required to maintain homeostatic and pristane-induced systemic BAFF levels and to sustain mature B cell pools in spleens and BMs. Although acting through different mechanisms, Nph, MO and DC sources of BAFF were each required for the development of auto-Abs in PIL mice.ConclusionsOur findings underscore the importance of considering the relative roles of specific myeloid BAFF sources and B cell niches when developing treatments for SLE and other BAFF-associated autoimmune diseases

CD22 is required for formation of memory B cell precursors within germinal centers.

CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens

CD22-deficient B cells fail to form CD38^hiCXCR4^hi GC B cells.

<p>Adoptive transfers and immunizations were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174661#pone.0174661.g001" target="_blank">Fig 1</a>. (<i>A</i>) Representative flow cytometric plots show CXCR4 and CD86 expression among NP-specific GL7⁺ splenic B cells gated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174661#pone.0174661.g002" target="_blank">Fig 2A</a> 7 days p.i. (<i>B</i>) Summary data from 2 independent experiments using 3 mice per group showing the frequency of NP-specific GL7⁺ LZ (CXCR4^loCD86^hi) B cells 7 days p.i. Each dot represents an individual animal. (<i>C</i>,<i>E</i>) Representative flow plots show the frequency of cells expressing CD38 (<i>C</i>) or CD38 and CXCR4 (<i>E</i>) among NP-specific GL7⁺ B cells 7 days p.i. (<i>D</i>,<i>F</i>) Summary data of CD38 (<i>C</i>) or CD38 and CXCR4 expression (<i>E</i>) from 3 independent experiments using 2–3 mice per group. Each dot represents an individual animal. (<i>G</i>) Histograms show PNA, CCR6 or CD95 expression on each population (P1-P4) of NP-specific GL7⁺ splenic B cells depicted in the diagram at left. Total (shaded), WT (solid line) and CD22^-/- (dashed line) B cells are shown. Data are representative of 2 independent experiments using 5–7 mice each. (<i>H</i>) Frequencies of WT (white circles) and CD22-/- (black circles) NP-specific GL7⁺CD38^hiCXCR4^hi splenic B cells are plotted over time. Data are from one experiment utilizing 7 mice/group at each time point. (<i>I</i>) Summary data depicting the frequency of CD38^hiCXCR4^hi GC B cells in WT and CD22^-/- mice (<i>E</i>) from 2 independent experiments using 4–6 mice per group. Each dot represents an individual animal.</p

CD22-deficient GC B cells are capable of Ag processing and presentation.

<p>Recipient mice were immunized with 50 micrograms NP-CGG and 6 days later injected i.v. with 5 micrograms NP-streptavidin-I-Ealpha or streptavidin-I-Ealpha. 18 h later mice were sacrificed and spleens analyzed by flow cytometry. (<i>A</i>) Representative analysis of peptide:MHCII expression among NP-specific GL7⁺ WT (solid lines) and CD22^-/- (dashed lines) B cells. Total B220⁺ cells are depicted by shaded histograms for reference. (<i>B</i>) Summarized data from experiment in (<i>A</i>). Each dot represents a single animal with the mean indicated by a horizontal line. Data are representative of 2 independent experiments using 3–7 mice each.</p

CD22-deficient B cells expand normally and undergo germinal center differentiation.

<p>Adoptive transfers and immunizations were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174661#pone.0174661.g001" target="_blank">Fig 1A/1B</a>. (A) Representative flow cytometry analysis showing the frequency of WT and CD22^-/- splenic B1-8^hi B cells 7 days p.i. Data from 3 independent experiments are summarized in (<i>B</i>). Each dot represents a single animal with the mean indicated by a horizontal line. (<i>C</i>) Representative flow cytometric plots show the frequency of NP-specific splenic B cells (gated as in <i>A</i>) that co-express GL7 and CD95 7 days p.i. Data from 3 independent experiments are summarized in (<i>D</i>) where each dot represents a single animal with the mean indicated by a horizontal line. (<i>E</i>) NP-specific B cells were assessed for cell death. Representative flow cytometry plots depict Annexin V binding and Mitotracker Red exclusion among live NP-specific GL7⁺ splenic B cells identified as in (<i>A</i>) 7 days p.i. (<i>F</i>) Summarized data from one of 3 independent experiments using 3 mice per group.</p

CD22^-/- B cells mount normal early TD Ab responses but do not form memory B cells or long-lived plasma cells.

<p>Splenocytes from WT or CD22^-/- B1-8^hi mice containing 2 x 10⁵ NP-specific B cells were adoptively transferred to B6 recipients 24 h prior to immunization with 50 micrograms NP-CGG in alum. (<i>A</i>) ELISA analysis of sera from WT (black squares) or CD22^-/- (white circles) recipients measuring anti-NP IgG1^a Abs on the indicated days. Data are from 3–5 mice per group per time point and are representative of 2 independent experiments. (<i>B</i>,<i>C</i>) The frequency of AFCs secreting NP-specific IgG1 Ab from spleen (<i>B</i>) or bone marrow (<i>C</i>) as determined by ELISPOT are plotted. Each dot represents a single animal with the mean indicated by a horizontal line. For <i>D</i>-<i>G</i>, splenoctyes containing 2 x 10⁵ NP-specific B cells from both WT and CD22^-/- B1-8^hi mice were adoptively transferred to Ly5.1 recipient animals that were then immunized as in <i>A</i>. (<i>D</i>) Representative flow cytometry analysis identifying NP-specific splenic B cells 125 days p.i. (<i>E</i>) Total number of NP-specific splenic B cells on day 42, 60 and 125 p.i. Each dot represents a single animal with the mean indicated by a horizontal line. 3 animals were used per time point. (<i>F</i>) Surface expression of the indicated marker was determined on total splenic B cells (shaded histogram) or WT B1-8^hi B cells (black line) from recipient mice immunized 125 days previously. Data are representative of 3 independent experiments using 3 mice per group. (<i>G</i>) Mean number of NP-specific B cells +/- SEM are plotted for immune mice (day 125 p.i.) before and 4 days after re-challenge with 20 micrograms soluble NP-CGG. One of two independent experiments using 3–4 mice/group is shown.</p