Fig 2: ?-secretase regulates plasma cells in mice.(a) Immunized (OVA–LPS in alum) C57/BL6 mice were treated with the ?-secretase inhibitor LY-411575-I or vehicle, and the surface display of BCMA in splenocytes was measured using flow cytometry 1 day later. BCMA expression on gated B220+CD138+ cells is shown, a representative example (left) and compiled data from all 17 analysed animals (mean, ***P<0.001, unpaired t-test; right). The black symbols on the right indicate the samples shown on the left. Closed histograms indicate isotype controls. (b) NZB/W mice pretreated with BrdU received the ?-secretase inhibitor LY-411575-I (red) or vehicle (blue) for 1 day. Surface expression of BCMA on all CD138+ plasma cells (PC) and the BrdU+ and BrdU- PC subgroups in the spleen and bone marrow (BM) was determined using flow cytometry. (c–e) Seven-day treatment of NZB/W mice with LY-411575-I. (c) BCMA surface expression in the spleen and BM on B220, and BrdU+ and BrdU- plasma cells was determined. (d) Absolute number of plasma cells, BrdU+ and BrdU- plasma cells in the spleen and BM. (e) Frequency (% of all cells in the organ) of plasma cells in the spleen and BM. Compiled data from 10 analysed animals per group (mean; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001, unpaired t-test).

Fig 3: ?-secretase regulates BCMA-mediated NF-?B activation.(a,b) HEK293T cells were transfected with full-length human BCMA or an empty vector. DAPT, APRIL (a) or BAFF (b) were added, and NF-?B activation was determined. Combined data of three independent experiments (mean±s.e.m., *P<0.05, paired t-test).

Fig 4: sBCMA is a decoy for APRIL in vitro.(a) A scheme of the ELISA is shown on the left; it detects BCMA–APRIL–FLAG (left panel) or BCMA–BAFF–FLAG (right panel) complexes, but neither BCMA nor APRIL–FLAG nor BAFF–FLAG alone alone (*P<0.05, paired t-test). sBCMA was derived from the supernatant of plasmacytoma cultured under serum-free conditions. Combined data of three independent experiments (mean±s.e.m.). (b) HEK293T cells were transfected with human BCMA and activated with APRIL (left panel) or BAFF (right panel). sBCMA (50 and 200 ng ml-1) was applied as indicated. sBCMA and control supernatant were obtained as mentioned above. BCMA-Fc (50 and 200 ng ml-1) was used as a positive control. Combined data of three independent experiments (mean±s.e.m., *P<0.05; **P<0.01 paired test). (c) Murine B cells were activated via anti-IgM and cultured for 2 days with APRIL in the presence or absence of sBCMA (200 and 400 ng ml-1). APRIL-induced survival was calculated as described in the Methods section. sBCMA was obtained from supernatant from HEK293T cells transfected with full-length BCMA (black bars). Control supernatant was obtained after transfection with an empty vector (white bars). sBCMA significantly inhibited APRIL-mediated survival (***P<0.001 and **P<0.01, paired t-test). Combined data of six independent experiments (mean±s.e.m.). (d) Illustration of the consequences of sBCMA shedding by ?-secretase: left: an active ?-secretase cleaves mBCMA. This reduces the number of membrane-bound BCMA molecules and releases sBCMA, which binds its ligand APRIL functioning as a decoy. Right: ?-secretase inhibitors (GSIs) result in elevated mBCMA on the surface and increased APRIL-mediated activation and survival.

Fig 5: Release of sBCMA requires active presenilin.(a–d) Presenilin-deficient MEF cells (PS-/-) were transduced with human BCMA (PS-/- BCMA) and then with wild-type PS1 (PS-/- BCMA PS1) or with a catalytically inactive mutant (PS-/- BCMA PS1-D385A). BCMA surface expression (a,b) and sBCMA release (d) were determined. In a, a representative experiment is shown; in b,d, mean±s.e.m. of four independent experiments is given (respectively, P=0.0313 and P=0.0033, paired t-test). (c) Cells used in a,b,d were analysed by immunoblotting for expression of full-length PS1 (PS1holo), for autoendoproteolysis of PS1 generating a C-terminal fragment (PS1CTF) reflecting an active state of the ?-secretase, for maturation of nicastrin (NCT) and for full-length BCMA (mBCMA).