γ-Secretase directly sheds the survival receptor BCMA from plasma cells

Abstract

Survival of plasma cells is regulated by B-cell maturation antigen (BCMA), a membrane-bound receptor activated by its agonist ligands BAFF and APRIL. Here we report that γ-secretase directly cleaves BCMA, without prior truncation by another protease. This direct shedding is facilitated by the short length of BCMA's extracellular domain. In vitro, γ-secretase reduces BCMA-mediated NF-κB activation. In addition, γ-secretase releases soluble BCMA (sBCMA) that acts as a decoy neutralizing APRIL. In vivo, inhibition of γ-secretase enhances BCMA surface expression in plasma cells and increases their number in the bone marrow. Furthermore, in multiple sclerosis, sBCMA levels in spinal fluid are elevated and associated with intracerebral IgG production; in systemic lupus erythematosus, sBCMA levels in serum are elevated and correlate with disease activity. Together, shedding of BCMA by γ-secretase controls plasma cells in the bone marrow and yields a potential biomarker for B-cell involvement in human autoimmune diseases.

Figure 1. sBCMA as a biomarker.

(a) sBCMA plasma concentrations were determined using ELISA in healthy controls (HC), patients with a clinically isolated syndrome (CIS) or MS, or other neurological diseases (OND). (b) sBCMA in the CSF was determined in patients with OND, CIS/MS or neuroborreliosis (NB) (***P<0.001, Kruskal–Wallis test followed by Dunn's Multiple Comparison Test). (c) sBCMA in the CSF correlated strongly with the intrathecal IgG production represented by the IgG Index. This correlation was evident when all analysed CSF samples were considered (P<0.0001, r=0.85) and in the MS/CIS group (P<0.0001, r=0.77, Spearman rank correlation, CIS/MS n=36; OND n=20, NB n=5). (d) sBCMA in the serum was determined with ELISA in HC, untreated (red) and treated (black) patients with SLE. sBCMA was elevated in SLE patients and in the untreated SLE patients compared with the treated patients (***P<0.001 and *P<0.05, Kruskal–Wallis test followed by Dunn's Multiple Comparison Test). (e) sBCMA in the serum of SLE patients correlated strongly with disease activity quantified with SLE disease activity index (SLEDAI; P<0.001; r=0.54, Spearman correlation). (f) sBCMA in the serum of SLE patients inversely correlated with the level of the complement factor C3 (P=0.0374, r=−0.29, Spearman correlation). Bars represent means.

Figure 2. sBCMA is released when B cells differentiate towards plasma cells and comprises the extracellular domain plus part of the transmembranous region of BCMA.

(a) Human purified B cells were activated for 5 days as indicated; IgG and sBCMA in the supernatant were quantified using ELISA. Combined data of three independent experiments (mean±s.e.m., P=0.0073, paired t-test). (b) PBMCs were stimulated with R848+IL-2 for 7 days. IgG and sBCMA in the supernatant were quantified using ELISA. Combined data of three independent experiments (mean±s.e.m., P=0.0227, paired t-test). (c–e) Human purified B cells were stimulated with CD40L+IL-21. (c) surface BCMA was measured using flow cytometry on unstimulated B cells, CD19⁺CD38⁻ cells and CD19⁺CD38⁺ cells. (d) Sorted CD38⁺ and CD38⁻ cells were cultured for another 24 h and the amount of shed sBCMA was measured using ELISA, combined data of two independent experiments. (e) Correlation between sBCMA release and surface expression of BCMA for a single replicate. (f,g) sBCMA was immunoprecipitated from supernatant of plasmacytoma cells (f, lanes 1, 2), serum (f, lane 3) and from supernatant of human purified B cells cultured with CD40L plus IL-21 (f, lane 4) with anti-BCMA monoclonal antibodies (mAbs) A7D12.2 (f, lanes 1 and 4) or C12A3.2 (f, lane 2) or goat-anti-BCMA (f, lane 3). Western blot analysis for BCMA (f) and silver staining of sBCMA immunoprecipitated from plasmacytoma supernatant (g) was performed. (h) The band at 6 kDa (from g) and sBCMA obtained using immunoprecipitation were analysed with mass spectrometry. The aa sequences of BCMA and peptides identified after tryptic (blue) or chymotryptic (red) digestion are shown. No peptide was detected with a C-terminal aa that was not a site for either tryptic or chymotryptic cleavage, indicating that the precise cleavage site of γ-secretase within the membrane needs to be identified.

Figure 3. γ-secretase inhibitor DAPT reduces release of sBCMA and enhances surface expression of BCMA on activated human B cell.

(a,b) Human B cells were differentiated into Ig-secreting cells via CD40L+IL-21. (a) Release of sBCMA on treatment with DAPT or TAPI-1 was measured using ELISA. sBCMA release was normalized to the amount of sBCMA shed under vehicle conditions, which was set as 100%. Combined data of three independent experiments (mean±s.e.m.). (b) These activated primary human B cells were subgrouped on the basis of expression of CD27 and CD38. A high surface expression of BCMA was seen on the CD27⁺⁺CD38⁺ subset. Surface expression of BCMA was enhanced using DAPT treatment (1 μM), while TAPI-I (50 μM) had little effect. (c,d) Human PBMCs were stimulated with R848+IL-2 for 7 days. (c) Release of sBCMA on treatment with DAPT or TAPI-1 was measured using ELISA. sBCMA release was normalized to the amount of sBCMA shed under vehicle conditions, which was set as 100%. Combined data of three independent experiments (mean±s.e.m.). (d) High surface expression of BCMA was seen on the CD19⁺CD38⁺ subset; this was further enhanced by DAPT (1 μM), while TAPI-I (50 μM) had little effect.

Figure 4. 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 (PS1_holo), for autoendoproteolysis of PS1 generating a C-terminal fragment (PS1_CTF) reflecting an active state of the γ-secretase, for maturation of nicastrin (NCT) and for full-length BCMA (mBCMA).

Figure 5. Release of sBCMA occurs without prior N-terminal trimming and is facilitated by the short extracellular domain of BCMA.

(a,b) HeLa cells were transfected with plasmids coding for full-length human BCMA or BCMA with an N-terminal FLAG and then cultured with increasing amounts of the γ-secretase inhibitor DAPT (0.02, 0.1 and 0.5 μM). Twenty-four hours after transfection supernatants were harvested and the released sBCMA was analysed using ELISA. In (a), ELISA wells were coated with anti-BCMA, and in (b) with anti-FLAG or a control IgG (anti-myelin oligodendrocyte glycoprotein (MOG) 8.18 C5), both were developed with anti-BCMA. Schemes of the ELISAs are shown on the right. Combined data of two independent experiments (mean±s.e.m.). (c–e) Human BCMA wild type (wt) or BCMA–BCMA with a doubled extracellular domain of BCMA were transfected into HEK293T cells. (c,d) Surface expression of BCMA was determined in the absence or presence of the γ-secretase inhibitor DAPT (1 μM). (d) The combined data of three experiments (P=0.0049, **P<0.01, unpaired t-test). (e) The effect of DAPT on the released sBCMA after transfection with BCMA wt or BCMA–BCMA (mean±s.e.m. of three experiments), P=0.0081, **P<0.01, unpaired t-test.

Figure 6. γ-secretase regulates BCMA-mediated NF-κB 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-κB activation was determined. Combined data of three independent experiments (mean±s.e.m., *P<0.05, paired t-test).

Figure 7. 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 γ-secretase: left: an active γ-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: γ-secretase inhibitors (GSIs) result in elevated mBCMA on the surface and increased APRIL-mediated activation and survival.

Figure 8. γ-secretase regulates plasma cells in mice.

(a) Immunized (OVA–LPS in alum) C57/BL6 mice were treated with the γ-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 γ-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).