B-lymphocyte stimulator/a proliferation-inducing ligand heterotrimers are elevated in the sera of patients with autoimmune disease and are neutralized by atacicept and B-cell maturation antigen-immunoglobulin

Abstract

Introduction: B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor (TNF) family that regulate B-cell maturation, survival, and function. They are overexpressed in a variety of autoimmune diseases and reportedly exist in vivo not only as homotrimers, but also as BLyS/APRIL heterotrimers.

Methods: A proprietary N-terminal trimerization domain was used to produce recombinant BLyS/APRIL heterotrimers. Heterotrimer biologic activity was compared with that of BLyS and APRIL in a 4-hour signaling assay by using transmembrane activator and CAML interactor (TACI)-transfected Jurkat cells and in a 4-day primary human B-cell proliferation assay. A bead-based immunoassay was developed to quantify native heterotrimers in human sera from healthy donors (n = 89) and patients with systemic lupus erythematosus (SLE; n = 89) or rheumatoid arthritis (RA; n = 30). Heterotrimer levels were compared with BLyS and APRIL homotrimer levels in a subset of these samples.

Results: The recombinant heterotrimers consisted mostly of one BLyS and two APRIL molecules. Heterotrimer signaling did not show any significant difference compared with APRIL in the TACI-Jurkat assay. Heterotrimers were less-potent inducers of B-cell proliferation than were homotrimeric BLyS or APRIL (EC(50), nMol/L: BLyS, 0.02; APRIL, 0.17; heterotrimers, 4.06). The soluble receptor fusion proteins atacicept and B-cell maturation antigen (BCMA)-immunoglobulin (Ig) neutralized the activity of BLyS, APRIL, and heterotrimers in both cellular assays, whereas B-cell activating factor belonging to the TNF family receptor (BAFF-R)-Ig neutralized only the activity of BLyS. In human sera, significantly more patients with SLE had detectable BLyS (67% versus 18%; P < 0.0001), APRIL (38% versus 3%; P < 0.0002), and heterotrimer (27% versus 8%; P = 0.0013) levels compared with healthy donors. Significantly more patients with RA had detectable APRIL, but not BLyS or heterotrimer, levels compared with healthy donors (83% versus 3%; P < 0.0001). Heterotrimer levels weakly correlated with BLyS, but not APRIL, levels.

Conclusions: Recombinant BLyS/APRIL heterotrimers have biologic activity and are inhibited by atacicept and BCMA-Ig, but not by BAFF-R-Ig. A novel immunoassay demonstrated that native BLyS/APRIL heterotrimers, as well as BLyS and APRIL homotrimers, are elevated in patients with autoimmune diseases.

Figure 1

Sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blot analysis of (a) BLyS/APRIL heterotrimer and (b) trypsinized (nonzippered) heterotrimer. APRIL, a proliferation-inducing ligand; BLyS, B-lymphocyte stimulator.

Figure 2

Size-exclusion chromatography with multiangle light-scattering mass distribution of purified HT. Red line, molecular weight species by static light scattering; blue line, BLyS/APRIL HT; APRIL, a proliferation-inducing ligand; BLyS, B-lymphocyte stimulator; HT, heterotrimer; MW, molecular weight.

Figure 3

(a) Biologic activity and (b) neutralization of BLyS/APRIL HTs compared with those of BLyS and APRIL in TACI-Jurkat proliferation assays. APRIL, a proliferation-inducing ligand; BAFF-R, BAFF receptor; BCMA, B cell-maturation antigen; BLyS, B-lymphocyte stimulator; EC₅₀, 50% effective concentration; HT, heterotrimer; IC₅₀, 50% inhibition concentration; Ig, immunoglobulin; nz, trypsinized trimer without the "zipper" domain; TACI, transmembrane activator and CAML interactor; zz, trimer containing the "zipper" trimerization domain.

Figure 4

(a) Biologic activity and (b) neutralization of BLyS/APRIL HTs compared with those of BLyS and APRIL in B cell-proliferation assays. BLyS, APRIL, and heterotrimers were used at concentrations of 1 nMol/L, 3 nMol/L, and 10 nMol/L, respectively. APRIL, a proliferation-inducing ligand; BAFF-R, BAFF receptor; BCMA, B cell-maturation antigen; BLyS, B-lymphocyte stimulator; CPM, counts per minute; EC₅₀, 50% effective concentration; HT, heterotrimer; IC₅₀, 50% inhibition concentration; Ig, immunoglobulin; TACI, transmembrane activator and CAML interactor.

Figure 5

Detection of BLyS/APRIL HTs by using a bead-based HT immunoassay. The assay has a broad detection range (~25 ng/ml to ~100 pg/ml; see standard curve, inset), with an LOQ of ~100-313 pg/ml in the presence of serum (EC₅₀~6 ng/ml). The average serum sample size required is 25 μl, and the total assay time is 2 hours. The assay does not detect BLyS or APRIL homotrimers. APRIL, a proliferation-inducing ligand; BLyS, B-lymphocyte stimulator; EC₅₀, 50% effective concentration; HT, heterotrimer; LOQ, limit of quantitation.

Figure 6

Serum levels of BLyS, APRIL, and HTs in healthy donors and patients with autoimmune diseases. (a) Serum concentrations of HTs (upper panel), BLyS (middle panel), and APRIL (lower panel) for each patient. Horizontal bars depict median values for each serum donor group: healthy donors (blue squares), patients with SLE (red triangles), and patients with RA (green inverse triangles). P values were determined by using Fisher's exact test. *P < 0.05, **P < 0.0001. (b) Bivariate plots showing serum levels of BLyS, APRIL, and HTs in healthy donors (blue squares), patients with SLE (red triangles), and patients with RA (green inverse triangles). For data plotting, samples with serum ligand levels below the LOQ were assigned values equal to half the LOQ for each assay (0.156, 0.39, and 1.0 ng/ml for HT, BLyS, and APRIL, respectively). APRIL, a proliferation-inducing ligand; BLyS, B-lymphocyte stimulator; HT, heterotrimer; LOQ, limit of quantitation; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus.