BAFF and MyD88 signals promote a lupuslike disease independent of T cells

Abstract

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies. However, the underlying cause of disease appears to relate to defects in T cell tolerance or T cell help to B cells. Transgenic (Tg) mice overexpressing the cytokine B cell-activating factor of the tumor necrosis factor family (BAFF) develop an autoimmune disorder similar to SLE and show impaired B cell tolerance and altered T cell differentiation. We generated BAFF Tg mice that were completely deficient in T cells, and, surprisingly, these mice developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did, however, require B cell-intrinsic signals through the Toll-like receptor (TLR)-associated signaling adaptor MyD88, which controlled the production of proinflammatory autoantibody isotypes. TLR7/9 activation strongly up-regulated expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), which is a receptor for BAFF involved in B cell responses to T cell-independent antigens. Moreover, BAFF enhanced TLR7/9 expression on B cells and TLR-mediated production of autoantibodies. Therefore, autoimmunity in BAFF Tg mice results from altered B cell tolerance, but requires TLR signaling and is independent of T cell help. It is possible that SLE patients with elevated levels of BAFF show a similar basis for disease.

Figure 1.

T cell abnormalities in BAFF Tg mice. T cell subsets were determined by FACS, as described in the Materials and methods. (A) Total splenic T cell numbers of CD4⁺ (top) and CD8⁺ (bottom). Naive (left) or effector–memory (right) T cells were assessed from 12-mo-old WT (circle) and BAFF Tg (square) mice. (B) Representative FACS plots of PLN FoxP3⁺ T reg cells from WT (left) and BAFF Tg (right) mice. The T reg cell population is indicated with a box. (C) Total numbers of T reg cells from WT (circle) and BAFF Tg (square) mice from thymus, spleen, and PLN. In A and C, symbols represent individual mice, and the mean for each group is indicated by a column. Significant P values are shown. n ≥ 7 per group.

Figure 2.

Similar splenomegaly, B cell hyperplasia, and elevated Ig levels in TΔ-BTg and Baff Tg mice. 12-mo-old WT (circle), TCR^−/− (diamond), BAFF Tg (square), and TΔ-BTg (triangle) mice were assessed for spleen size (A) and total numbers of T2-MZ, T2-Fo, MZ, and Fo B cell subsets in spleen and B1a, B1b, and Fo B cells in peritoneal lavage (PerC; B). (C) Fo and MZ B cells in peripheral blood (left) and PLN (right). Subsets were gated by FACS analysis as described in Materials and methods. (D) Basal Ig levels in TΔ-BTg mice and control mice. Symbols represent individual mice, and columns indicate the mean for each group. Significant P values are shown. n ≥ 7 per group.

Figure 3.

Increased switching to T cell–independent antigens and low-level switching to T cell–dependent antigens in TΔ-BTg mice. Titers of antigen-specific Ig from 12-wk-old WT (circle), TCR^−/− (diamond), BAFF Tg (square), and TΔ-BTg (triangle) mice were determined, 5 d after T cell–independent NP-Ficoll immunization (A) and 28 d after initial T cell–dependent immunization NP-OVA (B). Symbols represent individual mice, and the mean for each group is indicated by a column. n ≥ 5 per group. Significant P values are shown between TΔ-BTg v BAFF Tg and TΔ-BTg v TCR^−/− groups.

Figure 4.

Production of IgM and IgG, but not IgA, autoantibodies in TΔ-BTg mice. (A) ELISA was used to determine IgG, IgA, and IgM anti-ssDNA (top), anti-dsDNA (middle), and RF (bottom) in serum from 8-mo-old WT (circle), TCR^−/− (diamond), BAFF Tg (square), and TΔ-BTg (triangle) mice. Symbols represent individual mice, and the mean for each group is indicated by columns. Significant P values are shown. n ≥ 6 per group. (B) Representative staining of isotype-specific ANA and anti-dsDNA were determined by staining Hep-2 and C. luciliae slides, respectively, with serum from indicated mice.

Figure 5.

TΔ-BTg and BAFF Tg mice develop indistinguishable kidney and salivary gland pathology. In A, F, and G, the mice are WT (circle), TCR^−/− (diamond), BAFF Tg (square), and TΔ-BTg (triangle). Symbols represent individual mice, and the mean for each group is indicated by a column. (A) Proteinuria analysis of 12-mo-old animals. (B) HE staining of kidney tissue sections. BAFF Tg and TΔ-BTg sections show glomeruli separation (white arrows) and mononuclear cell infiltrate (black arrows). (C) IgG, IgA, and IgM (D) C3 deposition in the kidney of 8-mo-old mice. (E) HE staining of salivary gland tissue sections from BAFF Tg and TΔ-BTg sections show salivary gland destruction and lymphocyte infiltrate (black arrows). (F) Saliva flow after pilocarpine injection in 8-mo-old animals. (G) Total numbers of MZ-like B cells detected in CLN and salivary glands. (H) Isotype-specific Ig deposition in salivary gland of 8-mo-old mice. Significant P values are shown.

Figure 6.

TLR7/9, but not TLR7, ligands increase BAFF-R expression, and BAFF enhances TLR-mediated autoantibody secretion. (A) FACS-sorted Fo, MZ, B1a, and B1b B cells were stimulated with the following: unstimulated (solid black), CpG (TLR9; dashed black), R848 (TLR7; solid gray), and LPS (TLR4; dashed gray) for 24 h and analyzed for surface expression of BAFF-Rs by FACS, as indicated. Histograms are representative of three individual sorting and culture experiments. MZ (B) and B1 (C) BAFF Tg B cells were cultured with TLR ligands with (squares) or without (circles) BAFF for 6 d. Total anti-dsDNA antibody levels in culture supernatants were measured by ELISA. Symbols indicate individual cultures. In C and D, significant P values are shown between TLR-stimulated cultures with or without BAFF, between unstimulated and TLR stimulated (dashed line [x]) and between unstimulated with BAFF and TLR-stimulated with BAFF (solid line [y]).

Figure 7.

MyD88 signaling is essential for B cell activation and IgG autoantibody production in BAFF Tg mice. Lethally irradiated 6-wk-old BAFF Tg mice were reconstituted with MyD88^+/+ or MyD88^−/− or mixed Rag1^−/−MyD88^+/+ or Rag1^−/−MyD88^−/− BM, as indicated. (A) Histogram of B cell activation markers CD44 (top) and CD69 (bottom) of Fo (left) and MZ (right) splenic B cells from MyD88^+/+ (solid line) and MyD88^−/− (dashed line) BAFF Tg BM chimeras. (B) Anti-dsDNA and RF antibody production in WT mice reconstituted with MyD88^+/+ (circles) or MyD88^−/− (diamonds); BAFF Tg mice reconstituted with MyD88^+/+ (squares; light gray bar); or MyD88^−/− (triangles; light gray bar); or BAFF Tg mice reconstituted with Rag1^−/−MyD88^+/+ (squares; dark gray bar); or Rag1^−/−MyD88^−/− (triangles; dark gray bar). IgG, IgA, and IgM (C) and C3 (D) deposition in the kidney of BAFF Tg mice reconstituted with MyD88^+/+, MyD88^−/−, Rag1^−/−MyD88^+/+, or Rag1^−/−MyD88^−/− BM, as indicated. Significant P values are shown.

Figure 8.

Model of BAFF-induced, T cell–independent autoimmune disease. (Stage 1) Excess levels of BAFF expand self-reactive MZ and B1 B cells (24). (Stage 2) BAFF signals promote TLR activation after internalization of autoreactive B cell receptors bound to either dsDNA or to immune complexes containing nucleic acids. (Stage 3) Positive feedback of this activation exists, with BAFF increasing TLR7/9 expression and TLR7/9 ligands stimulating BAFF-R expression. (Stage 4) BAFF enhances TLR signals, which lead to the production of IgG2c and IgG2b antibodies. Autoantibodies deposit in the kidney and promote inflammation through complement fixation.