FcγRIIB regulation of BCR/TLR-dependent autoreactive B-cell responses

Abstract

Crosslinking of Fc γ receptor II B (FcγRIIB) and the BCR by immune complexes (IC) can downregulate antigen-specific B-cell responses. Accordingly, FcγRIIB deficiencies have been associated with B-cell hyperactivity in patients with systemic lupus erythematosus and mouse models of lupus. However, we have previously shown that murine IgG2a-autoreactive AM14 B cells respond robustly to chromatin-associated IC through a mechanism dependent on both the BCR and the endosomal TLR9, despite FcγRIIB coexpression. To further evaluate the potential contribution of FcγRIIB to the regulation of autoreactive B cells, we have now compared the IC-triggered responses of FcγRIIB-deficient and FcγRIIB-sufficient AM14 B cells. We find that FcγRIIB-deficient cells respond significantly better than FcγRIIB-sufficient cells when stimulated with DNA IC that incorporate low-affinity TLR9 ligand (CG-poor dsDNA fragments). AM14 B cells also respond to RNA-associated IC through BCR/TLR7 coengagement, but such BCR/TLR7-dependent responses are normally highly dependent on IFN-α costimulation. However, we now show that AM14 FcγRIIB(-/-) B cells are very effectively activated by RNA IC without supplemental IFN-α priming. These results demonstrate that FcγRIIB can effectively modulate both BCR/TLR9 and BCR/TLR7 endosomal-dependent activation of autoreactive B cells.

Figure 1. R2^- B cells display an enhanced response to GAMIG but not to F(ab′)₂ anti-IgM or TLR ligands

B cells from AM14 R2⁺ and R2^- mice (top panels) and from non-transgenic R2⁺ and R2^- mice (bottom panels) (R2⁺ (●); R2^- (□)) were stimulated with increasing concentrations of F(ab′)₂ fragment of goat anti-mouse IgM (F(ab′)₂), intact goat anti-mouse IgM (GAMIG), LPS, 1826 and R848, and proliferation determined by [³H] thymidine incorporation. Shown are mean±SD of a representative experiment of four (top panels) and mean±SEM of five independent experiments (bottom panels).

Figure 2. R2⁺ and R2^- AM14 B cells fail to respond to protein ICs and respond comparably to chromatin ICs

(A) R2⁺ AM14 (●), R2^- AM14 (□) and 20.8.3 (◊) splenic B cells were stimulated with protein ICs formed by combining increasing concentrations of biotinylated BSA (BSA-Bio) and 1.5 μg/ml anti-biotin IgG2a. (B) R2⁺ (●) and R2^- (□) AM14 B cells were stimulated with increasing concentrations of PL2-3. In all panels, proliferation was determined at 24 h by [³H] thymidine incorporation. Shown are mean±SEM of four independent experiments.

Figure 3. R2^- AM14 B cells respond to dsDNA fragment ICs containing non-optimal TLR9 ligands through a TLR9-dependent mechanism

(A) Clone 11 ICs, SenP1 ICs and CGneg ICs were preformed by combining 1D4 (5 μg/ml) with increasing concentrations of Bio-dsDNA fragments and then added to R2⁺ or R2^- AM14 B cells. Results shown are the mean±SEM of three (SenP1 and Clone 11 IC) or five (CGneg IC) independent experiments. * = p<0.05, and *** = p<0.001 indicate significant differences between R2⁺ and R2^- B cells as calculated using the unpaired Student t test. (B) R2^- AM14 B cells were stimulated with SenP1 ICs, Clone 11 ICs (C11), ODN 1826 (CpG), or Pam3CysK₄ (P3CysK) in the absence (black bars) or presence of TLR9 inhibitor INH-18 (checkered bars) or control INH-48 (open bars). Results shown are the mean±SEM of three independent experiments. In all panels, proliferation was measured at 24 h by [³H]-thymidine incorporation.

Figure 4. FcγRIIB downregulates AM14 B cell responses to RNA ICs in the absence and presence of IFNα

R2⁺ (●) and R2^- (□) AM14 B cells were stimulated with increasing concentrations of IgG2a anti-RNA BWR4 in the absence (left panel) or the presence (right panel) of 300 U/ml of IFNα. Proliferation was measured by [³H] thymidine incorporation. Results shown are mean±SEM of three independent experiments. * p<0.05; ** p<0.01 and *** p<0.001 indicate significant differences between R2⁺ and R2^- B cells as calculated using the unpaired Student t test.