B cell-derived IL-6 initiates spontaneous germinal center formation during systemic autoimmunity

Abstract

Recent studies have identified critical roles for B cells in triggering autoimmune germinal centers (GCs) in systemic lupus erythematosus (SLE) and other disorders. The mechanisms whereby B cells facilitate loss of T cell tolerance, however, remain incompletely defined. Activated B cells produce interleukin 6 (IL-6), a proinflammatory cytokine that promotes T follicular helper (T_FH) cell differentiation. Although B cell IL-6 production correlates with disease severity in humoral autoimmunity, whether B cell-derived IL-6 is required to trigger autoimmune GCs has not, to our knowledge, been addressed. Here, we report the unexpected finding that a lack of B cell-derived IL-6 abrogates spontaneous GC formation in mouse SLE, resulting in loss of class-switched autoantibodies and protection from systemic autoimmunity. Mechanistically, B cell IL-6 production was enhanced by IFN-γ, consistent with the critical roles for B cell-intrinsic IFN-γ receptor signals in driving autoimmune GC formation. Together, these findings identify a key mechanism whereby B cells drive autoimmunity via local IL-6 production required for T_FH differentiation and autoimmune GC formation.

Figure 1.

IFN-γ synergizes with B cell activation signals to promote IL-6 production by mouse and human B cells. (A) Representative FACS plots showing CD45.1⁺ Was^−/− versus CD45.2⁺ Was^−/−Ifngr^−/− B cells in GC and non-GC B cell compartments. (A, left) Gated on CD19⁺ B cells. (A, right) Gated on PNA⁺FAS⁺ GC B cells (top) and PNA⁻FAS⁻ non-GC B cells (bottom). Number equals percentage within gate. (B) Selection of CD45.1⁺ Was^−/− versus CD45.2⁺ Was^−/−Ifngr^−/− B cells into the GC compartment. NS, not significant. (C) IL-6 from splenic WT (black), Tbx21^−/− (gray), and Ifngr^−/− (white) B cells cultured for 48 h with anti–IgM, R848, anti–CD40 and/or IFN-γ. (D) IL-6 production by stimulated mouse B cells with or without ruxolitinib or tofacitinib (500 nM). (E) IL-6 in WT (black) and Stat1^−/− (white) B cells stimulated as indicated. (F) Surface PNA binding in WT (left) and Stat1^−/− (right) splenic B cells stimulated as indicated. (G) IL-6 intracellular staining at 72 h in cultured human B cells, gated as CD38⁻CD27⁻ “naïve” (white) or CD38⁺CD27⁻ “GC” (black) B cells. (H) IL-6 mean fluorescence intensity by intracellular staining in human CD38⁺CD27⁻ B cells stimulated for 24 h. (I) IL-6 from human B cells stimulated for 72 h with or without ruxolitinib (500 nM). (B–E and G–I) Error bars indicate means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, by two-tailed Student’s t test (B); by one-way ANOVA and Tukey's multiple comparison test (C–E and I); or by paired two-tailed Student’s t test (G and H). (H) Data shown as paired analysis of different stimulation conditions from individual human donors. (A and B) Data representative of three independent Was^−/− versus Was^−/−Ifngr^−/− competitive chimeras. (C-H) Data representative of at least two independent experiments.

Figure 2.

B cell–derived IL-6 promotes systemic inflammation in SLE. (A) Serum IL-6 levels in WT, Was^−/−, and B cell–intrinsic Was^−/−Il6^−/− chimeras. (B–D) Spleen weight (B), number of splenic CD4⁺ T cells (C), and CD11b⁺GR1⁺ neutrophils (D) in indicated chimeras. (E) Representative FACS plots (gated on CD4⁺ T cells) showing naive (CD44^loCD62L^hi) and effector/memory (CD44^hiCD62L^lo/hi) CD4⁺ T cells. The percentages are those within the gate. (F) Number of naive and effector/memory CD4⁺ T cells. (A–F) Error bars indicate means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, by one-way ANOVA and Tukey's multiple comparison test. Data are representative of four WT (white, n = 11), four Was^−/− (black, n = 13), and two B cell–intrinsic Was^−/−Il6^−/− (gray, n = 6) independent chimeras.

Figure 3.

B cell IL-6 production initiates spontaneous, autoimmune GCs. (A and B) Representative FACS plots (gated on splenic CD4⁺ T cells) showing PD1⁺CXCR5⁺ T_FH cell percentage (left) and total number (right) of splenic T_FH cells in indicated chimeras. (C) Representative FACS plots (gated on splenic CD19⁺ B cells) showing loss of PNA⁺GL7⁺ GC B cells after B cell–intrinsic Il6^−/− deletion. (D) Percentage (left) and total number (right) of splenic GC B cells in indicated chimeras. (E) Representative splenic sections stained with B220 (red), PNA (green), and CD3 (blue). Bar, 150 µm. (F and G) Anti–dsDNA (F) and anti–Sm/RNP (G) IgM, IgG, and IgG2c autoAb at 12 (F) and 24 (G) wk after transplant. (H) Representative overlaid FACS plots showing PNA⁺FAS⁺ GC phenotype in Qβ-VLP⁺ (red) versus Qβ-VLP⁻ (blue) B cells in Was^−/− (left) and B cell–intrinsic Was^−/−Il6^−/− (right) chimeras, 12 d after Qβ-VLP immunization. Percentages are Qβ-VLP⁺ B cells within the PNA⁺FAS⁺ gate. (I) Percentage of PNA⁺FAS⁺ B cells in Qβ-VLP⁺ (black) versus Qβ-VLP⁻ (white) B cells in indicated chimeras. (B, D, F, G, and I) Error bars indicate means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significant, by one-way ANOVA and Tukey's multiple comparison test. (A–G) Data representative of four WT (white, n ≥ 10), four Was^−/− (black, n ≥ 12), and two B cell–intrinsic Was^−/−Il6^−/− (gray, n = 6) independent chimeras. (H and I) Representative FACS plots (H) and combined data (I) from two independent, B cell–intrinsic, chimera experiments (Was^−/− [n = 4] and Was^−/−Il6^−/− [n = 5]).

Figure 4.

Development of IC glomerulonephritis requires B cell–derived IL-6. (A–D) Immunofluorescence staining for glomerular (A) IgM; (B) IgG; (C) IgG2c; (D) C3. (Left) Representative images. (Right) Intensity of glomerular immunofluorescence staining. (E, left) Representative kidney sections stained with periodic acid–Schiff. (E, right) Glomerular inflammation score. (A–E) Data representative of ≥2 independent chimeras, including WT (white, n = 3 [A–D], n = 4 [E]), Was^−/− (black, n = 5 [A–E]), and B cell–intrinsic Was^−/−Il6^−/− (gray, n = 5 [A–D], n = 4 [E]) chimeras. Scoring by observers blinded to genotype. Error bars indicate means ± SEM. Bars, 50 µm. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA and Tukey's multiple comparison test.