Lack of T cells in Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease

Abstract

Act1 is a negative regulator of B-cell activation factor of the TNF family (BAFF) and CD40L-induced signaling. BALB/C mice lacking Act1 develop systemic autoimmunity resembling systemic lupus erythematosus (SLE) and Sjögren's syndrome (SjS). SLE and SjS are characterized by anti-nuclear IgG autoantibody (ANA-IgG) production and inflammation of peripheral tissues. As autoantibody production can occur in a T-cell dependent or T-cell independent manner, we investigated the role of T-cell help during Act1-mediated autoimmunity. Act1-deficiency was bred onto C57Bl/6 (B6.Act1(-/-) ) mice and B6.TCRβ(-/-) TCRδ(-/-) Act1(-/-) (TKO) mice were generated. While TCRβ/δ-sufficient B6.Act1(-/-) mice developed splenomegaly and lymphadenopathy, hypergammaglobulinemia, elevated levels of ANA-IgG, and kidney pathology, TKO mice failed to develop any such signs of disease. Neither B6.Act1(-/-) nor TKO mice developed SjS-like disease, suggesting that epigenetic interactions on the BALB/C background are responsible for this phenotype in BALB/C.Act1(-/-) mice. Interestingly, BAFF-driven transitional B-cell abnormalities, previously reported in BALB/C.Act1(-/-) mice, were intact in B6.Act1(-/-) mice and largely independent of T cells. In conclusion, T cells are necessary for the development of SLE-like disease in B6.Act1(-/-) mice, but not BAFF-driven transitional B-cell differentiation.

Figure 1

Lupus-like hypercellularity in B6.Act1^−/− mice is dependent on T-cell help. (A-F) C57Bl/6 (WT), B6.Act1^−/−, TCRβ/δ^−/− and TKO (TCRβ/δ^−/−Act1^−/−) mice were generated and analyzed for the development of splenomegaly and lymphadenopathy at 32-40 weeks of age. Data are presented as (A, E) mg tissue as well as (B, F) total cells isolated. (C, D) In addition, total numbers of B and T lymphocytes were calculated from spleen samples. Samples were collected over 12 months and evaluated at the time of harvest. Each symbol represents one individual mouse and the horizontal line signifies the mean. **p<0.01, ***p<0.001; two-tailed Mann-Whitney test. # denotes data showing a trend towards statistical difference: p < 0.1.

Figure 2

Lupus-like serological abnormalities in B6.Act1^−/− mice are dependent on T-cell help. Serum samples were obtained from the indicated 28-32 week old mice and analyzed for levels of total (A) IgM, (B) IgG, (C) IgG₁ and (D) IgG_2c as well as (E) ANA: anti-chromatin IgG and (H) IgM, (F) anti-histone IgG and (I) IgM, and (G) anti-dsDNA IgG and (J) IgM. Serum was collected and stored over a 12 month period. All samples were run at the same time to minimize assay to assay variation. Each symbol represents one individual mouse and the horizontal line signifies the mean. * p<0.05, **p< 0.01, ***p<0.001; two-tailed Mann-Whitney test. # denotes data showing a trend towards statistical difference: p<0.1.

Figure 3

T-cell deficient B6.Act1^−/− mice are protected from glomerulonephritis and IgG-IC deposition in kidney glomeruli. (A-C) Kidneys were harvested from 32-40 week old WT, TCRβ/δ^−/−, B6.Act1^−/− and TKO mice and either fixed in 10% formalin or quick frozen in OCT as described in Materials and Methods. (A) Formalin-fixed samples were embedded in paraffin and 5 μm sections were stained with hematoxylin/eosin. Data shown represent the average of four individual mice per genotype. The mesangial cell index is given by number of mesangial cells/glomerulus and normalized to levels in WT mice. (B, C) Quick-frozen samples were sectioned (5μm) and stained for the presence of IgG (red, (B)) or IgM (red, (C)) and complement factor C3 (green) by immunofluorescence staining. Pictures shown are representative of results from three individual mice per genotype. Two independent sections were analyzed per mouse representing >20 glomeruli/genotype. All immunofluorescence stainings were performed at the same time and pictures were taken using identical camera settings to allow for direct comparison. IgG and IgM staining per glomerulus was measured based on the density (red) per glomerulus per mouse and is displayed after normalization to the levels in WT mice (WT = 1.0).

Figure 4

B6.Act1^−/− mice fail to develop SjS-like disease. (A) Submaxillary glands were harvested from 16-18 week old mice and the weight was measured. (B) Serum levels of anti-SSB/La autoantibodies were measured in WT, B6.Act1^−/−, TCRβ/δ^−/− and TKO mice (n = 6-10 per strain). Sera were obtained as described in the legend of Figure 2. Each symbol represents one individual mouse and the horizontal line signifies the mean. (C) Frozen submaxillary glands were stained for deposition of IgG (green) and evaluated by immunofluorescence staining. All immunofluorescence stainings were performed at the same time and pictures were taken using identical camera settings to allow for direct comparison. Pictures are representative of 3 independent sections per strain.

Figure 5

B6.Act1^−/− mice develop B-cell hyperplasia, elevated levels of T2 and T3 transitional B cells and increased numbers of plasma cells. 17-20 week old WT (n = 8), TCRβ/δ^−/− (n = 12), B6.Act1^−/− (n = 5), and TKO (n = 12) mice were sacrificed and splenic levels of B-cell subsets were determined by flow cytometry. Absolute numbers of (A) immature (AA4.1⁺B220⁺) and mature B cells (AA4.1⁻B220⁺), (B) mature B-cell subsets (MZ: CD21⁺CD23^lowB220⁺ and FM: CD21^lowCD23^highB220⁺), (C) CD138⁺B220^low plasma cells, and (D) immature transitional B-cell subsets (T1:AA4.1⁺B220⁺CD23⁻IgM^high, T2: AA4.1⁺B220⁺CD23⁺IgM^high and T3: AA4.1⁺B220⁺ CD23⁺IgM^+/low) were calculated per mouse. Samples were obtained over a 12 month period and analyzed at the time of harvest. Each symbol represents levels in one individual mouse and the horizontal line signifies the mean. (E) The ratio of T2:T1 and T3:T1 were calculated based on the total number of cells (D). Each bar represent the mean +/− SEM. n= 5-12 per strain as described above. *p<0.05; **p<0.01; *p<0.001; two-tailed Mann Whitney test. # denotes data showing a trend towards statistical difference: p < 0.1.

Figure 6

TCRβ/δ-deficient mice express increased levels of serum BAFF, but no difference in levels of BAFF-R and TACI expression. (A) WT, TCRβ/δ^−/−, B6.Act1^−/− and TKO mice were sacrificed at 16-18 weeks of age and levels of BAFF-R and TACI was evaluated on T1 (B220⁺AA4.1⁺CD23^low) and T2/T3 (B220⁺AA4.1⁺CD23^high) immature B-cell subsets. Samples were obtained over a 12 month period and analyzed at the time of harvest. Data shown were pooled from 6-8 mice analyzed per strain. (B) Levels of serum BAFF was detected in 17-20 week old mice by ELISA as described in the Materials and Methods. Sera were obtained and stored over a 12 month period. All samples were run at the same time to minimize assay to assay variation. n = 10 (WT); n = 9 (B6.Act1^−/−); n = 9 (TCRβ/δ^−/−); n = 11 (TKO). *p<0.05; **p<0.01; ***p<0.001; two-tailed Mann Whitney test.

Figure 7

B6.Act1^−/− as well as TCRβ/δ-deficient mice develop significantly increased levels of MZ B cells. Splenic MZ B cells (B220⁺CD21^highCD23^lowIgM^high) were identified in 16-18 week old WT (n = 7), TCRβ/δ^−/− (n = 11), B6.Act1^−/− (n = 5), and TKO mice (n = 11) by flow cytometry and total number of cells were enumerated. The mean number of cells (×10⁶) is added above the graph for easier assessment. Samples were obtained as described in the legend of Figure 5. *p<0.05; **p<0.01; two-tailed Mann Whitney test.

Figure 8

Model of the involvement of BAFF, IL-17A and T cells during immature B-cell differentiation and plasma cell generation. WT mice (upper left quadrant): Immature B-cell (IMB) survival and differentiation is under the control of Act1 ( ) negatively affecting BAFF and CD40L and positively affecting IL-17A induced signaling (reviewed in [9]). TCRβ/δ^−/− mice (lower left panel) express increased levels of BAFF by a yet unknown mechanism, resulting in elevated IMB survival and differentiation into marginal zone (MZ) and follicular mature (FM) B cells. Due to the lack of T cells (CD40L), however, no Ig class switching takes place and antibodies present are IgM only. B6.Act1^−/− mice (upper right quadrant) have normal levels of BAFF, but increased responsiveness due to the lack of Act1 [1]. This drives IMB survival and differentiation into MZ and FM B cells [2]. Furthermore, as Act1-mediated negative regulation of CD40 signaling is lacking, the formation of IgG-secreting autoreactive plasma cells (PC) is increased. Combined deficiency of T cells and Act1 (lower right quadrant) results in both high levels of BAFF and BAFF hyperresponsiveness [1] driving the accumulation of MZ and FM B cells, but due to the lack of CD40L-expressing T cells, still inhibited Ig class switching. IL-17 is induced during inflammatory conditions and further enhances B-cell survival, proliferation and differentiation in WT mice [37]. Lack of T cells or Act1 will each separately abrogate this pathway.