Complement C4A Regulates Autoreactive B Cells in Murine Lupus

Abstract

Systemic lupus erythematosus (SLE) is a severe autoimmune disease mediated by pathogenic autoantibodies. While complement protein C4 is associated with SLE, its isoforms (C4A and C4B) are not equal in their impact. Despite being 99% homologous, genetic studies identified C4A as more protective than C4B. By generating gene-edited mouse strains expressing either human C4A or C4B and crossing these with the 564lgi lupus strain, we show that, overall, C4A-like 564Igi mice develop less humoral autoimmunity than C4B-like 564Igi mice. This includes a decrease in the number of GCs, autoreactive B cells, autoantibodies, and memory B cells. The higher efficiency of C4A in inducing self-antigen clearance is associated with the follicular exclusion of autoreactive B cells. These results explain how the C4A isoform is protective in lupus and suggest C4A as a possible replacement therapy in lupus.

Keywords: B cell tolerance; Complement C4; Murine model; SLE.

Figure 1.. Generation of C4A and C4B Gene-Edited Mouse Strains

(A) Simplified representation of C4A and C4B mouse generation using Crispr-Cas9 technology. sgRNA target in green, mutations in red, isotypic residues modulating covalent binding affinities of C4 in bold, and isotypic region in capital letters. (B and C) Assessment of C4- and C3-coated immune complexes (ICs) in vitro. WT, C4A, C4B, or C4^−/− mouse serum was incubated with an artificial IC. Left panels, representative dot plot; right panels, C3 and C4 mean fluorescence intensity (MFI) on ICs evaluated by flow cytometry. Means ± SEMs. One dot represents 1 mouse. n > 6 for each strain; 1-way ANOVA with Tukey’s test. (D and E) C4 puncta quantification on FDC networks in spleen. (D) Representative image of spleen immunostained for FDCs (anti-CD21 Ab, 7E9 clone, blue) and mouse C4 (16D2 clone, red). Scale bar represents 200 μm. (E) The number of C4 puncta per μm² of FDC network was quantified using Cell Profiler software. Means ± SEMs. One dot represents 1 field of view; n > 9; 3 mice per group. (F and G) Analysis of hemolytic activity of C4^−/−, WT, C4A, and C4B serum. Mean ± SEM (F); 1 dot represents the average value of 8 mice ± SD. H₂O was considered 100% of lysis. Human serum was used as a positive control. Two-way ANOVA with Tukey’s test. (G) Results for 1/30 dilution; Means ± SEMs; 1 dot represents 1 mouse; 1-way ANOVA with Tukey’s test. ****p < 0.0001. See also Figure S1.

Figure 2.. Absence of C4A Leads to Impaired Regulation of Autoreactive B Cells in Periphery

(A and B) Analysis of autoreactive B cell population (B220⁺ Id⁺) in peripheral blood of C4A 564Igi (n = 5) and C4B 564Igi (n = 8) by flow cytometry. (A) Representative dot plots. (B) Percentage of Id⁺ B cells within the total B220⁺ B cell population. Means ± SEMs; Mann-Whitney test; 1 dot represents the average percentage for 1 strain. (C–E) Analysis of autoreactive Id⁺ B cells within the total B220⁺ B cell population in secondary lymphoid organs by flow cytometry. (C) Proportion of splenic Id⁺ B cells within the total B220⁺ B cell population from 8, 15, and 20 wo C4A and C4B 564Igi mice. Means ± SEMs; Mann-Whitney test; each dot represents the average of n > 8 mice. (D) Proportion of Id⁺ B cells within the total B220⁺ B cell population in spleen (top panel) and skin-draining LNs (bottom panel) from 8 and 15 wo C4A, C4B, C4^+/+, and C4^−/− 564Igi. Means ± SEMs; 1-way ANOVA with Tukey’s test; each dot represents 1 mouse; n > 4 for each strain. (E) Gating strategy to discern immature (i, B220⁺ AA4.1⁺), transitional (t, B220⁺ AA4.1^int), and mature (m, B220⁺ AA4.1⁻) splenic B cell subpopulations. (F) Proportions of Id⁺ B cells within immature, transitional, and mature splenic B cell subpopulations of 15 wo C4A, C4B, C4^+/+, and C4^−/− 564Igi. Means ± SEMs; 1-way ANOVA with Tukey’s test; each dot represents 1 mouse; n > 6. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S2.

Figure 3.. GC Structures Are More Frequent and Larger in C4B 564Igi Mice Than in C4A 564Igi Mice

(A) Splenic GC B cell population (B220⁺ CD38^dim/− GL7^high) analyzed by flow cytometry in 15 wo C4A, C4B, and C4^+/+ 564Igi. Means ± SEMs; 1-way ANOVA with Tukey’s test; 1 dot represents 1 mouse; n > 6. (B–D) Analysis of GC structures by immunohistochemistry. (B) Representative images of spleen follicles. Marginal zone macrophages: CD169 (red), B cell zone: B220 (blue), T cell zone: CD3 (green) and GC: GL7 (white); the scale bar represents 100 μm. (C) Frequency of GL7⁺ follicles. Means ± SEMs; 1-way ANOVA with Tukey’s test; 1 dot represents 1 mouse; n > 8 for each 564Igi strain. (D) Quantification of individual GL7⁺ area using ImageJ software. Means ± SEMs; 1-way ANOVA with Tukey’s test; 1 dot represents 1 GL7⁺ area; n > 7 animals for each 564Igi strain. (E) Serum anti-nucleoli IgG titers measured by ELISA. Means ± SEMs; 1 dot represents 1 mouse; n > 8. *p < 0.05, **p < 0.01, ****p < 0.0001). See also Figure S3.

Figure 4.. Reduction of Autoreactive Memory-like B Cells in C4A 564Igi

(A) Experiment timeline. (B) Gating strategy to identify memory-like B cells (B220⁺ EYFP⁺ GL7⁻ CD138⁻). (C) Analysis of autoreactive memory-like B cell population (B220⁺ YFP⁺ CD138⁻ GL7⁻) by flow cytometry. Means ± SEMs; 1-way ANOVA with Tukey’s test; 1 dot represents 1 mouse; n > 3. *p < 0.05. See also Figure S4.

Figure 5.. C4A Is More Efficient Than C4B in Inducing Clearance of Apoptotic Cells In Vivo and Driving Effective Follicular Exclusion of Id⁺ B Cells

(A) Experiment timeline. (B) Representative dot plot of apoptotic stages analyzed by flow cytometry using propidium iodide (PI) and annexinV (AnnV) staining (a [live cells]: AnnV⁻ PI⁻; b [early apoptosis]: AnnV⁺ PI⁻; c [late apoptosis]: AnnV⁺ PI⁺). (C and D) Analysis of apoptotic cell uptake by F4/80⁺ macrophages. (C) Representative dot plots of apoptotic cell uptake by macrophages (pH-Rhodo⁺) for all of the strains. (D) Frequency of macrophages ingesting apoptotic bodies, analyzed by flow cytometry and relative to WT mice. Means ± SEMs; each dot represents 1 mouse; n > 10 over 6 independent experiments; 1-way ANOVA with Tukey’s test; *p < 0.05; ***p < 0.005. (E and F) Analysis of Id⁺ B cell follicular exclusion at 20 wo. (E) Representative image of splenic follicles. Follicular B cells: IgD (blue), T cell zone: CD3 (white), Id⁺ B cells: anti-idiotype (9D11 clone) (red). The scale bar represents 100 μm. The dashed lines determine B cell follicles. (F) Follicular exclusion, expressed as percentage of follicular B cell zone occupied by Id⁺ B cells, was quantified using Cell Profiler software. Means ± SEMs; each dot represents 1 mouse; n > 5; 1-way ANOVA with Tukey’s test; *p < 0.05; ****p < 0.001. See also Figure S5.

Figure 6.. C4A Lessens Autoantibody Production in the 564Igi Lupus Model

(A) Volcano plot comparison of median IgG signal intensities in C4A 564Igi (n = 8) relative to C4B 564Igi (n = 10) mice. Log2(fold change) appears on the x axis. Significance (presented as −10 × log10[p value]) is on the y axis. The p value was obtained running multiple unpaired t tests corrected with the Holm-Sidak method. One dot represents 1 self-Ag. (B–D) Dosage of anti-Ro60, -BPI, and -CTGF IgG titers in serum of 15–20 wo C4^−/−, C4A, C4B, and C4^+/+ 564Igi by ELISA. Means ± SEMs; 1 dot represents 1 mouse; n > 4; 1-way ANOVA with Tukey’s test; *p < 0.05. See also Figure S6 and Table S6.