Measuring plasma C4D to monitor immune complexes in lupus nephritis

Abstract

Objective: Because currently available assays that measure circulating immune complexes (ICx) are suboptimal, a novel assay was recently developed measuring C4d, a stable product of activation of the classical complement pathway. The present study aimed to establish the value of measuring plasma C4d levels in a longitudinal cohort of patients with severe refractory SLE who were treated with a combination therapy of rituximab with belimumab (RTX+BLM).

Methods: Fifteen patients with SLE who were treated with RTX+BLM in a phase 2A, open label study were included to sequentially measure plasma C4d levels and correlated to well-established markers of ICx-formation, that is, autoantibodies against double-stranded (ds) DNA, autoantibodies against C1q and proteinuria. The performance of plasma C4d measurements, C4 measurements and the ratio of C4d over C4 (C4d:C4) was evaluated.

Results: After establishing that on RTX+BLM treatment kinetics of C4d levels was distinct from traditional C3 and C4 levels, we found strong correlation of C4d:C4 with anti-dsDNA (R=0.76, p<0.001) and anti-C1q (R=0.65, p<0.001) autoantibody levels, which outperformed both stand-alone C4 and C4d levels. Additionally, changes in C4d:C4 over time correlated strongly with changes in proteinuria (R=0.59, p<0.001) as well as anti-dsDNA (R=0.46, p=0.003) and anti-C1q (R=0.47, p=0.002).

Conclusion: In patients with severe SLE, plasma C4d levels in relation to C4 levels is useful for longitudinal monitoring after RTX+BLM treatment to reflect amelioration of classical complement activation by ICx as well as proteinuria.

Keywords: C4d; complement; immunoglobulin-mediated membranoproliferative glomerulonephritis; lupus nephritis; systemic lupus erythematosus.

Figure 1

Significant association of plasma C4d levels with traditional, surrogate markers for immune complex formation. (A). Levels of C4d and C4 were measured simultaneously from which the C4d:C4 ratio was derived and correlated to circulating levels of autoantibodies directed against double stranded (ds) DNA and C1q and proteinuria (measured by the urine protein:creatinine ratio (UPCR)). The correlation plots of the ratio of C4d:C4 illustrate strong correlation with levels of anti-dsDNA and anti-C1q autoantibodies (R=Spearman’s correlation coefficient). The table reports the correlation coefficients separately for C4d, C4 and C4d:C4 where strong correlations (R≥0.5) are depicted in dark grey filled boxes; moderate correlation (0.3