Donor-specific HLA antibody-mediated complement activation is a significant indicator of antibody-mediated rejection and poor long-term graft outcome during lung transplantation: a single center cohort study

Abstract

Complement-mediated allograft injury, elicited by donor-specific HLA antibodies (DSA), is a defining pathophysiological characteristic of allograft damage. We aimed to study DSA-induced complement activation as a diagnostic marker of antibody-mediated rejection (AMR) and a risk stratification tool for graft loss in the context of lung transplantation (LT). We identified 38 DSA-positive patients whose serum samples were submitted for C3d deposition testing via the C3d assay. Among these 38 patients, 15 had AMR (DSA^Pos AMR^Pos ). Results were reported for each patient as the C3d ratio for each DSA, the immunodominant DSA, and the C3d ratio for all DSA present in a sample (C3d ratio_SUM ). DSA^Pos AMR^Pos patients had higher C3d ratio_SUM values (58.66 (-1.32 to 118.6) vs. 1.52 (0.30 to 2.74), P = 0.0016) and increased immunodominant C3d ratios (41.87 (1.72 to 82.02) vs. 0.69 (0.21 to 1.19), P = 0.001) when compared with DSA^Pos AMR^Neg patients. Specificity and calculated positive predictive value of the immunodominant C3d ratio and BCMsum tests for AMR diagnosis were both 100% (CI = 17.4-100) in this cohort. Worst graft survival was associated with both immunodominant C3d ratio ≥4 or C3d ratio_SUM ≥10 or BCMsum >7000, suggesting that the antibody composition and/or strength are the principal determinants of an HLA DSA's capacity to activate complement.

Keywords: antibody-mediated rejection; complement; donor-specific HLA antibodies; lung transplant.

Figure 1

Study population: flow chart

Figure 2. Comparison of BCM and C3d ratio for each DSA bead for each DSA+ patient between DSA^PosAMR^Pos and DSA^PosAMR^Neg patients

(A) Each dot represents BCM value for a single bead. Only DSA beads with BCM>500 are represented. Beads of DSA^PosAMR^Pos patients (n= 85) have significantly higher BCM than DSA^PosAMR^Neg patients (n=45) (mean±SD respectively 4814±5407 vs. 2060±1908, p=0.0024 Mann-Whitney). Only beads from DSA^PosAMR^Pos patients had a BCM >7000 (dashed line). (B) Each dot represents the C3d ratio value for a single bead. Only DSA beads with a C3d ratio >1 are represented. Beads of DSA^PosAMR^Pos patients (n=81) have significantly higher C3d ratios than beads of DSA^PosAMR^Neg patients (n=49) (mean±SD respectively 21.84±49.88 vs. 1.5±0.48, p<0.001 Mann-Whitney). Only beads from DSA^PosAMR^Pos patients had a C3d ratio >4 (dashed line). Data graphed as mean ± CI 95.

Figure 3. Correlation Between BCM & C3d Ratio in Lung Transplant Recipients with Respect to AMR Diagnosis

The BCM and C3d ratio were plotted for each DSA bead with white squares for DSA^PosAMR^Neg patients and gray circles for DSA^PosAMR^Pos patients. Correlation between BCM and C3d ratio was calculated for each group of patients. R² and p values are reported.

Figure 4. Use of Immunodominant C3d MFI and C3d ratio_SUM Values to Predict AMR Diagnosis

(A) The immunodominant C3d value was graphed for each patient; each symbol is the value of a single immunodominant DSA for a given patient. DSA^PosAMR^Pos patients had significantly higher immunodominant C3d ratios than DSA^PosAMR^Neg patients (mean±SD: 41.9 ±72.5 vs. 0.7±1.1, p= 0.0010). Immunodominant C3d ratio >4 (indicated by a dashed line) was only found in DSA^PosAMR^Pos. (B) The C3d Ratio_SUM was calculated for each patient in each group; each symbol represents the sum total of C3d activation for the given patient. DSA^PosAMR^Pos patients had significantly higher C3d Ratio_SUM (mean±SD: 58.7±108.3 vs. 1.5±2.8, p= 0.0016). C3d Ratio_SUM >10 (indicated by a dashed line) was only found in DSA^PosAMR^Pos. DSA^PosAMR^Neg patients, white squares, n=23; DSA^PosAMR^Pos patients, gray circles, n=15. Mann-Whitney analysis was performed, p values as depicted in each graph.

Figure 5. C3d Ratio is Associated with Poor Graft Survival

Kaplan-Meier curves were used to determine graft survival with respect to an immunodominant C3d ratio threshold of 4 (A), a C3d ratio_SUM threshold of 10 (B), a BCM_SUM threshold of 7000 (C).

Figure 6. Detection of Complement Activating DSA Prior to Episodes of AMR

Sera from DSA^PosAMR^Neg patients at the peak DSA and from DSA^PosAMR^Pos patients prior to the rejection episode (ranging 13–581 days before rejection) were assessed for BCM_SUM (A) (n=23 and n=10 respectively) and C3d ratio_SUM (B) (n=23 and n=10 respectively). Mann-Whitney analysis was performed, p values as indicated.