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 (DSAPos AMRPos ). 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 ratioSUM ). DSAPos AMRPos patients had higher C3d ratioSUM 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 DSAPos AMRNeg 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 ratioSUM ≥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.

Conflict of interest statement

Disclosures

Dr. Antoine Roux has conflicts of interest to disclose as described by Transplant International: he served as a consultant for Novartis France (concerning CMV in solid organ transplantation). The other authors have no conflicts of interest to disclose as described by the Transplant International journal. C3d reagents were provided by Immucor® (Lifecodes, Norcross, GA, USA) for this study.

© 2018 Steunstichting ESOT.

Figures

Figure 1

Study population: flow chart

Figure 2. Comparison of BCM and C3d ratio for each DSA bead for each DSA+ patient between DSAPosAMRPos and DSAPosAMRNeg patients

(A) Each dot represents BCM value for a single bead. Only DSA beads with BCM>500 are represented. Beads of DSAPosAMRPos patients (n= 85) have significantly higher BCM than DSAPosAMRNeg patients (n=45) (mean±SD respectively 4814±5407 vs. 2060±1908, p=0.0024 Mann-Whitney). Only beads from DSAPosAMRPos 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 DSAPosAMRPos patients (n=81) have significantly higher C3d ratios than beads of DSAPosAMRNeg patients (n=49) (mean±SD respectively 21.84±49.88 vs. 1.5±0.48, p<0.001 Mann-Whitney). Only beads from DSAPosAMRPos 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 DSAPosAMRNeg patients and gray circles for DSAPosAMRPos 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 ratioSUM 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. DSAPosAMRPos patients had significantly higher immunodominant C3d ratios than DSAPosAMRNeg 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 DSAPosAMRPos. (B) The C3d RatioSUM was calculated for each patient in each group; each symbol represents the sum total of C3d activation for the given patient. DSAPosAMRPos patients had significantly higher C3d RatioSUM (mean±SD: 58.7±108.3 vs. 1.5±2.8, p= 0.0016). C3d RatioSUM >10 (indicated by a dashed line) was only found in DSAPosAMRPos. DSAPosAMRNeg patients, white squares, n=23; DSAPosAMRPos 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 ratioSUM threshold of 10 (B), a BCMSUM threshold of 7000 (C).

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

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