Human cytomegalovirus (HCMV) infection is a risk factor for renal allograft loss, particularly among HCMV donor positive (D+) patients with acute rejection, but the underlying pathogenesis remains unclear. We investigated the role of CMV antibodies in allograft injury, using an acutely rejecting murine kidney transplant model.Methods
Murine CMV (MCMV) D-/R- and D+/R- kidney transplants were performed using BALB/c donors and C57BL/6 (B6) recipients (R), with or without cyclosporine (CsA) immunosuppression. Intragraft immunoglobulin (Ig) or complement C3 were quantitated by immunofluorescent (IF) staining (counts/high power field [hpf], average of 5 fields) using Image J (https://imagej.net). Graft injury was graded by a veterinary pathologist blinded to sample identity, using a published 24-point scale. Serum anti-MCMV antibodies were quantitated by ELISA. Intragraft B cells (CD45+/CD19+/B220+) were identified by flow cytometry. To deplete complement, some D+/R- transplants were treated with cobra venom factor (CVF). To determine whether anti-CMV antibodies deposit into D+ allografts, D+/R- transplants using C57BL/6-Igh-6tm1Cgn (B cell deficient) recipients were treated with no serum, noninfected B6 serum, or MCMV immune B6 serum. For each experimental group, 3-6 transplants were analyzed (means ± SEM) using the Student’s t-test or one-way analysis of variance.Results and Discussion
Among non-immunosuppressed mice, Ig and C3 were observed in allografts as early as day 3 post-transplant, and were greater in D+/R- compared to D-/R- grafts (Ig, 192 ± 29 vs. 36 ± 10/hpf, p<0.01; C3, 45 ± 9 vs. 5 ± 3/hpf, p= 0.04). CsA-immunosuppressed D+/R- mice examined at day 14 post-transplant had detectable serum anti-CMV antibodies, greater intragraft Ig compared to D-/R- grafts (150 ± 14 vs. 33 ± 8/hpf, p<0.01), greater graft damage (12.9 ± 0.6 vs. 9.6 ± 1.4, p=0.04), and more abundant B cell infiltrates (346504 ± 59060 vs. 91756 ± 37027 cells/g, p= 0.01).Results and Discussion
Complement-depleted D+/R- recipients had undetectable intragraft C3 deposition, less histologic graft injury (damage score 8.6 ± 1.4 vs. 14.2 ± 0.5, p=0.03), but similar Ig deposition compared to recipients without CVF treatment (200 ± 42 vs. 157 ± 28/hpf, p=n.s.), suggesting that complement contributes to MCMV associated graft damage.Results and Discussion
Among B cell deficient D+/R- recipients, Ig deposition was more abundant in MCMV antibody-treated animals (175 ± 40/hpf) compared to those receiving no serum (0 ± 0/hpf) or nonimmune serum (51 ± 21/hpf) (p<0.01). C3 staining was more intense in mice receiving MCMV immune serum (61 ± 14/hpf) compared to those receiving no serum (2 ± 0.7/hpf) and nonimmune serum (6 ± 5.6/hpf), indicating that MCMV complement-fixing antibodies can bind to infected allograft tissue.Conclusions
In this acutely rejecting murine model, complement-fixing MCMV antibodies can deposit into D+/R- renal allografts, and are associated with allograft damage.Conclusions
NIH R01AI101138. NIH NIDDK 1P30 DK079337. NIH NIDDK DK64400. The Research Institute at Nationwide Children's Hospital.