1Rheumatology and Clinical Immunology Unit, University Hospital Münster, Münster, Germany;2Division of Pediatric Infectious Diseases, Dept. of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, United States;3Dept. of Pathology, University of Alabama at Birmingham, Birmingham, AL, United States;4Division of Cardiothoracic Surgery, Dept. of Surgery, University of Alabama at Birmingham, Birmingham, AL, United States;5Dept. of Genetics, University of Alabama at Birmingham, Birmingham, AL, United States;6Center for Vaccines and Immunity, Dept of Pediatrics, Nationwide Children's Hospital and The Ohio State University, Columbus, OH, United States.
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IntroductionHuman 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.MethodsMurine 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 DiscussionAmong 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).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.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.ConclusionsIn this acutely rejecting murine model, complement-fixing MCMV antibodies can deposit into D+/R- renal allografts, and are associated with allograft damage.NIH R01AI101138. NIH NIDDK 1P30 DK079337. NIH NIDDK DK64400. The Research Institute at Nationwide Children's Hospital.