Complications Infections & Malignancies (Videos Available)

Wednesday July 04, 2018 from 09:45 to 10:45

Room: N-117/118

527.2 Impact of complement fixing antibodies in murine cytomegalovirus infected renal allografts

Masako Shimamura, United States

Associate Professor
Center for Vaccines and Immunity
Nationwide Children's Hospital


Impact of Complement Fixing Antibodies in Murine Cytomegalovirus Infected Renal Allografts

Ute Saunders1, Mao Li2, Bo Chen3, Lingling Guo4, Trenton R Schoeb5, Masako Shimamura6.

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

Introduction: 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 ( 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).
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.
Conclusions: In 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.

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