Noninvasive Imaging of Acute Renal Allograft Rejection by Ultrasound Detection of Microbubbles Targeted to T-lymphocytes in Rats

Abstract

Purpose: We propose CD3-antibody-mediated contrast-enhanced ultrasonography using human T-lymphocytes for image-based diagnosis of acute allograft rejection (AR) established in a rat renal transplantation model.

Materials and Methods: 15 minutes after tail vein injection of 30 × 10⁶ human T-lymphocytes, contrast media/microbubbles conjugated with an anti-human CD3 antibody was applied to uni-nephrectomized 10-week-old allogeneically transplanted male rats (Lewis-Brown Norway (LBN) to Lewis, aTX) and ultrasound was performed to investigate the transplanted kidney as well as the native kidney. In vivo results were confirmed via immunohistochemical stainings of CD3 after post mortem dissection. Syngeneically transplanted rats (LBN to LBN, sTX), rats with ischemia/reperfusion injury (IRI, 45 min. warm ischemia), and rats subjected to acute cyclosporin A toxicity (CSA) (cyclosporine 50 mg/kg BW for 2 days i. p.) served as controls.

Results: Accumulation of human T-lymphocytes was clearly detected by antibody-mediated sonography und was significantly increased in allografts undergoing AR (5.41 ± 1.32 A. U.) when compared to native control kidneys (0.70 ± 0.08 A. U.). CD3 signal intensity was low in native kidneys, sTX (0.99 ± 0.30 A. U.), CSA (0.10 ± 0.02 A. U.) and kidneys with IRI (0.46 ± 0.29 A. U.). Quantification of the ultrasound signal correlated significantly with the T-cell numbers obtained by immunohistochemical analysis (R2 = 0.57).

Conclusion: Contrast-enhanced sonography using CD3-antibodies is an option for quick and highly specific assessment of AR in a rat model of renal transplantation.

Zusammenfassung

Ziel: Evaluation des Kontrastmittel-gestützten Ultraschalls unter Verwendung humaner T-Lymphozyten und anti-CD3-markierter Microbubbles zur Detektion und Differentialdiagnostik der akuten renalen Abstoßung im Transplantationsmodell der Ratte.

Material und Methoden: 30 × 10⁶ humane T-Zellen wurden uninephrektomierten, allogen nierentransplantierten Ratten (LBN auf Lewis) am 4. postoperativen Tag (POD4) injiziert. 15 min später erfolgte die i. v. Gabe humanspezifischer anti-CD3 Antikörper-markierter Microbubbles. Die Akkumulation humaner T-Zellen und gebundener Microbubbles wurde mittels Kleintierultraschall im Nierentransplantat und der Kontrollniere festgestellt und quantifiziert. Als Differentialdiagnose dienten syngen transplantierte Nieren (LBN auf LBN), Nieren mit warmem Ischämie/Reperfusionsschaden und Nieren mit akuter Calcineurin-inhibitortoxizität. Die Signalstärke der Ultraschallmessung wurde mit der Nierenhistologie (Banff Score, CD3 Färbung) korreliert.

Ergebnisse: Ratten mit akuter Abstoßung zeigten am POD4 eine signifikante Akkumulation humaner T-Zellen und Microbubbles in der allogenen Transplantatniere (5,41 ± 1,32 A. U., p < 0,05, n = 4 – 10 in allen Gruppen) im Vergleich zur gesunden Eigenniere (0,70 ± 0,08 A. U.). Syngen transplantierte Nieren ohne Rejektion (0,99 ± 0,30 A. U.), Nieren mit akuter Cyclosporin A Toxizität (0,10 ± 0,02 A. U.), bzw. akutem Ischämie/Reperfusionsschaden (0,46 ± 0,29 A. U.) wiesen keine erhöhte Anhäufung auf. Die Signalstärke des kontrastmittelgestützten Ultraschalls korrelierte in allen Nierenschädigungsmodellen signifikant mit der histologischen Quantifizierung des inflammatorischen Infiltrates.

Schlussfolgerungen: Mithilfe von T-Zellen und Antikörper-markierter Microbubbles ermöglicht die Kontrastmittel-gestützte Sonographie eine frühe und spezifische Diagnose der akuten Nierentransplantatabstoßung.

Key words

diagnostic radiology - ultrasound - molecular imaging - microbubbles - transplantation

^* Contributed equally

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