Organ transplantation is a lifesaving treatment for patients with end-stage organ failure. However, despite potent immunosuppression, graft rejection remains a major problem, at least partly due to a lack of effective tools for patient stratification and the monitoring of therapeutic effectiveness. Clinical immune rejection is currently monitored by biopsies, which are invasive and provide no spatiotemporal information. Therefore, we explored the feasibility of non-invasive immuno-PET imaging for monitoring (and predicting) graft rejection and therapeutic efficacy after solid organ transplantation. 

To study transplantation, we used the well-established DBA/2 to C57BL/6 mouse heart transplant model in which allografts reliably reject 7-9 days after transplantation. Specifically, our study included naïve mice, syngeneic heart recipients, untreated allogeneic heart recipients, allogeneic heart recipients treated with widely used CTLA4-Ig (i.p. 0.25 mg, on post-operative day 0), and allogeneic heart recipients treated with our mammalian target of rapamycin (mTOR)-inhibiting nanotherapeutics (i.v. 5.0 mg/kg, on post-operative days 0, 2, and 5).¹ Graft survival of the various groups is shown in Figure 1A. To monitor the immune response to transplantation, we imaged these groups at various time points post-surgery using ¹⁸F-FDG PET to monitor glucose uptake, an ⁸⁹Zr-labeled CD11b-targeting nanobody to monitor myeloid cell dynamics,^2,3 and an ⁸⁹Zr-labeled CD8-targeting specific nanobody to track T cells.⁴ PET imaging was performed one hour after ¹⁸F-FDG administration and 24 hours after nanobody administration. The animals were sacrificed directly after PET imaging, and their tissues analyzed by gamma counting to validate our imaging results.

Our findings demonstrate the potential of our immuno-PET imaging in transplantation. Specifically, ¹⁸F-FDG PET imaging on post-operative day six showed a significant increase in tracer uptake in the graft, spleen, and bone marrow compared to naïve, syngeneic, and treated groups (Figure 1B). PET imaging of CD8⁺ cells on day six showed a significant increase in PET signal intensity in the grafts of allogeneic mice, while this was not present in the treated and control groups (Figure 1C). Interestingly, signal intensity increases on post-operative day 20 for the group treated with mTOR-inhibiting nanotherapeutics, which start to reject around day 50, suggesting that graft rejection can might be detectable by PET imaging several weeks before this is possible by ultrasound. Our FDG and CD8 imaging results were corroborated by ex vivo gamma counting (not shown) and we are currently analyzing data obtained using the CD11b-specific nanobody.

In conclusion, immuno-PET imaging is an effective approach for monitoring organ transplant rejection and therapeutic efficacy. In the near future, test immuno-PET imaging in a kidney transplant mouse model, augment our PET imaging and gamma counting results with immunohistochemistry, and initiate a clinical study aimed at PET imaging kidney transplant recipients using a CD8-targeting minibody (ImaginAb).

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Figure 1. DBA/2 hearts were transplanted in C57BL/6 mice that were untreated (allogeneic), treated with mTOR-inhibiting nanotherapeutics, or treated with CTLA-Ig. We also included naïve and syngeneic (C57BL/6 to C57BL/6) recipients, n=6 A) Graft survival of the various groups as measured by ultrasound. B) ¹⁸F-FDG uptake in the graft and spleen as a function of time. ¹⁸F-FDG was i.v. injected, and the animals were imaged 2 hours later. C) ⁸⁹Zr-CD11b-nanobody uptake in the spleen as a function of time. The radiolabeled nanobody was i.v. injected, and the animals were imaged 24 hours later. D) ⁸⁹Zr-CD8-nanobody uptake in the graft as a function of time. The radiolabeled nanobody was i.v. injected and the animals were imaged 24 hours later. Data were analyzed using t-tests (^*p < 0.05; ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001. The data for some groups is still incomplete, as we are still actively processing our obtained data. 

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