Jacinta Jacob et al, Molecules, 2023
Regulatory T cells (Tregs) are a promising candidate cell therapy to treat autoimmune diseases and aid the longevity of transplanted solid organs. Despite increasing numbers of clinical trials using human Treg therapy, important questions pertaining to their in vivo fate, distribution, and function remain unanswered. Treg accumulation in relevant tissues was found to be crucial for Treg therapy efficacy, but existing blood-borne biomarkers are unlikely to accurately reflect the tissue state. Non-invasive Treg tracking by whole-body imaging is a promising alternative and can be achieved by direct radiolabelling of Tregs and following the radiolabelled cells with positron emission tomography (PET). The goal was to evaluate the radiolabelling of polyclonal Tregs with 89Zr to permit their in vivo tracking by PET/CT for longer than one week using the Mediso NanoScan PET/CT instrumentation. [89Zr]Zr(oxinate)4 was used as the cell-labelling agent and successful radiolabelling efficiency of human Tregs spanning 0.1–11.1 Bq 89Zr/Treg cell was achieved, which would be compatible with PET tracking beyond one week. The 89Zr-Tregs were characterised, assessing their phenotypes, and it was found that they would not tolerate these intracellular 89Zr amounts, as they failed to survive or expand in a 89Zr-dose-dependent manner. Moreover, PET imaging revealed signs of 89Zr-Treg death after adoptive transfer in vivo. In summary, 89Zr labelling of Tregs at intracellular radioisotope amounts compatible with cell tracking over several weeks did not achieve the desired outcomes, as 89Zr-Tregs failed to expand and survive. Consequently, we conclude that indirect Treg labelling is likely to be the most effective alternative method to satisfy the requirements of this cell tracking scenario.
Results from nanoScan® PET/CT
Survival of 89Zr-labelled Tregs in Immunodeficient Mice
The detection and longevity of radiolabelled 89Zr-Tregs in vivo was investigated, as well as the cell distribution in immunodeficient animals, as they are frequently the basis for establishing humanized mouse/xenograft models. The animals received 5 million 89Zr-Tregs (0.1 Bq 89Zr per cell) co-administered with 5 million HLA-A2-negative CD25- human peripheral blood mononuclear cells (PBMC’s) into BRG mice via their tail veins. This reflected conditions that were previously established and optimized. The total amount of radioactivity administered was 0.7 MBq 89Zr. Whole‑body PET/CT imaging was performed using nanoScan PET/CT. A total of 45 min after 89Zr-Treg administration, CT images were acquired (55 kVp tube voltage, 1200 ms exposure time, 360 projections), and 60 min after adoptive cell transfer, PET images were acquired (30 min scans), as well as 48hr and 120hr later. PET/CT data were reconstructed using Tera‑Tomo™ 3D SPECT reconstruction technology (Mediso Medical Imaging Systems Ltd.) with corrections for attenuation, detector dead time, and radioisotope decay in place as needed. All images were analyzed using VivoQuant software (inviCRO Ltd.). The total activity in the whole animal (excluding the tail) at the time of tracer administration was defined as the injected dose (ID). Image-based ROI analysis of relevant organs was performed, and radioactivity data were expressed as %ID/mL.
Figure 1. In vivo imaging of 89Zr-Tregs. (A–C) PET/CT overlay images of a representative BRG mouse that had received HLA-A2-negative 89Zr-Tregs (0.1 Bq/cell; 5 × 106; 0.7 MBq total radioactivity administered) with HLA-A2-positive Teffs admixed (5 × 106). Time points refer to the time passed after adoptive cell transfer. Maximum intensity projection images and sagittal sections of one of n = 3 mice are shown. The images are from the same mouse across (A–C). The inset in (C) shows 2-fold zoom of the pelvic area, clearly visualizing widespread and strong PET signals in various bones. (D) Quantitative ROI analysis of relevant tissues from the representative mouse shown in (A–C). n = 3 mice were used in this experiment and three different bone types analyzed per mouse.
The results obtained by in vivo PET/CT imaging highlighted that the administered 89Zr-Tregs initially behaved as expected for intravenously administered cells, i.e., they accumulated in the lungs at 1 hr post administration and then largely cleared the lungs and redistributed elsewhere within the body in a day. Importantly, Treg accumulation in bones was not expected. Moreover, they significantly increased over time across different bones. In contrast, 89Zr signals from bones are generally associated with free 89Zr that is rapidly mineralized, therefore this indicated 89Zr efflux from 89Zr-Tregs (and thereby the loss of the ability to track Tregs) or the death of 89Zr-Tregs with concomitant release of free 89Zr after 5 days post-administration.
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