Summary     

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.

• The PET/CT data revealed ⁸⁹Zr-Tregs were found mainly in the lungs and liver, one-hour post-administration (Figure 1A).
• After 48 hours, ⁸⁹Zr signals had decreased in the lungs, indicative of active ⁸⁹Zr-Treg clearance from the lungs. ⁸⁹Zr signals were at this point predominantly in the liver alongside minor signals in shoulder and knee joints (Figure 1B,D).
• Five days post-administration, although most radioactivity remained in the liver, large ⁸⁹Zr signals were found in different bones, including the spine (Figure1C,D); see also inset depicting the pelvic area).
• Quantitative image analysis showed ⁸⁹Zr signals increased in various bones over time across different animals (Figure1D).

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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