Radiolabelling of Polyclonally Expanded Human Regulatory T Cells (Treg) with 89Zr-oxine for Medium-Term In Vivo Cell Tracking

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). Our goal was to evaluate the radiolabelling of polyclonal Tregs with Zr-89 to permit their in vivo tracking by PET/CT for longer than one week with current preclinical PET instrumentation. We used [Zr-89]Zr(oxinate)(4) as the cell-labelling agent and achieved successful radiolabelling efficiency of human Tregs spanning 0.1-11.1 Bq Zr-89/Treg cell, which would be compatible with PET tracking beyond one week. We characterized the Zr-89-Tregs, assessing their phenotypes, and found that they were not tolerating these intracellular Zr-89 amounts, as they failed to survive or expand in a Zr-89-dose-dependent manner. Even at 0.1 Bq Zr-89 per Treg cell, while Zr-89-Tregs remained functional as determined by a five-day-long effector T cell suppression assay, they failed to expand beyond day 3 in vitro. Moreover, PET imaging revealed signs of Zr-89-Treg death after adoptive transfer in vivo. In summary, Zr-89 labelling of Tregs at intracellular radioisotope amounts compatible with cell tracking over several weeks did not achieve the desired outcomes, as Zr-89-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.