The manufacturing process of regulatory T (Treg) cells for clinical application begins with the positive selection of CD25þ cells using superparamagnetic iron oxide nanoparticle (SPION)-conjugated anti-CD25 antibodies (spCD25) and immunomagnetic cell separation technology. Our findings revealed that the interaction of spCD25 with its cell target induced the internalization of the complex spCD25–interleukin-2 receptor. Accumulation of intra- cellular spCD25 triggered oxidative stress, causing delayed Treg expansion and temporary reduction in suppressor activity. This activation delay hindered the efficient generation of clinically competent cells. During this early phase, Treg cells exhibited elevated mitochondrial superoxide and lipid peroxidation levels, with a concomitant decrease in mitochondrial respiration rates. The results uncovered the increased mitochondrial unfolded protein response. This protective, redox-sensitive activity is inherent in Tregs when contrasted with homologous, spCD25-treated, conventional T cells. Although the temporary effects of spCD25 on clinically competent cells did not impede their use in a safety/feasibility pilot study with kidney transplant recipients, it is reasonable to anticipate a potential reduction in their therapeutic efficacy. The mechanistic understanding of the adverse effects triggered by spCD25 is crucial for improving the manufacturing process of clinically competent Treg cells, a pivotal step in the successful implementation of immune cell therapy in transplantation.

Electron Microscope. Unlabeled control (Kit-Treg) cells and spCD25-labeled Treg cells were fixed 3.9% paraformaldehyde and 3.5% glutaraldehyde at 4°C for 1 hour followed by 3 washes in sucrose for 5 minutes. The cells were dipped with 1% OsO₄ at 4°C for 1 hour. Cells were then resuspended in 0.1 M buffer and dehydrated at 4°C in an ascending series of ethanol baths and finally twice in propylene oxide at room temperature. The cells were infiltrated overnight in a 1:1 mixture of propylene oxide and epoxy resin. The next day, the cells were resuspended in fresh resin and polymerized for 48 hours at 60°C. Selected areas were thin sectioned on a microtome at 60-80 nm, mounted on copper 300 mesh grids and then stained with uranyl acetate and lead citrate. Grids were examined using a FEI Talos F200X transmission electron microscope (TEM). Inverted high-angle annular dark-field images of scanning TEM (HAADF-STEM) of intracellular spCD25 particles and energy-dispersive X-ray spectroscopy (EDS) spectra were acquired using inbuilt software and digital camera.

Prussian blue stains. Treg and Tconv cells were stained with the Prussian blue reaction to identify the presence of intracellular ferric iron following the reported procedure. Histologic images were acquired on a Nikon Ti-U Microscope (Nikon, Tokyo, Japan) with 60X objectives.