DOI: 10.5061/dryad.v9s4mw76d
title: Data from: Effect of mitochondrial oxidative stress on regulatory T cell manufacturing for clinical application in transplantation: Results from a pilot study

description: 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.
description: <strong>Electron Microscope</strong>. 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%
 OsO<sub>4 </sub>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.
 <strong>Prussian blue stains.</strong> 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. 
description: # README for Imaging Dataset ## Dataset Overview This dataset contains
 electron microscopy images used for structural characterization of the
 **CD25** sample at various imaging modes and detector settings. All image
 filenames encode descriptive parameters including sample ID, thickness,
 imaging technique, and detector type. To ensure interoperability, `.HEIC`
 files (from camera capture) should be converted to `.png` format before
 analysis. These have been provided in the folder "Cell pictures in
 png format.zip" --- ## File Inventory and Descriptions | File Name |
 Description | | ------------------------------------------------- |
 ---------------------------------------------------------------------------------------------------- | | **CD25\_70nm\_STEM\_HAADF\_0006\_HAADF\_TEM.png** | STEM image of CD25, 70 nm section. Image #0006 collected using HAADF detector; includes TEM overlay. | | **CD25\_70nm\_STEM\_HAADF\_0006\_HAADF.png** | Same sample and section (70 nm), HAADF-STEM image without TEM overlay. | | **CD25\_70nm\_STEM\_HAADF\_0007\_HAADF\_TEM.png** | Image #0007 from the 70 nm CD25 section, HAADF-STEM with TEM overlay. | | **CD25\_70nm\_STEM\_HAADF\_0007\_HAADF.png** | Image #0007, HAADF-STEM only (no overlay). | | **IMG\_6730.HEIC** | Camera-captured image of specimen setup (e.g., grid alignment). | | **IMG\_6732.HEIC** | Sample overview image showing microscope stage positioning. | | **IMG\_6733.HEIC** | Setup image highlighting region of interest before data acquisition. | | **IMG\_6736.HEIC** | Image documenting instrument calibration setup. | | **IMG\_6737.HEIC** | Reference photo for stage or tilt orientation. | | **IMG\_6738.HEIC** | Context image for sample holder or instrument control view. | | **IMG\_6740.HEIC** | Overview of electron microscopy setup or beam alignment stage. | | **IMG\_6786.HEIC** | Additional reference image captured during data collection session. | | **IMG\_6788.HEIC** | Contextual image for secondary sample mounting. | | **IMG\_6803.HEIC** | Laboratory photo showing specimen context or preparation. | | **IMG\_6804.HEIC** | Additional documentation image of specimen configuration. | | **IMG\_6805.HEIC** | Capture showing sample grid prior to imaging. | | **IMG\_6806.HEIC** | Final setup image recorded before main data acquisition. | &gt; **Note:** `.HEIC` (High Efficiency Image Container) is a proprietary format.\ &gt; For full interoperability, please convert all `.HEIC` files to `.png` while retaining the same filenames.\ &gt; These HEIC files document sample setup, preparation, and imaging context rather than raw microscopy data. --- ## Naming Convention Each structured filename follows: ``` [SampleID]_[Thickness]_[MicroscopyType]_[ImagingMode]_[ImageNumber]_[Detector][_OptionalTag].png ``` | Element | Meaning | Example | | -------------- | ------------------------------------------------------ | ------------------------------------------------ | | SampleID | Biological or material sample identifier | CD25 | | Thickness | Sample section thickness (nanometers) | 70nm | | MicroscopyType | Technique used | STEM = Scanning Transmission Electron Microscopy | | ImagingMode | Contrast or mode | HAADF = High-Angle Annular Dark Field | | ImageNumber | Sequential acquisition number | 0006, 0007 | | Detector | Detector or channel used | HAADF, BF | | \_TEM | Optional suffix indicating a TEM overlay image | 
 
 | | .png | File format (converted from `.HEIC` for compatibility) | 
 
 | ## File Format Notes * Original camera images were `.HEIC`. * All
 microscopy images were saved as `.png` to preserve fidelity and improve
 software compatibility. * No rescaling or intensity normalization was
 performed during conversion. * Metadata (acquisition date, microscope
 settings) retained where possible. --- ## Data Notes * The HAADF and TEM
 images correspond to the same regions at similar magnification, allowing
 correlation between structural and compositional contrast. * The `.HEIC`
 series (IMG_6730–IMG_6806) provides contextual and setup documentation for
 reproducibility and record-keeping. --- ## Recommended Usage Open `.png`
 images in: * **FIJI / ImageJ** * **Python** (`matplotlib.image`, `Pillow`,
 or `OpenCV`) * **MATLAB** * **Any standard image viewer** If you need to
 include `.HEIC` files, convert them first (e.g., using `heif-convert` or
 macOS **Preview → Export → PNG**). --- ## Citation If you use this
 dataset, please cite: &gt; Gedaly R, Orozco G#, Lewis L. J, Valvi D,
 Chapelin F, Khurana A, Hidalgo G. E, Shmookler A, Tripathi A, Zhang C,
 Zwischenberger J. B, Marti F.\ &gt; *Effect of mitochondrial oxidative
 stress on Regulatory T Cell manufacturing for clinical application in
 transplantation: results from a pilot study.*\ &gt; **American Journal of
 Transplantation**, (2025).