Receptor-Mediated SPION Labeling of CD4⁺ T Cells for Longitudinal MRI Tracking of Distribution Following Systemic...

Tracking T cells in vivo using MRI is a major challenge due to the difficulty of labeling these non-phagocytic cells with a sufficient contrast agent to generate a detectable signal change. In this study, we explored CD4-Superparamagnetic iron oxide nanoparticles (SPION), which is commonly used in magnetic cell sorting, as a potential receptor-mediated, specific CD4⁺ T cell MRI labeling agent. We optimized the labeling protocol for maximal CD4⁺ cell labeling and viability. Cell health was confirmed with trypan blue assay, and labeling efficacy was confirmed with Prussian blue staining, transmission electron microscopy, and MRI of labeled cell pellets. Key cell functionality was assessed by flow cytometry. Next, CD4-SPION-labeled T cells or unlabeled T cells were delivered via intravenous injection in naïve mice. Liver MRIs pre-, 24 h, and 72 h post-T cell injection were performed to determine in vivo tracking ability. Our results show that CD4-SPION induces significant attenuation of T2 signals in a concentration-dependent manner, confirming their potential as an effective MRI contrast agent. In vitro, analyses showed that CD4⁺ T cells were able to uptake CD4-SPION without affecting cellular activity and key functions, as evidenced by Prussian blue staining and flow cytometric analysis of IL-2 receptor and the IL-7 receptor α-chains, CD69 upregulation, and IFN-γ secretion. In vivo, systemically distributed CD4-SPION-labeled T cells could be tracked in the liver at 24 and 72 h after injection, contrary to controls. Histological staining of tissue sections validated the findings. Our results showed that SPION CD4⁺ T cell sorting coupled with longitudinal MR imaging is a valid method to track CD4⁺ T cells in vivo. This safe, specific, and sensitive approach will facilitate the use of SPION as an MRI contrast agent in clinical practice, allowing for non-invasive tracking of adoptive cell therapies in multiple disease conditions.