(E-175) CD4 cell-sorting microbeads as MRI contrast agents to track cell fate in vivo

Introduction::

Since the first successful organ transplant in 1954 [1], there have been ongoing efforts to ensure immunological acceptance of foreign tissues into the body. Previous methods, like immunosuppressive therapies, offer temporary effectiveness, but ultimately still result in tissue rejection [2]. However, regulatory T-cell (T-reg) therapy shows promise to halt the aggressive responses of other immune cells. Prior research has shown that T-regs can be successfully used alongside tissue transplantation, thereby preventing acute and chronic rejection [2].

               To understand the success of these T-reg cells, it is important to be able to track them in vivo. Imaging methods such as bioluminescence and radiolabeling have numerous limitations [3]. A more clinically relevant method of viewing these cells should be considered, one such example being MRI. If the T-cells are labeled with an iron-oxide contrast agent, the magnetic property of the beads allows for relative quantitation in vivo by measuring the T2 values before and after injection. But there are questions as to whether the beads can be internalized by the cell, and do not just reside on the cell membrane. However, there is now evidence that cell-sorting microbeads like CD25-targeted microbeads can be readily internalized in T-reg cells [3].

Materials and Methods::

The objective of this study was to see an apparent difference in T2 values when tracking CD4+ tagged cells in vivo via MRI and if we could further confirm cell internalization of the microbeads. T-cells were isolated from blood samples of anonymous human donors using an easysep kit. CD4+ T cells were expanded in RPMI media supplemented with 10% fetal bovine serum (FBS). Subsequently, 1.4*10⁸ CD4+ cells were labeled with CD4 microbeads in 6-well plates and 1.4*10⁸ remained unlabeled. Twenty million CD4 microbead-labeled or unlabeled cells were injected into five 8-10 week old female nude mice respectively, and MRI was performed at 0, 24 and 72 hours post-injection. Samples of cells were then prepped for electron microscopy.

Results, Conclusions, and Discussions:: MRI studies confirmed quantifiable signal decrease in mice receiving CD4-labeled T cells compared to mice receiving unlabeled T cells as shown in figure 1. At pre-scan, the p-value of the difference in means was determined to be p=0.19, indicating no difference at the start of the experiment. At 24 hours, the p-value decreased to p=0.082. After 72 hours, the p-value significantly decreased to 0.048. This indicates that CD4 microbeads are a promising candidate for T cell labeling and tracking in vivo. Longitudinal repeated measures analyses, under the general framework of mixed-effects models, are in progress to establish significance between both groups and all time points.

     Electron microscopy images showed indications of microbead presence inside the T cell cytoplasm as shown in figure 2. Additionally, the intensity spectrum revealed a Fe-K peak, and from that Fe presence could be confirmed (figure 5). Therefore, there is enough evidence to suggest the internalization of these CD4 microbeads.

There is still much to be learned of microbeads use for MRI cell tracking applications and we will continue to observe and track various T-cell phenotypes in vivo. This will open knowledge for the future of tracking immune cells for organ transplant, and possibly other disease systems and processes in the body. The closer immune cells can be predicted to locations, the better one can understand and modulate the immune response and harness it for biomedical applications.

Acknowledgements (Optional): :

References (Optional): :

      [1] “The History of Organ Donation and Transplantation.” UNOS, United Network for Organ Sharing, 9 Sept. 2022, unos.org/transplant/history/.
[2] Chaudhry, Sulemon et al. “Immune Cell Trafficking to the Liver.” Transplantation vol. 103,7 (2019): 1323-1337. doi:10.1097/TP.0000000000002690
[3] Khurana A, Marti F, Powell D.K, Brandon A.J, Dugan A, Gedaly R, Chapelin F. Cell sorting microbeads as an alternative agent for magnetic resonance imaging. Scientific Reports, (2022).