Introduction

Rapid evolution and prevalent use of magnetic resonance imaging (MRI) in the clinic has fueled research in the development of contrast agents for specific cell tracking applications. Safe and efficient labeling of non-phagocytic cell types such as T cells nonetheless remains challenging. Magnetic-activated cell sorting (MACS) is a common method for cell population separation from tissue homogenates. The technique relies on targeting cell surface markers with specific antibodies bound to superparamagnetic nanoparticles also called ‘microbeads’. When placed in a high gradient magnetic field, the labeled cells remain trapped while unlabeled cells elute. Labeled cells can be collected by removing the magnetic field. Our hypothesis is that the microbeads used for cell separation have magnetic properties pertinent to cell labeling and tracking applications. We compared the MR signal effects of MACS microbeads (CD25) and in vitro activation beads (CD3/28) to the current preclinical standard ferumoxytol. Thereafter, we investigated in vitro labeling efficiency of regulatory T cells (Tregs) with MRI and histopathologic confirmation.

Methods

Triplicate samples of half and one million Treg were labeled overnight (16 hours) with 10 µl CD25 microbeads per million Tregs. After incubation, excess microbeads were removed by three washes followed by centrifugation. Triplicate samples of unlabeled half and one million T cells were also prepared. All cell samples were resuspended in 100 µl agarose gel (1% dilution in PBS) and transferred to PCR tubes for MR imaging. MRI images were acquired on a preclinical Bruker 7T imaging system (Bruker, Billerica, MA) using a mouse birdcage body coil (Bruker). Axial images of the cell samples were acquired using a Turbo RARE sequence with parameters TR/TE= 2500/33 ms, Matrix size = 128×128, FOV = 28×28 mm², Slice thickness = 0.7 mm, Averages = 2, Slice number = 16 and a T2map Multi Slice Multi Echo (MSME) sequence with TR = 2200 ms and TEs ranging from 7 to 90 ms, Matrix size = 96×96, FOV = 28×28 mm², Slice thickness = 0.7 mm, Averages = 1, Slice number = 16. T2 values were extracted using the system’s inbuilt Image Sequence Analysis (ISA T2vtr) software by defining circular ROIs over each tube and averaged over 3 slices.

Results

We tested the respective magnetic properties of CD3 and CD25 microbeads compared to cell tracking standard Ferumoxytol (Figure 1) and agarose gel controls. Both microbeads exhibit T2* effects with increasing dosage, with CD3 microbeads showing a weaker effect (Figure 1A&B). Regarding T2* relaxation curves, both agarose and CD3 microbeads exhibit linear relaxation within the echo time range selected (Figure 2), indicating long inherent T2* and limited impact of CD3 microbeads on MRI signal. However, ferumoxytol and CD25 microbeads display comparable logarithmic patterns (Figure 2), demonstrating CD25 microbeads’ ability to generate strong signal to noise on MR images. Overnight Treg cell labeling with CD25 microbeads shows positive intracellular Prussian Blue staining, indicating successful uptake of CD25 microbeads (Figure 3A) without viability impairment (94% viable cells, p<0.01 compared to unlabeled controls). CD25-labeled T cells and unlabeled controls do not exhibit positive staining (Figure 3C&D), signifying that CD25 needs to be present at the surface of the cells to enable internalization of the microbead. Intracellular labeling with CD3 microbeads was unsuccessful, possibly due to the large size of the bead and was therefore excluded from further testing. Likewise, Treg labeling with ferumoxytol is ineffective (Figure 3B), likely due to the non-phagocytic nature of T cells. CD25 microbead-labeled Tregs (Figure 4A, 1-2) displayed decreased signal on T2-weighted MR images compared to unlabeled cells (Figure 4A, 3-4) and agarose controls (Figure 4A, 5), consistent with adequate labeling with a paramagnetic iron oxide. T2 relaxation time maps (Figure 5B) yielded quantitative T2 signal decay. The T2 of half million Tregs (T2₍₂₎ = 62.67 ms ±0.67) is significantly lower and about three-fourth as high as the T2 of unlabeled T cells (T2₍₄₎ = 80.89 ms ±2.34) and agarose alone (T2₍₅₎ = 78.11 ms ±2.59, P = 0.0001 and 0.0006 respectively). T2 signal is further shortened in one million labeled Treg samples (T2₍₁₎ = 54.22 ms ± 0.51) and significantly lower than the half million labeled Treg samples (P=0.0001).

Discussion and conclusion

The CD25 microbeads that serve as MRI contrast agents in this project are approved for clinical research. CD25 microbeads alone displayed strong MR T2 effects, comparable to the standard Ferumoxytol, which is also used ‘off label’ for cell tracking applications. Equivalent CD34 microbeads are FDA approved as a humanitarian use device, which should fast-track approval for clinical ‘off-label’ use of CD25 microbeads. This study demonstrates that the CD25 microbeads used for magnetic sorting of regulatory T cells constitute an excellent cell labeling agent for subsequent MR imaging. The specific antibody binding promotes internalization of the magnetic bead, leading to strong MR effects, which have so far been unattainable with other iron oxides. This novel cellular tracking method will be key in tracking the homing and fate of Tregs in inflammatory pathologies and solid organ transplantation. Given the use of this agent in the existing clinical Treg sorting workflow, this method offers ease of use for Treg labeling during transplant surgery given time and sterility constraints.

Acknowledgements

This work was supported by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1TR001998 and by the Shared Resource Facilities of the University of Kentucky Markey Cancer Center P20CA177558.

References

No reference found.