Antibody-mediated cell labeling of peripheral T cells with micron-sized iron oxide particles (MPIOs) allows single cell detection by MRI

Abstract

Labeling cells with iron oxide is a useful tool for MRI based cellular imaging. Here it is demonstrated that peripheral rat T cells can be labeled in whole blood, in vitro, with streptavidin-coated micron-sized iron oxide particles (MPIOs), achieving iron concentrations as high as 60 pg iron per cell. This is 30 times the amount of labeling reported with ultrasmall particles of iron oxide (USPIOs). Labeling was mediated by use of a biotinylated anti-CD5 antibody, which is specific for peripheral T cells. Such labeling allowed the in vitro detection of single lymphocytes by MRI, using conditions well suited for in vivo animal work. Electron microscopic analysis demonstrated that MPIOs remained largely extracellular after labeling, with some evidence of intracellular uptake. Cell viability and early and late cytokine release studies showed no significant differences between labeled and unlabeled cells. Therefore, the use of MPIOs for achieving high iron concentrations for cellular MRI is potentially an effective new modality for non-invasive imaging of lymphocytes.

1)

Plot of % T cell labeling versus antibody concentration. The red curves are for 50 beads per cell in the labeling procedure, the blue curves are for 5 beads per cell and the blue curves are for 1 bead per cell. The black curve is for 0 beads per cell. Solid lines are means of the flow cytometry measurements; dashed lines are the manual counts. Note that the X axis is logarithmic.

2)

Example flow cytometry reports for samples without (A) and with MPIO labeling (B). This report is from cells labeled with 0.4 times saturating amounts of antibody and 5 beads per cell. Gate A is the less granular T cells, gate E is the more granular T cells. Panel C is the fluorescence measured for gate A without cell labeling. Panel D and E are the fluorescence measurements from gate A and E, respectively for labeled cells. (F) Representative labeled cell from gate E, MPIOs are green, nucleus is stained blue with DAPI. (G) Representative cell from gate A. Each T cell measures approximately 10 microns in diameter.

3)

Electron microscopy of labeled T cells. Particles were found sticking to cellular processes (A-C), in concave extracellular cavities (D-F) and sometimes in intracellular compartments (G). Panels H and I are expansions of two particles from the cell in Panel G (dashed boxes).

4)

ELISA cytokine assays for A) Interleukin-2 and B) Interferon-gamma production and release. The four conditions are 1) cells alone, 2) cells labeled only with anti-CD5 antibody (AB alone), 3) cells incubated only with MPIOs (MPIO alone), and 4) cells first labeled with the antibody, then incubated with the MPIOs (AB + MPIO).

5)

Panel A is an MRI slice from the labeled lymphocyte phantom showing isolated, punctate, dark contrast spots. The barscale is in millimeters. Panel B is a mean plot profile through 12 spots, measuring the linear size and percent contrast generated by labeled lymphocytes in the MRI experiments performed here.