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In vivo magnetic resonance imaging of immune cells in the central nervous system with superparamagnetic antibodies¹

ISTVAN PIRKO^*,2, AARON JOHNSON^*,,2, BOGOLJUB CIRIC, JEFF GAMEZ^*, SLOBODAN I. MACURA, LARRY R. PEASE^, and MOSES RODRIGUEZ^*,,3

^* Departments of Neurology,
Immunology, and
Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota, USA

³Correspondence: Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905, USA. E-mail: Rodriguez{at}mayo.edu

SPECIFIC AIMS

We developed an MRI technique to image immune cell location and homing in vivo to the central nervous system (CNS). Superparamagnetic antibodies specific for cell surface markers allowed imaging of CD4+ T cells, CD8+ T cells, and Mac1+ cells in the CNS of mice infected with Theiler’s murine encephalomyelitis virus (TMEV) and in mice with experimental autoimmune encephalomyelitis (EAE).

PRINCIPAL FINDINGS

1. In vivo MRI detects immune cells in viral and autoimmune models of multiple sclerosis
We evaluated the efficacy of a new method to image immune cells in the CNS of animals chronically infected with TMEV. Superparamagnetic anti-CD4, anti-CD8, anti-Mac1, and irrelevant control anti-human CD19 antibodies were administered intravenously into both healthy (noninfected) and chronically infected IFN-R^–/– mice 16–20 h before MRI. IFN-R^–/– mice were used in this assay due to their capacity to have severe CNS pathology during TMEV infection.

We compared cell-specific MRI images to immunohistochemistry to prove the validity of this new technique. Superparamagnetic anti-CD8, anti-CD4, and Mac-1 antibodies were each administered to six chronically infected IFN-R^–/– mice 1 day before imaging. Immediately after MRI, animals were killed. Their brains were removed, frozen, and processed for immunohistochemistry.

In six animals injected with anti-CD8 antibodies, we acquired T1 and T2* weighted 3-dimensional image sets to compare with immunohistochemistry (Fig. 1 ). The location of CD8 cells on immunohistochemistry (Fig. 1A ) corresponded well with the low signal areas on the T2* weighted image (Fig. 1C ) or with high signal areas on the T1 weighted image (Fig. 1B ). A composite image (Fig. 1D ) derived by dividing the T1 image by the T2* image using an image algebra algorithm made it possible to clearly delineate the area of the brain with CD8+ T cell infiltration. We conclude from these experiments that the in vivo MRI technique we have described using superparamagnetic antibodies accurately identifies the location and distribution of CD8+ cells in the CVNS.

View larger version (40K): [in this window] [in a new window]   Figure 1. Distribution of CD8+ T cells in CNS of TMEV-infected mice. A) By immunohistochemistry CD8+ T cells are easily recognized as dark clusters (arrows). B) On T1 weighted imaging, the labeled structures are hyperintense, making their identification easier. C) On a corresponding T2* weighted scan, anti-CD 8 USPIO-labeled antibodies are visualized as low signal areas. D) Composite image showing distribution of CD8+ T cells. From T2* (B) and T1 (C) weighted images, a mask was generated by dividing the T1 image by the T2* image using an image algebra algorithm. Since the labeled lesions are bright on T1 and dark on T2*, the generated mask had high intensity areas corresponding to the lesions. The mask image was color coded and a Gaussian filter was applied for edge smoothing. Finally, it was superimposed on the original T1 weighted image.

We analyzed using the in vivo technique the distribution of CD4+ T cells (Fig. 2 A) and macrophages (Fig. 2D ) in IFN- receptor knockout mice after Theiler’s virus infection. Four animals were studied per cell type. Analysis of immunostaining revealed similar patterns of CD4+ T cells (Fig. 2B ) and macrophage cell clusters (Fig. 2E ) as determined using T1 weighted MR images. Higher magnification of lesions demonstrated both by in vivo MRI and immunohistochemistry revealed the presence of CD4+ T cells (Fig. 2C ) and macrophage cells (Fig. 2F ). There was precise correlation of specific inflammatory cell types with the patterns predicted by MR imaging. We conclude from these experiments that the in vivo MRI technique was highly specific for immune cells infiltrating the CNS and highly reproducible.

View larger version (83K): [in this window] [in a new window]   Figure 2. Distribution of CD4+ T cells and Mac1+ cells in TMEV-infected mice. Live TMEV-infected mice were imaged with MRI after intravenous injection with superparamagnetic antibody specific for A) CD4 or D) Mac-1. A 3-dimensional spin echo sequence was used for this acquisition. The slices were reconstructed from the 3-dimensional data set to match the histology slides. CNS tissue sections from the same mice imaged were immunostained with B) anti-CD4 antibody or E) anti-Mac1 antibody. Higher magnification of tissue (boxed area) immunostained with C) anti-CD4 antibody or F) anti-Mac1 antibody reveals the presence of CD4+ T cells and Mac1-positive cells. Arrows on MRI images A and D point to areas of high signal indicating the presence of USPIO-labeled antibodies in those regions. These areas corresponded precisely to areas of CD4+ T cell and macrophage infiltration in CNS tissue sections shown in panels B and E, respectively.

We labeled in vivo by MRI CD4+ T cells infiltrating the CNS of SJL/J mice (n=6 mice) with EAE. After 20 days of EAE induction, the location of CD4+ T cells was determined by our MRI method. We also demonstrated that a negative control superparamagnetic anti-human CD19 antibody caused only minimal to no enhancement in the MRIs of these animals with diffuse CD4+ T cell infiltration of the brain. We conclude that the in vivo MRI technique can be used to image inflammatory cells in virus-induced and autoimmune-mediated murine models of multiple sclerosis.

2. In vivo MRI technique delineates the time course of CD8+ T cell infiltration in the CNS of TMEV-infected C57BL/6 mice
We studied whether this in vivo MRI technique could be used to monitor the infiltration of T cells in the CNS over time. We used C57BL/6 mice infected with TMEV. These mice develop acute encephalitis on day 3 after infection, which peaks on day 7. We monitored CD8+ T cells in the CNS in vivo by MRI.

No CD8+ T cells are detected before TMEV infection. The MRI technique detected a strong CD8+ T cell signal 3 and 7 days after infection. No CD8+ T cell signal was detected on days 21 and 45, when the virus is cleared from the CNS. We conclude that the in vivo MRI technique using superparamagnetically labeled antibodies can follow the progression of immune cell infiltration into the CNS over time.

CONCLUSIONS

The kinetics of immune cell homing to the target tissue is of great scientific interest. Dynamic processes using conventional methods (immunohistochemistry, flow cytometry) can only be addressed by analyzing tissue samples at different time points from multiple animals. Our novel and powerful cell-specific MR imaging method is useful for imaging immune cell dynamics of a living animal. Most important, this technology can be used to visualize any cell in a living organism with unique surface antigens, provided that a monoclonal antibody is available and the antibody has access to the cells of interest via the bloodstream.

Previously defined in vivo lymphocyte imaging strategies required cell harvesting and ex vivo labeling. These cells needed to be labeled with USPIOs before transfer. Our technique requires no additional ex vivo steps of cell labeling.

The superparamagnetic antibodies used in this study can be detected on T1 weighted images as areas of high signal. Therefore, these agents are ideal for accurate imaging in clinical practice. We observed T1 relaxation shortening effects of superparamagnetic antibodies. This contradicts the usual dogma of only T2* and T2 effects by these agents. The T1 relaxation shortening was likely related to the high local concentration of USPIO-s at the target site of interest. However, chemical modification after homing cannot be excluded. Previous studies have demonstrated that USPIOs in the size of 10–50 nm are optimal for MR imaging. Above 50 nm the particles are much less likely to leave the bloodstream via fenestrated capillaries. The particles used in our method were 30–50 nm in size and were conjugated to IgG subclasses. We conclude that the small size of the particles as well as the conjugation to dimeric IgG molecules contributed to the success of our study.

A clear limitation of the study is the potential immunomodulatory effect of the applied contrast material. Long acquisition times may represent a problem in animal imaging that can be managed by using inhalational anesthetics.

USPIOs are completely biodegraded in 7 days. Because of this property, USPIOs are gaining acceptance in multiple clinical applications. Superparamagnetic antibodies similar to the ones we used are approved for selecting and enriching human CD34-positive hematopoietic progenitor cells from leukapheresis products. These cells have been shown to reconstitute all lineages of immune cells when reinfused into patients that have received myeloablative chemotherapy for cancer. Because we have used superparamagnetic antibodies similar to those approved for human use, it is possible that the in vivo MRI technique to image immune cells in the CNS described here could be readily developed for human use.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1124fje

² Both authors contributed equally to this manuscript.

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