Normal T-cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivator-peptide-derived superparamagnetic nanoparticles

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Abstract

The present study analyzed the feasibility of using magnetic resonance imaging (MRI) to monitor T-cell homing in vivo after loading T cells with superparamagnetic iron oxide (CLIO) nanoparticles derivatized with a peptide sequence from the transactivator protein (Tat) of HIV-1. T cells were isolated from C57BL/6 (B6) mice and loaded with 0, 400, 800, 1600, or 8000 ng/ml of FITC conjugated CLIO-Tat (FITC-CLIO-Tat). There was a dose-dependent uptake of FITC-CLIO-Tat by T cells. Stimulation of FITC-CLIO-Tat loaded T cells with anti-CD3 (0.1 μg/ml) plus IL-2 (5 ng/ml) elicited normal activation and activation-induced cell death (AICD) responses, and normal upregulation of CD69, ICAM-1 (CD54), L-selectin (CD62L), and Fas. The FITC-CLIO-Tat loaded T cells (3×107) were transferred intravenously (i.v.) into B6 mice and the in vivo MRI of mice was acquired using a spin-echo pulse sequence at 4.7 T with a Bruker Biospec system. Homing of T cells into the spleen was observed by a decrease in MRI signal intensity within 1 h after the transfer, which remained decreased for 2–24 h after transfer. These homing data were confirmed by FACS analysis and biodistribution analysis using 125I-CLIO-Tat. Thus, T cells can be efficiently loaded with FITC-CLIO-Tat without interfering with their normal activation and AICD, or homing to the spleen, and the biodistribution of FITC-CLIO-Tat loaded T cells can be monitored in vivo over time by MRI.

Introduction

A key aspect of the immune system is its cell migration, which involves extensive and continual redistribution of the T cells to different anatomic sites Potsch et al., 1999, De Becker et al., 2000. Although the T-cell response has been analyzed extensively in vitro, studies of the dynamic redistribution of the T cells have been restricted by the available methodologies to analysis of a limited number of anatomic sites at a limited number of time points. A non-invasive method of tracking the distribution of the T cells over time, and particularly in the first 72 h of the immune response, would be extremely informative.

The possibility of monitoring T cell migration through detection by magnetic resonance imaging (MRI) has been advanced by the recent progress in the development of superparamagnetic particles Bulte et al., 1996, Weissleder et al., 2000. It has been shown that T cells can be loaded with a superparamagnetic particle and imaged in vitro at the single cell level (Dodd et al., 1999). Loading of the T cells has now been facilitated by coating the particle with peptide sequences of the transactivator protein (Tat) of HIV-1. This protein has the desirable characteristic of being internalized by cells when present in the extracellular milieu Vives et al., 1997, Mischiati et al., 1999, Rusnati et al., 2000. The Tat peptide sequence can be linked to a small (5 nm) monocrystalline superparamagnetic iron oxide (IO) core coated with cross-linked (CL) aminated dextran, yielding CLIO-Tat nanoparticles. This nanoparticle exhibits enhanced stability and cell permeability, and enables entry of the superparamagnetic particles into cells at 10- to 100-fold greater efficiency than that achieved using previously (Josephson et al., 1999). Further, the CLIO-Tat particles can be labeled with FITC (FITC-CLIO-Tat) enabling detection by FACS analysis and visualization by microscopy, as well as high resolution MRI (Lewin et al., 2000). The CLIO-Tat nanoparticles have been reported to allow in vivo tracking and recovery of progenitor cells (Lewin et al., 2000). It has not yet been established, however, whether such methods can be used to follow migration of T cells in vivo without interfering with their normal function and responsiveness.

Activation of the T-cells normally results in upregulation of the expression of adhesion molecules, including L-selectin (CD62L) and ICAM-1 (CD54), which promote migration and homing to sites of inflammation Smith, 1993, Lawrence et al., 1995, Tangemann et al., 1998. In addition, the expression of the activation induction molecule, CD69, which represents the earliest lymphocyte activation antigen, is upregulated Lopez-Cabrera et al., 1993, Ziegler et al., 1994, Lauzurica et al., 2000. The majority of T cells then undergo activation-induced cell death (AICD) through the process of apoptosis involving the upregulation of Fas on the cell surface within 24–72 h of the activation event Golstein et al., 1991, Green and Scott, 1994.

To determine if loading of the T cells with CLIO-Tat superparamagnetic particles affects their function, we analyzed FITC-CLIO-Tat loaded T cells for activation, determined by expression of CD69; upregulation of the adhesion molecules, ICAM-1 and L-selectin; and apoptosis, as determined by FACS analysis of Fas and the apoptotic stain, 7-amino-actinomycin D (7-AAD)Philpott et al., 1996, Herault et al., 1999. The expression of these activation and adhesion molecules and the process of AICD in response to stimulation with anti-CD3 plus interleukin-2 was the same for the FITC-CLIO-Tat loaded cells as for the unloaded cells.

On transfer of the loaded T cells into C57BL/6 (B6) mice, imaging from 0 to 24 h after transfer indicated there was T-cell migration to the spleen within 1 h of transfer, which is the pattern of distribution that has been previously observed for T cell after intravenous administration (Bradley et al., 1999). The in vivo distribution of T cells in space and time determined by MRI analysis correlated with the spatial and temporal distribution of T cells in mice using two different biodistribution models: 125I-labeled CLIO-Tat loaded T cells and Thy1.1+ Thy1.2 T cells loaded with CLIO-Tat and injected into wild type B6 mice. Homing of T cells in vivo was further confirmed by the presence of FITC-CLIO-Tat in the spleen of the recipient mice as determined by FACS analysis. These results suggest that the labeling of T cells with superparamagnetic CLIO-Tat particles provides a potential method to monitor in vivo T-cell responses using MRI.

Section snippets

Mice

C57BL/6 (B6) and the Thy1.1+ congenic strain of B6 mice (Ogimoto et al., 1991) were purchased from the Jackson Laboratory (Bar Harbor, ME). The cages, bedding, water, and food were sterilized, and the mice were handled with aseptic gloves. Mice were housed in groups of three or four mice per cage for the duration of the experiment and all care was in accordance with institutional guidelines. All mice were female at 12 weeks of age.

Synthesis of CLIO-Tat

Aminated CLIO was prepared as described Josephson et al., 1999,

Loading of T cells with FITC-conjugated CLIO-Tat nanoparticles

To first determine the optimal dose for loading of T cells with FITC-conjugated cross-linked iron oxide-dextran coated-Tat nanoparticles (FITC-CLIO-Tat), T cells isolated from the spleen of B6 mice were incubated with different concentrations (0, 400, 800, 1600, and 8000 ng/ml) of FITC-conjugated CLIO-Tat in culture medium. The presence of FITC-CLIO-Tat in the loaded T cells was then determined using FACS analysis. There was a dose-dependent increase in FITC loading of primary T cells and 97%

Discussion

The low sensitivity of MRI contrast agents has hampered the use of MRI to efficiently monitor in vivo T-cell trafficking (Dodd et al., 1999). The development of superparamagnetic particles has been shown to provide a significant increase in sensitivity in the detection of single cells or cell suspensions by MRI. MRI of cells using paramagnetic contrast agents relies on the diffusion of the contrast agent through the cell membrane. However, most cells do not take up nanoparticles efficiently and

Acknowledgements

We thank Dr. T. Rogers and Mr. M. Spell of the FACS Core Facility at UAB for operating the FACS. We also thank Dr. F. Hunter for the critical review of the manuscript and Ms. Linda Flurry for excellent secretarial work. We thank Mrs. Leigh Millican of UAB's High Resolution Imaging Facility for the help with Electron microscopy and the Comprehensive Cancer Center's Radio Labeling Shared Facility. We thank Mr. Dan Clark for technical support in operation of MRI. This work is supported by NIH

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