Elsevier

Cytotherapy

Volume 12, Issue 2, 2010, Pages 238-250
Cytotherapy

Functional assessment of human dendritic cells labeled for in vivo 19F magnetic resonance imaging cell tracking

https://doi.org/10.3109/14653240903446902Get rights and content

Abstract

Background aims

Dendritic cells (DC) are increasingly being used as cellular vaccines to treat cancer and infectious diseases. While there have been some promising results in early clinical trials using DC-based vaccines, the inability to visualize non-invasively the location, migration and fate of cells once adoptively transferred into patients is often cited as a limiting factor in the advancement of these therapies. A novel perflouropolyether (PFPE) tracer agent was used to label human DC ex vivo for the purpose of tracking the cells in vivo by 19F magnetic resonance imaging (MRI). We provide an assessment of this technology and examine its impact on the health and function of the DC.

Methods

Monocyte-derived DC were labeled with PFPE and then assessed. Cell viability was determined by examining cell membrane integrity and mitochondrial lipid content. Immunostaining and flow cytometry were used to measure surface antigen expression of DC maturation markers. Functional tests included bioassays for interleukin (IL)-12p70 production, T-cell stimulatory function and chemotaxis. MRI efficacy was demonstrated by inoculation of PFPE-labeled human DC into NOD-SCID mice.

Results

DC were effectively labeled with PFPE without significant impact on cell viability, phenotype or function. The PFPE-labeled DC were clearly detected in vivo by 19F MRI, with mature DC being shown to migrate selectively towards draining lymph node regions within 18 h.

Conclusions

This study is the first application of PFPE cell labeling and MRI cell tracking using human immunotherapeutic cells. These techniques may have significant potential for tracking therapeutic cells in future clinical trials.

Introduction

Dendritic cells (DC) are often referred to as the ‘professional’ antigen-presenting cells of the immune system because of their unique ability to initiate and regulate primary T-cell responses (1). DC are strategically positioned throughout the body at anatomical sites common to pathogen entry, such as the skin, gastrointestinal tract and respiratory tract (1). In the periphery, they capture antigen and quickly become activated in response to pathogen-derived ‘danger signals’, as well as to endogenous factors released into the tissue environment during the assault (2,3). Acting as a bridge between innate and adaptive immunity, activated DC carry the pathogen-derived information from the periphery to draining lymph nodes, where they present processed antigen in the context of their major histocompatibility complex (MHC) class I and II molecules to activate naive CD8+ and CD4+ T cells, respectively (i.e. signal 1) (1). Along with this antigen-specific signal 1, DC also provide additional co-stimulatory and polarizing factors (signals 2 and 3, respectively) that regulate the magnitude and type of immune response needed to match effectively the particular character of the pathogen and the affected tissue (1,4).

Because of the unique and central role of DC in the immune response, DC therapeutics have covered a wide range of medical interests during the past two decades (5). The ability of DC to induce strong cell-mediated immune responses by promoting the effector functions of T helper (Th)-1 cells, cytotoxic T lymphocytes (CTL) and natural killer (NK) cells has positioned them at the fore-front of therapeutic applications in the setting of chronic diseases such cancer (5,6) and human immunodeficiency virus (HIV) infection (7). In contrast, because they promote the activity of regulatory T cells (Tregs), DC also play a critical role in the development of immune tolerance and immunosupression (8) and thus have been a major focus of research in areas of autoimmunity as well as organ transplantation (9).

In recent years, there have been many DC-based clinical trials initiated, most notably in the setting of cancer, where activated DC are loaded with tumor antigens and utilized as anti-cancer vaccines (5,6). While protective and therapeutic responses to tumors have been observed widely in animal models (10., 11., 12.), results from human clinical trials have been mixed (6,13). As Engell-Noerregaard et al. (6) reported in a comprehensive review of 38 published clinical studies for the treatment of cancer (6,13), there are many variables to account for such inconsistencies in clinical outcomes, including the particular type of DC used, the methods used for DC generation and activation, the type and source of antigen used, and the route of DC administration (5,6,14). Therefore, there is a critical need to limit this variability by developing standardized methods for the preparation of effective DC vaccines, their delivery to the patient and their functional assessment once administered (15).

One critical aspect influencing the therapeutic effectiveness of antigen-carrying DC is their requirement to enter draining lymph nodes, the place where they elicit robust adaptive T-cell responses (13). While the maturation status and route of administration can theoretically be used to predict the fate and effectiveness of the administered DC (16), in practice the therapeutic outcomes can be highly patient-specific. Non-efficacy is often attributed to aberrant DC trafficking or inefficient nodal delivery. Hence the ability to non-invasively monitor the actual trafficking and survival of DC in every patient is paramount (15).

A number of methods have been used to monitor transplanted cells in vivo. Optical approaches, using fluorescently or bioluminescently tagged cells (17,18), are powerful tools in certain pre-clinical settings but generally ineffective for visualizing deep structures at high resolution because of the opacity of most mammalian tissues. Radioisotope cell tagging, using positron emission tomography (PET) or single-photon emission computed tomography (SPECT) detection, is a highly sensitive technique but has limited spatial resolution (19,20) and is complicated by the requirement of inoculation and handling of radioactive materials in the clinic. Moreover, labeled cells can only be detected for a limited time period because of a finite half-life of the radioisotope.

Alternatively, magnetic resonance imaging (MRI) techniques allow for high-resolution three-dimensional (3-D) imaging and are widely used clinically. One MRI-based technique that has been studied extensively for in vivo tracking of administered cells employs superparamagnetic iron oxide (SPIO) nanoparticles (21., 22., 23.). This method has been proven useful in the clinic for monitoring human DC following their injection into lymphoid tissue (21). However, a challenge often encountered with metal ion-based contrast agents is that the large proton (1H) background signal present in soft tissues makes it difficult to identify unambiguously regions containing labeled cells throughout the body. It may be necessary to interpret subtle changes in gray-scale contrast, or measured relaxation rates, in regions believed to contain labeled cells. Consequently, cell quantification in regions of interest (ROI) is experimentally complex, perhaps requiring two MRI scans, both pre- and post-inoculation.

In this study we explored labeling human DC ex vivo with a commercially available perfluoropolyether (PFPE) tracer agent that can be used for tracking administered cells using 19F MRI or magnetic resonance spectroscopy (MRS), either in vivo or in excised tissues. Because mammalian tissues have negligible 19F content, this PFPE-based MRI technology has the benefit of yielding positive contrast images that are highly selective for labeled cells. Moreover, the PFPE reagent does not require transfection agents to label cells efficiently, even for non-phagocytic cell types, thereby minimizing cell toxicity and cell culture manipulation. While previous studies have demonstrated PFPE labeling in either immortalized cell lines or primary rodent cells (24., 25., 26., 27.), we report here the first studies using this technology platform to label and track therapeutically relevant primary cell types generated from human subjects. We demonstrate that 19F MRI is an effective way to track human DC in vivo post-administration in a xenograft model. Importantly, we also show that PFPE labeling has no significant impact on the viability, phenotype and functional properties of the cells, making this an attractive approach for exploring clinical applications. These findings provide evidence that PFPE cell tracking may be able to improve the effectiveness of monitoring cell therapy delivery in patients.

Section snippets

Media and culture reagents

The clinically applicable serum-free AIM-V medium (Invitrogen Inc., Grand Island, NY, USA) was used to generate DC. Iscove's modified Dulbecco's medium (IMDM) (Cellgro Mediatech, Herndon, VA, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Invitrogen) was used as an assay base medium. RPMI-1640 (Cellgro Mediatech) was used as the standard DC wash buffer throughout the study. The following recombinant human cytokines were used throughout the study: granulocyte-macrophage

Human DC can be labeled effectively with a 19F MRI tracer agent

Using conventional NMR spectroscopy, we were able to demonstrate that human DC can be labeled effectively with PFPE (Figure 1). A representative 19F NMR spectrum (Figure 1A) displayed a single peak from the intracellular PFPE at −91.58 p.p.m. in addition to the TFA reference at −76.00 p.p.m. From the integrated areas of these two peaks we calculated the mean 19F/cell of the DC (Figure 1B). In Figure 1B, the 19F content/cell is shown to increase monotonically with increasing PFPE added to

Discussion

In recent years there has been significant progress in the field of cancer immunotherapy. Clinical research has evolved from implementing ‘passive’ immunotherapeutic approaches, such as the infusion of large numbers of effector NK cells or antigen-specific CTL, to more ‘active’ immunotherapies where attempts are made to induce specific immune responses and effector cell functions within the patient. Active immunotherapies often involve administering relatively small numbers of antigen-loaded DC

Acknowledgments/disclosure of interests

The authors thank Vinod Kaimal and Patrick McConville from MIR, and Lisa Pusateri and Gayathri Withers from Carnegie Mellon University, for their technical assistance. We also thank Charlie O'Hanlon for logistical help and critical comments. The authors BMH, AB, ADN and RBM are employed by Celsense Inc., the supplier of the 19F MRI tracer agent used in this study. This work was supported in part by the National Institutes of Health (SBIR RAI078602A and R01 CA134633).

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