Abstract

Positron emission tomography (PET) is a repeatable, noninvasive, analytical imaging modality that provides three-dimensional images of mammalian biological processes in vivo. This study compares established naïve and memory T-cell trafficking patterns during anti-tumor responses to gauge the potential of PET imaging technology as a cancer therapy tool. Naïve T-cells are directly harvested from transgenic OT-1 mice expressing ovalbumin-specific T-cells, while memory T-cells are collected from OT-1 splenocyte recipients immunized with irradiated ovalbumin expressing cells. Harvested naïve and memory splenocytes are retrovirally transduced with the msv1-sr39tk-ires-gfp PET reporter gene, and then adoptively transferred into antigen-specific tumor-bearing hosts. These hosts are subjected to several PET scans to monitor the migration kinetics of naïve and memory T-cells. MicroPET imaging provides in vivo visualization of T-cell localization at cognate tumor sites; it also provides a demonstration of memory T-cells’ faster homing responses and better effectiveness at retarding tumor growth as compared to naïve T-cells. Memory T-cells are also seen to persist in local lymphoid and lung tissues, a phenomenon not observed in paired naïve T-cell systems. Flow cytometry analysis corroborate PET observations. Study results support previous findings and direct a course toward future applications on anti-tumor therapies in clinical settings.

Introduction

Cancer is the second leading cause of death in both men and women in the United States (Center for Disease Control and Prevention 2004). Clearly, there is a need for effective cancer therapy. Effective therapy, however, is only possible with the essential tools needed to generate those therapies. Positron emission tomography (PET) holds the promise of such an effective tool.

PET is an imaging modality that uses molecular probes to provide three-dimensional models of mammalian biochemical and biological processes in vivo. Target compounds of biochemical processes are labeled with such positron-emitting radioisotopes as oxygen (¹⁴O and ¹⁵O), nitrogen (¹³N), and carbon (¹¹C); fluorine (¹⁸F) substitutes for non-positron emitting hydrogen. Tagged probes serve as direct markers to assay targeted biochemical processes at the level of transcription (Phelps 2000, Yu et al. 2000). By externally tracking the migration of these molecular probes with PET, reaction rates are determined (Phelps 2000).

In addition to kinetics, PET reporter gene/probe systems allow quantification of gene expression in vivo. This study applies ex vivo methods of gene delivery in which host target cells are extracted, transduced with virions containing a specific gene, and subsequently redelivered to the host. Reporter-gene/reporter-probe systems rely on a number of variables including transduced cell counts, levels of gene expression per cell, known attenuation probe signals through tissue, and clearance properties of probes from non-transduced tissues (Yu et al. 2000). Visualization of PET reporter probes depends on probe retention, which results from molecular modification by translated PET reporter gene proteins, a method employed in this study.

Typical PET assays initially inject positron-labeled probes into the subject; subsequent PET scans reveal probe concentration in tissues as percent injected dose per gram of tissue (%ID/g) and labeled products over time. The system operates three-dimensionally with a 7.8 cm axial and a 19 cm transaxial field of view. It is composed of 168 lutetium oxyorthosilicate crystal (LSO) detector modules (Tai et al. 2001). Time course measurements of plasma probe concentration demonstrate probe delivery to tissue. An image of the process under study is obtained after these data are processed through equations describing probe transport and reaction processes. Probes are delivered in low mass amounts, ranging from pico- to femtomoles per gram, to prevent considerable interference with other biological processes in the system (Yu et al. 2000). Tomographic images are derived from probe retention within target cells. Therefore, this imaging is done by indirect tracking of PET-marked reporter gene cells as sequestered products result from selective reaction of probes with reporter gene products (Yu et al. 2000, Cherry et al. 1997).

While PET scanners provide large-scale imaging, MicroPET scanners enable imaging of small animals using the principles of PET technolog. MicroPET scanners accommodate small animal sizes with approximately 8 mm³ volumetric resolution (Yu et al. 2000, Dubey et al. 2003). Repeat imaging of the same animal permits for kinetic analysis on cell migration, localizationm and expansion. This provides tomographic images allowing digital reconstruction of marked T-cell distribution and consequent determination of signal intensity differences within a tissue (Dubey et al. 2003).

This study utilizes a herpes simplex virus type-1 thymidine kinase mutant (HSV1-sr39tk) as the PET reporter gene with two PET reporter probes: 9-[4-[¹⁸F]fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and 2-[F-18]fluoro-2-deoxy-D-glucose (FDG). This system is based on principles of phosphorylation, capitalizing on the relaxed substrate specificity of viral thymidine kinase (TK) to phosphorylate glucose and nucleoside derivatives unlike endogenous mammalian TK (Gentry 1992). Radioactive molecular probes readily diffuse across cellular membranes and become phosphorylated upon entering a cell expressing viral TK. The phosphorylated product is subsequently trapped within the cell, with the positron-emission serving to directly mark labeled cells. HSV1-sr39TK is a mutant viral TK generated through random sequence mutagenesis (Black et al. 1996), which demonstrates a greater sensitivity and affinity to molecular probes than HSV1-TK (Gambhir et al. 2000). FHBG is used to trace the location, proliferation and time variation of marked cells. Non-phosphorylated FHBG is removed by hepatobiliary and renal clearance, which generates a background signal in the gut and bladder. Cells rapidly metabolizing glucose uptake FDG, and enzymes in the glycolysis pathway phosphorylate this glucose derivative to trap the phosphorylated product within the cell. Positron-emission from this molecular probe enables the detection of glucose metabolic activity to show viability, size, localization and metabolic activity of tumors (Dubey et al. 2003). Thus, molecular probes and reporter genes together provide the tools necessary to comparatively monitor known differential migration kinetics of naïve and memory T-cell populations in vivo.

Materials & Methods

Animals: Four- to six-week-old male and female immunocompetent C57BL/6 and immuno-incompetent RAG-/- knockout murine models were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained according to the guidelines of the University of California Animal Research Committee. OT-1 mice, bearing transgenes for expression of an ovalbumin-specific T-cell receptor (TCR), were carried on the RAG11-/- (Mombaerts et al. 1992) background to assure exclusive T-cell expression of the TCR transgene (Cabrone et al. 1989, 1992; Moore et al. 1988).

Cell Lines and Culture Conditions: The lymphoma cell line EL4 was obtained from American Type Culture Collection (ATCC # TIB-39), and is cultured in complete RPMI-1640 (BioWhittaker, MD) supplemented with 10% FBS (Omega Scientific, CA), 100 µg/ml penicillin, 292 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 1 percent ß-mercaptoethanol, all supplied by Gibco in New York. The EL4 derived E.G7-OVA cell line (Moore et al. 1988) (ATCC # CRL-2113) is cultured in similar complete media with the addition of 0.4 mg/ml of G418 (Sigma) for selection. The EL4 and E.G7-OVA cell lines are tumorigenic, generating foreign and cognate antigenic tumors in murine models. Foreign antigenic tumors are unrecognized by the murine immune system, whereas cognate antigenic tumors are recognized by the murine immune system and will consequently mount an intensive memory T-cell response to rapidly clear the system of the tumor. The human embryonic kidney cell line 293T (ATCC # CRL-11268) is cultured in complete Dulbecco’s MEM (Gibco, NY) supplemented with 10 percent FBS (Omega Scientific, CA), 100 µg/ml penicillin, 292 µg/ml streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate, all supplied by Gibco, New York. The 293T cell line is a highly transfectable derivative of the 293 cell line utilized to generate virions for transducing splenocyte target cells.

Results

Characterizing naïve and memory T-cells: Naïve and memory T-cells were fluorescently labeled with a-tetramer and a-CD8 monoclonal antibodies to verify OT⁺ phenotypes where tetramer antibodies present OVA peptides (SIINFEKL) in the context of MHC class one (H-2k^b). Double positive cell populations indicate the presence of OT⁺ cells, which are CD8⁺ T-cells capable of recognizing the SIINFEKL peptide sequence. CD62L and CD44 cell surface expression are utilized to further characterize the OT⁺ T-cells. Previous work has shown that memory cells typically demonstrate a low expression of CD62L and a high expression of CD44, while naïve cells exhibit a high expression of CD62L and a low expression of CD44 (Walker et al. 1995). Flow analysis illustrates similar results (data not shown), suggesting that naïve and memory T-cells had the expected surface markers for their respective differentiated states.

Monitoring naïve and memory T-cells in vivo: Upon determining anticipated cell surface marker expression, tumor cells and immune cells were adoptively transferred to immuno-incompetent hosts (Figure 1). Immune hosts recognize cognate antigens (E.G7); foreign antigens (EL4) serve as a negative control. This adoptive transfer reconstitutes the immune system of the immuno-incompetent host with CD8⁺ T-cells marked with the PET reporter gene and CD4⁺ T-cells, which help during an immunological response to antigen.

As early as Day One from post-adoptive transfer, memory T-cells are observed at E.G7 tumor sites, with no such signal detectable in paired naïve systems. By Day Four, memory T-cells have migrated to cervical lymph nodes with signals intensified in the nodes. By Day Eight, memory T-cells have accumulated in the lungs. Again, similar results are not seen in naïve systems.

To evaluate whether or not undetected signal in the naïve system resulted from tumor inviability, tumor growth was assessed with [¹⁸F]FDG scans on the indicated time points (Figure 2). At six days post-tumor induction, signals are detectable from both shoulders of tumor recipients in both naïve and memory systems. By Day 10, elevated signals are detected from EL4 shoulders in memory systems with little signal detected on E.G7 shoulders. In contrast, both EL4 and E.G7 tumors emitted high signal levels in naïve systems on Day 10, indicating viable tumors.

MicroPET detection of T-cell localization and cytotoxicity is corroborated with flow analysis. Immediately following MicroPET signal detection, subjects with the most intense signals were analyzed by flow. At Day One, signals are only detected from memory systems (Figure 2) and 1.6% OT⁺TK⁺ cells are found in the spleen (Figure 3). At Day Four, signals are detected in memory cervical lymph nodes, suggesting a migration away from the spleen as shown in the decrease from 1.6% to 0.01% OT⁺TK⁺ cells in spleen tissue. In contrast, naïve systems only exhibit 0.2% OT⁺TK⁺ cells in the spleen on Day Four, suggesting initial migration of naïve T-cells beginning at Day Four post-adoptive transfer. By Day Eight, lymph node and lung signals are detectable in memory systems, which are supported with 1.7% OT⁺TK⁺ cells in lymph nodes and 1.5% OT⁺TK⁺ cells in lung tissue. In contrast, on Day Eight, naïve cervical lymph nodes contained 0.02% OT⁺TK⁺ cells.

Discussion

Positron emission tomography may be an effective tool in the discovery of novel cancer therapies. To begin testing PET for such purposes, this study examines whether or not MicroPET imaging technology can be used to compare trafficking patterns between naïve and memory T-cell populations during anti-tumor responses. The following conclusions have been reached: MicroPET provides in vivo visualization of T-cell localization at tumor sites with memory T-cells demonstrating faster homing responses, more effective tumor growth retardation, and persistence in local lymphoid and lung tissue–phenomena not observable in paired naïve T-cell systems. This indicates that MicroPET is capable of comparatively distinguishing the differential migration kinetics of memory and naïve T-cell populations. The differential response rates observed in this study support previous work (Tough et al. 1999).

MicroPET imaging with [¹⁸F]FHBG demonstrates T-cell migration to cognate tumor in memory systems. These images reveal rapid memory T-cell localization kinetics suggesting faster responses than that of naïve T-cells. Surface-bound homing receptors expressed on memory T-cells facilitate this accelerated migration toward antigenic sites (Butcher and Picker 1996, Sprent and Surh 2002). Furthermore, naïve cells traffic at a diminished rate as their activation requires additional steps. This, in turn, slows their proliferation and differentiation into effector cells. The earlier effector cells reach antigenic sites during response, the fewer cells they must neutralize to eradicate the tumor. Naïve T-cells are thus less effective at combating similar challenges compared to memory cells.

While MicroPET imaging with [¹⁸F]FHBG detects T-cell migration, imaging with [¹⁸F]FDG demonstrates a gradual reduction in cognate tumor size in memory systems (Figure 2). Elevated signals on Day 10 detected from EL4 shoulders in memory systems and little signal detected from E.G7 shoulders, suggest tumor regression of cognate antigenic tumor (E.G7). In contrast, both EL4 and E.G7 tumors emitted high signal levels in naïve systems on Day 10, indicating viable tumors and ineffective tumor regression. Interestingly, [¹⁸F]FDG images from the memory group subject shows undetectable radioactive signal in the brain, contrary to the naïve subject. Organs typically metabolizing high amounts of glucose, such as the brain, tend to retain high amounts of radioactive [¹⁸F]FDG probe. An abnormal lack of brain signal, however, results from a limitation of the processing program that displays the image in lateral sections of the subject. As each adoptive transfer of tumorigenic cells incurs a degree of variability, some tumors proliferate at varying lengths across the shoulder. Thus, a section of the naïve subject demonstrating extensive glucose uptake in the tumors is proximal to the plane of the brain; a section of the memory subject demonstrating intense glucose uptake in the tumors is distant from the plane of the brain.

Flow cytometry on T-cell migration-specific tissue corroborates labeled T-cell migration. Flow cytometry on each tissue sample supports signals detected by the MicroPET imaging system. While cell percentages are not high in either naïve or memory samples, it is noteworthy to examine relative differences between naïve and memory systems. On Day Four, naïve and memory OT⁺TK⁺ cells in spleen demonstrate a sharp contrast. This is similarly observed between naïve and memory cervical lymph node OT⁺TK⁺ cell percentages. These observations suggest that memory T-cells home and migrate to tumor sites at a more rapid rate than naïve T-cells.

Considering that MicroPET exhibits the differential, biological functions of naïve and memory T-cells, we next examine whether or not the quantification of signal intensity in terms of cell numbers is possible. A collaborator pursued further characterization of MicroPET and developed an algorithm to relate signal intensities to absolute cell numbers (personal communication with Helen Su). This expansion of MicroPET enables not only qualitative analysis of in vivo biological processes, as presented here, but also the abstraction of quantitative data.

The differential migration kinetics of naïve and memory T-cells supports previous work. However, previous studies have not shown evidence of memory T-cell migration toward the lungs after tumor eradication i (Tough et al. 1999) (Figure 2). Currently, the biological significance of this phenomenon is not clear. It is anticipated that future studies will encompass monitoring T-cell populations (post-antigenic challenge) to identify novel memory T-cell migration toward the lungs. In addition, these results support future applications in developing anti-tumor therapies for clinical cancer research.

Acknowledgements

Many thanks to Dr. Carrie Miceli for the gift of the OT-1 mice and Dr. Owen Witte for the MSCV-sr39TK-IRES-GFP retroviral construct. Special thanks to Dr. Helen Su for assistance in lung extractions and flow analysis. I am thankful to Dr. Waldemar Ladno and Judy Edwards for their assistance with MicroPET imaging and also to the chemists and cyclotron crew for production of radioisotopes. Flow cytometry was performed in the UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Facility that is supported by National Institutes of Health awards CA-16042 and AI-28697, and by the JCCC, the UCLA AIDS Institute, and the David Geffen School of Medicine at UCLA. This work was supported by NIH/NCI P50-CA86306, the Ruzic Foundation, and the Jonsson Comprehensive Cancer Center.