Download Bioluminescence imaging of lymphocyte trafficking in vivo

Experimental Hematology 29 (2001) 1353–1360
Bioluminescence imaging of lymphocyte trafficking in vivo
Jonathan Hardya, Matthias Edingerb, Michael H. Bachmanna,
Robert S. Negrinb, C. Garrison Fathmanc, and Christopher H. Contaga
a
Departments of Pediatrics, Microbiology & Immunology, and Radiology, Stanford University School of Medicine,
Stanford, Calif., USA; bDivison of Bone Marrow Transplantation, Department of Medicine, Stanford University School of Medicine,
Stanford, Calif., USA; cDivision of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, Calif., USA
Lymphocytes are highly mobile cells that travel throughout the body in response to a tremendous variety of stimuli. Revealing lymphocyte trafficking patterns in vivo is necessary for a
complete understanding of immune function, as well as cell-cell and cell-tissue interactions in
immune development and in response to insult. Although the location of cell populations in
various tissues at any given point in time may be revealed by techniques such as flow cytometry and immunofluorescence, these methods are not readily amenable to the assessment of dynamic cell migration patterns in vivo. In the past 5 years, technologies for imaging molecular
and cellular changes in living animals have advanced to a point where it is possible to reveal
the migratory paths of these vitally important cells. Here, we review one advancement in cellular imaging, in vivo bioluminescence imaging, which addresses the problem of lymphocyte
tracking. This imaging strategy has the potential to elucidate the temporal patterns of immune
responses and the spatial distribution of lymphocytes within the body. © 2001 International
Society for Experimental Hematology. Published by Elsevier Science Inc.
From their origin in the bone marrow to their final destination in peripheral locations, the movement of lymphocyte
populations through the lymphoid organs and to their tissue
targets remains elusive. Despite the advent of extremely
useful reagents for the detection of lymphocyte populations,
and even with the analysis of antigen-specific T cells using
major histocompatibility complex (MHC) tetramers [1,2],
visualization of the dynamic flow of lymphocyte trafficking
has been limited. Monitoring of these cells in vivo is difficult due to the relatively small numbers within a given lymphocyte population, overlapping subsets, and the highly
mobile nature of these cells.
For decades, the migration of lymphocytes has been followed using blood and tissue samples and a variety of tools.
Imaginative, and sometime heroic, efforts have yielded much
information, but the analyses of these processes in single animals over time has not previously been feasible. Methods that
employ reagents for staining specific lymphocyte populations
in tissue samples can be restricted by sampling limitations or
the need to sacrifice the animals in the study. The removal of
blood and tissue samples eliminates the contextual influences
of living organs and tissues, with a resulting gap in temporal
Offprint requests to: Christopher H. Contag, Ph.D., Stanford University
School of Medicine, S230 Grant Building, Stanford Medical Center, Stanford, CA 94305;E-mail: [email protected]
knowledge. The tremendous demand for information pertaining to in vivo cell migration patterns has led to such techniques as cryosectioning whole animals and imaging the distribution of labeled lymphocyte populations within the
sections [3]. Valuable insights have been gleaned from such
studies and they provide a solid basis for extending the static
analyses to those that assess dynamic changes. Despite formidable obstacles, serial images of lymphocyte localization in
response to disease states may yet be obtained, revealing the
true dynamics of the immune response. Combining advances
in immunology with in vivo molecular and cellular imaging
techniques has already proven fruitful, and further technological advancements are certain to occur. The techniques that
are being directed at these problems are either variations of
those used in medical practice, or novel methods that are well
suited for studying molecular and cellular processes in vivo
in laboratory animals.
PET and SPECT imaging
The radioactive emission of radionuclides can be detected
and imaged in living animals with scintillation cameras or
other devices, serving to locate the radiolabeled substances
in the body. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are
two nuclear medicine techniques [4] with possible applica-
0301-472X/01 $–see front matter. Copyright © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.
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J. Hardy et al./Experimental Hematology 29 (2001) 1353–1360
tion to studying immune cell populations in vivo [5,6]. Nuclides such as 18F, 15O, 13N, and 124I that emit positrons are
used in PET imaging, which takes advantage of the pairs of
coincident 511 keV photons emitted by positron-electron
collisions to decrease background and scattered signal. Radionuclides that emit photons are utilized in SPECT, and
their emission can be directly imaged using gamma cameras
that can be rotated around a subject to detect emission from
multiple angles and determine the location of the emitter in
three-dimensional space. The spatial resolution of PET and
SPECT are well within limits that would yield informative
data, about 1 mm at optimum [7], and it is certainly possible
that these modalities could be used to locate exogenously
radiolabeled lymphocytes in animals and humans. The deep
penetration of the high-energy emission from radionuclides
and the three-dimensional images offer significant advantages for animal studies with direct application to clinical
imaging. Miniaturization of clinical instrumentation for use
on common animal models such as the mouse has been accomplished [8–10], and research applications for this instrumentation have been reported [11,12]. The limitations of
imaging radionuclide emission of exogenously labeled cells
are the relatively short half-lives of some of the isotopes
used, the dissociation of detectable signal from the target
cell population, some constraints in radiochemistry, and the
loss of signal due to dilution during cell division. To overcome the dilution problem and limitations due to short halflives of the isotopes, several reporter genes that concentrate
radiolabeled compounds in expressing cells have been described [13–15]. This strategy holds significant promise for
immune cell trafficking studies and may have clinical utility. The use of reporter genes for labeling of cells in this
manner overcomes the problems of diluting the radiolabel
and the short half-lives of isotopes, since the reporter replicates with cell division and the isotope can be administered
repeatedly prior to image acquisition. These advancements
permit the long-term analyses that are necessary for understanding lymphocyte biology.
MRI
Magnetic resonance imaging (MRI) has also been adapted
for small-animal imaging, and is a modality where translation to clinical imaging is possible. In this technique, the
subject is placed in a strong static magnetic field. Nuclei of
certain elements such as spin-1/2 hydrogen have a magnetic
moment that aligns preferentially along the direction of the
magnetic field. Irradiation with a radio frequency magnetic
field at the proper frequency (the resonance condition)
causes transitions between energy states of the nuclei, resulting in net excitation of the nuclear spin system. Relaxation of the spins back to the lower energy state generates a
signal (the nuclear magnetic resonance signal) that is characteristic of the nuclei and their chemical and physical environment. With suitable magnetic gradient coils and Fourier
Transform signal processing, these signals can be localized
to produce an MR image. Many refinements of MRI have
been made that permit images of unprecedented quality and
resolution for the detection of tumors and a wide variety of
other medical applications. Recently, MRI has been employed with the intent of localizing lymphocytes in vivo
[16]. In this study, the lymphocytes were labeled with iron
beads, which exhibit a MRI signal, linked to peptides derived from the HIV Tat protein to facilitate cellular uptake.
These peptides efficiently labeled lymphocytes, and individual cells labeled with this strategy were detectable within
bone marrow samples. Whole, living animals were not imaged in this study, but the possibility remains tantalizing.
Optical imaging techniques
The relative opacity of tissue permits the transmission and
piping of light through the body, and thus small amounts of
light emitted from weak internal biological sources escape
the scattering and absorbing environment of the body and
can be detected externally. As significant amounts of light
(on average, 10-fold per centimeter of tissue) are lost due to
absorption and scattering, this method is best suited to the
study of small laboratory animals such as mice and rats
[17]. Fluorescent signals can be detected in vivo [18–21]
and the use of optical dyes such as fluorescein permits the
reisolation of labeled cells from animals using flow cytometry. As an example of using dyes for tracking studies, exogenously labeled lymphocytes have been imaged in the rat
eye in vivo using a scanning laser opthalmoscope [22]. Unfortunately, light of the wavelengths that are needed to excite many of these fluors, as well as the wavelength of emission, are blue and green and do not penetrate deeply into
and out of mammalian tissues [23,24]. These techniques
are, therefore, best applied to the study cells in culture or at
superficial tissue sites in vivo. Advancements in optical detectors that employ confocal or two-photon detection modalities will greatly improve the utility of the in vivo detection of fluorescent signals as the excitation is accomplished
with longer wavelengths with deeper tissue penetration.
Red light penetrates tissues more efficiently than green
or blue emission; dyes that absorb and fluoresce in the red,
such as indocyanin green, have been well described and
used in animal models and in humans for detection of tumor
nodules using light in vivo [25–27]. Some of these dyes
have been coupled to proteolytic cleavage sites in juxtaposition to quenchers providing a molecular “light switch” that
is activated by specific protease activity, and cleavage of
such molecules in vivo has been used to assess protease activity in living animal models of neoplastic disease [21].
There are many applications in which optical imaging offers
significant advantages over other modalities, especially in
animal models of human biology and disease [17,28,29].
Despite the benefits of using fluorescent and other exogenous tags, cell trafficking studies employing these ap-
J. Hardy et al./Experimental Hematology 29 (2001) 1353–1360
proaches can be limited, as previously discussed, by the duration of the signal as it is diluted through cell division and
differentiation. Using reporter genes that are integrated into
the genome and whose detectable signal is inextricably
linked to the metabolic activity of the target cell population
can circumvent problems of loss of signal due to dilution,
as well as the confounding detection of signals that have
dissociated from the viable target cell population. Optimal
reporter genes for in vivo cell trafficking studies should
encode well characterized gene products with deeply penetrating emission and a high signal-to-noise ratio. Accessible
and versatile detectors with good sensitivity for detecting
the activity of the gene product with relative ease are desirable for accelerating immune cell trafficking studies and enabling more sophisticated biological assessment.
In vivo bioluminescence imaging
Although no single imaging modality can provide an investigator with all of the necessary information pertaining to a
biological system, imaging strategies that use genetically
tagged cells that express bioluminescent reporter proteins
offer the advantages of fine temporal analyses, labeling versatility, accessible instrumentation, and a signal-to-noise ratio that provides high sensitivity of detection. Weak internal
sources of biological light, bioluminescence, can be externally detected using sensitive charge-coupled device (CCD)
cameras as low-light detection systems. This imaging modality, known as bioluminescent imaging (BLI), employs
light-emitting proteins, known generally as luciferases, for
real-time in vivo detection of tagged cells, thus enabling the
type of temporal analysis in single animals that is necessary
to image lymphocyte trafficking. Given that there are very
few sources of light in mammalian cells and bioluminescent
proteins do not require an excitation source, the background
in BLI is nearly nonexistent. Luciferases have been studied
for decades and are thus well characterized and readily applied to a variety of biological questions; this approach has
been used to monitor infectious diseases, gene expression
patterns, and tumor cell proliferation in vivo [17,30,31].
Luciferases are a family of proteins produced by luminescent organisms such as Photinus pyralis, the common
American firefly, that produce visible light from substrates
known generically as luciferins. These reactions require
high-energy molecules such as ATP, and thus light production is coupled to metabolic activity of the cells expressing
the reporter gene. Genes coding for luciferases are useful
tools for molecular and cellular biologists and, due to the
penetration of bioluminescent light through mammalian tissues, have more recently been shown to be valuable for in
vivo analyses [32]. The luciferase (luc) gene of Photinus
pyralis or other genes encoding bioluminescent proteins,
can be inserted behind the regulatory elements of various
genes (promoters), thus becoming a reporter for expression of
a given gene. Reporter gene constructs that employ syn-
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thetic, or natural, promoters that are constitutively expressed in
a variety of cell types can be used to label immune cell populations such that the cells may remain labeled despite
physiologic changes that may occur in vivo.
The luciferin that is a substrate for the Photinus pyralis
luciferase is a small, water-soluble molecule that quickly
penetrates the membranes of cells in tissues following intraperitoneal or intravenous injection, even crossing the bloodbrain and placental barriers [33,34]. Limited penetration of
luciferin into prolactin-secreting cells has been reported
[35]; however, this appears not to be a general problem
[30,36]. Luciferin is easily administered intraperitoneally
together with anesthetic for imaging. The toxicity of this
molecule, while not yet fully evaluated, appears to be low.
Evidence for low toxicity has been obtained in a number of
studies [17,30,33,36–39], and most notably its repeated administration to newborn mice results in no discernable effects on the health of the animals (Contag et al., unpublished results). The distribution of luciferin to tissues has
been examined in a limited study [30], and more complete
pharmacologic studies are underway in several laboratories.
Strong correlations between in vivo signal intensity with
relative luminescence of ex vivo assays, and other measures, has been obtained at a number of tissue sites, suggesting that, under the conditions reported, luciferin is present
in excess in brain, fetal, and all other tissues that have been
examined [33,34,40].
The instrumentation for low-light imaging is comparatively inexpensive to purchase, operate, and maintain and
does not require the use of radionuclides, offering the opportunities of decentralized imaging facilities and thus enabling accelerated investigation of biological processes in
vivo [41]. The sensitive CCD detectors that are required to
capture transmitted photons have been widely used in biological imaging, and several camera architectures are available for imaging of extremely weak optical signals. Cooling
the CCD detector to temperatures of 100 to 120C is an
effective means of reducing thermal noise and results in extremely sensitive CCD cameras that can detect light over a
broad spectral range, including the wavelengths that are
more readily transmitted through living tissue (650–1100
nm, [41]). At present, CCD detectors of this format offer
greater sensitivity to light transmitted through living tissues
(100-fold) than cameras that employ other architectures
such as those that employ image intensifiers. This is, in
large part, due to the broad spectral range of cooled CCDs
and hence sensitivity to red light [41].
For BLI the animal is anesthetized and the substrate, luciferin, is injected, usually intraperitoneally, at the time that
anesthesia is administered. After a fixed time, usually 15
minutes, the animal is placed in a light-tight chamber upon
which a cooled CCD camera has been mounted [41]. Grayscale reference images of the study subjects are obtained
under weak illumination using camera settings that are optimal for this image (small lens aperture and short exposure
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J. Hardy et al./Experimental Hematology 29 (2001) 1353–1360
times of typically 1 second). A second image is then acquired, in the absence of an external light source, collecting
the light that is generated internally and transmitted through
the animal’s tissues. To obtain this bioluminescent image the
lens aperture is maximum, and integration times as short as
one second (even a fraction of a second) or as long as five
minutes, depending on signal intensity, are used. In cooled
CCD cameras, pixel size can be increased by “binning,”
which serves to increase the number of photons represented
at each point in the low-light image. Binning refers to combining a number of individual small pixels into a larger
pixel, which is useful for reducing the background due to
read noise and rapidly producing images with good contrast.
Binning, however, results in a loss of resolution. When the
signal intensity is high, binning is not necessary and the full
resolution of the CCD camera can be preserved. Pixel binning of 10 10 is generally used to acquire the bioluminescent image; however, binning of 2 2 can also be used for
study subjects with intense signals. The number of photons
collected at each pixel is stored digitally for each exposure.
These data can be presented in pseudocolor and overlayed
on the grayscale reference image to localize the signals to
specific tissue or organ sites. The raw data of actual photon
count that have been stored digitally can be recalled for
analysis and reanalysis.
The most serious limitation of BLI is the absorption and
scattering of light by mammalian tissue, which reduces both
the sensitivity and resolution of any optical method. The
primary absorber in tissue is hemoglobin, and imaging of
blood-rich organs, such as the liver and spleen, can be less
efficient than more peripheral sites with less hemoglobin.
As hemoglobin absorbs primarily in the blue-green region
of the spectrum, much of the yellow-green emission of the
firefly enzyme is absorbed by mammalian tissue [41]. The
recent cloning of a red-emitting luciferase from the railroad
worm [42] and the isolation of mutants of firefly luciferase
that emit in the red [43] may be of help in this regard. Background signals in BLI can result from the long-lived fluorescence (phosphorescence) of contaminants on fur and
plant-derived fluors in food, but these signals are generally
very low and are readily subtracted as background.
BLI has been applied to the in vivo study of neoplastic
disease [31,34]. Cancer cells are relatively easy to manipulate in culture, and were among the first targets of luciferase-based in vivo imaging technology [40]. When
HeLa cells were transfected with luciferase, their growth at
several different anatomical sites could be easily measured
[31]. As a model of minimal disease, the small numbers of
cells present early in the study were visible also, indicating
that BLI would be useful for studying therapies that address the minimal disease states that occur early in the disease course or after reduction of the tumor mass by other
therapies [40]. The ability to detect small numbers of cancer cells in whole-body images has also been useful in localizing micrometastasis in animal models of human cancer [29].
As bioluminescence images can be rapidly obtained from multiple animals simultaneously, drug efficacy and the kinetics of
tumor relapse could be readily evaluated in several study
groups [40]. This provides great insight into the early events of
tumor growth and response to novel therapeutic strategies such
as transfer of cytotoxic lymphocyte populations [40].
BLI in cancer immunotherapy
The effects of an anti-cancer treatment that utilizes lymphocytes expanded ex vivo have been evaluated using BLI. Cytokine-induced killer (CIK) cells, comprised largely of
CD3CD56 cells (NK T cells) were introduced into mice
bearing a luciferase-expressing tumor. CD3CD56 cells display antitumor activity against a variety of tumors in culture
and in vivo [44,45]. Sweeney and coworkers have demonstrated the effect of this immune cell therapy on tumor burden
over time using BLI [40]. As CIK cells are CD3, it has been
possible to redirect the activity of these cells using antibody
molecules with two distinct binding domains, one for CD3 and
the other for a tumor-associated antigen (Sheffold, Contag, and
Negrin, results under consideration for publication). These experiments were also performed in SCID mice using luciferaseexpressing tumor cells, in this case a human ovarian cancer cell
line that overproduces the surface protein HER2/neu (SKOV3luc). Bispecific antibodies directed toward CD3 and the
HER2/neu oncogene increased the therapeutic efficacy of the
CIK antitumor treatment, presumably due to increased adherence of CIK cells to the tumor cell targets. The quantification
of emitted photons over time provides information about the
kinetics of response in individual animals.
It is especially exciting that tumors below the size that can
be readily identified and recovered for histological examination at autopsy can be visualized. In this manner, image data
can be used to direct tissue sampling for the ex vivo assays,
leading to improved analyses and more predictive animal models of human neoplastic disease. In the imaging studies used to
examine the anti-tumor activity of CIK cells, the target was labeled and not the effectors; therefore the distribution of immune cells still needed to be analyzed by traditional methods,
most of which involve sacrifice of the animal subjects at various time points. By labeling the effector population rather than
the tumor, the dynamic migration and redistribution of these
lymphocytes could be observed in vivo. Such experiments are
currently underway and may reveal the properties of CIK cells
that allow them to find and clear tumor cells. Labeling of immune cell populations is performed by transducing the cells in
vitro [46], which is often inefficient when using peripheral
blood mononuclear cells (PBMCs) as the target cells.
Lymphocyte trafficking
The specific genetic labeling of effector lymphocytes in
vitro permits localization of these cells in animals to be imaged by BLI. When CD4 T-cell hybridomas or primary T
cells specific for known peptide antigens are transduced
J. Hardy et al./Experimental Hematology 29 (2001) 1353–1360
with retroviral vectors that encode multifunctional reporter
genes (see below), they can be followed as they migrate to
locations determined by their antigenic specificity. Employing a joint inflammation model of arthritis, T-cell hybridomas from transgenic mice that express a T-cell receptor specific for a type II collagen peptide (CII) home to the joints
[47]. This localization can be observed by BLI when the
specific T cells are transduced with a luciferase reporter
gene and injected into mice intraperitoneally or intravenously (Fig. 1). The process of homing can be imaged in individual mice over time, revealing initial localization
(within 5 minutes) in the lung, with subsequent localization
(at 24 hours) in lymph nodes, followed 48–96 hours later
by signals from sites of inflammation, in this case the inflamed joints. When the T cells are derived from mice transgenic for a T-cell receptor (TCR) that recognizes myelin basic protein (MBP), signal was retained in MBP-immunized
mice for at least nine days, and the labeled cells were observed to localize to the central nervous system (CNS) (Fig.
2) [37]. Interestingly, MBP-specific T cells also transiently
homed to inflamed joints in the arthritis model, indicating
nonspecific migration to sites of inflammation. The MBPspecific cells were only transiently retained in these sites,
Figure 1. Trafficking capacity of CII-specific CD4 T-cell hybridomas.
CII-immunized DBA/1 LacJ mice with severe arthritis (A and B) received
CII-specific CD4 T-cell hybridomas expressing a GFP-luciferase reporter
gene (1 106 cells per mouse) intravenously. The images were obtained
on day 3 (A) and on day 5 (B). The color scale represents luminescent signal intensity, with blue indicating the least intense and red the most intense
light originating from the transduced cells in the animals. The range of signal intensity represented by the color scheme is indicated by the scale for
each image. The data indicate concentration of the labeled cell population
to the joints of the affected hind limbs and the tail. This figure was adapted
from Nakajima et al. [47].
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whereas CII-specific cells remained in inflamed joints for
over seven days [47]. When the joints are preinflammatory
clinically, the nonspecific localization is not observed.
These data indicate that lymphocytes migrate nonspecifically to sites of inflammation but are retained in areas where
they encounter the “relevant” antigen, confirming long-held
models of lymphocyte migration and function.
These experiments represent the beginning of a field that
is certain to expand to include the analysis of many models
of autoimmunity and infection. The possibilities are manifold, including the determination of the kinetics of CTL migration and possibly even memory-cell distribution and reactivation. By understanding the trafficking patterns of
lymphocytes it may be possible to deliver therapeutic or immune modulatory proteins to diseased tissues as was demonstrated in the arthritis model [47].
One of the problems that might arise in the analysis of
lymphocyte migration is the background of circulating cells
in the blood. Placing luciferase genes behind appropriate promoters may restrict the expression of the reporter to desired
circumstances or locations. Activation markers might be a
target for this procedure and could reveal areas of lymphocyte activation. Effector genes such as the granzymes also exhibit expression patterns of this type. In the study of lymphocyte function, the analysis of gene expression patterns by
every means possible may be necessary. For greater versatility, multifunctional reporters that exhibit different properties
can be employed. Fusions of luciferase with green fluorescent
protein (GFP) [48], or related proteins, allow analyses with
CCD imaging, flow cytometry, and immunohistopathology,
greatly increasing the utility of the reporter gene construct. A
critical advance in this regard is the development of transgenic mice that express luciferase in all their tissues, thus
serving as universal donors for transplantation and trafficking
studies. Stem cells from such mice may be introduced into
appropriate recipients and followed throughout the life of the
animal, providing unprecedented possibilities of analyses. In
an initial attempt at creating such a universal labeled donor
mouse, Bachmann et al. (MHB, PK, CHC, unpublished) produced transgenic mice in which a cytomegalovirus (CMV)
immediate early promoter directed expression of a GFPluciferase fusion gene (Fig. 3) [49–51]. These mice were luminescent in several tissues such as heart and pancreas, and
could be used as donors of labeled tissues [38] and stem
cells. However, as they do not express GFP in hematopoietic tissues (Scheffold, Edinger, Bachmann, unpublished data),
cells of these lineages could not be identified by fluorescence.
Using transgenic animals that express the multifunctional
reporter genes in lymphoid tissues and sorting the donor cells
for such properties as antigen specificity using MHC tetramers, immune responses and memory could be examined by
imaging. The migration of other populations, such as dendritic cells, might also be imaged over time using transplants.
Of decided advantage would be the ability to track two or
more populations, using several different luciferases. The use
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J. Hardy et al./Experimental Hematology 29 (2001) 1353–1360
Figure 2. MBP-specific transduced CD4 T cells traffic to the CNS. Splenocytes from MBP TCR Tg mice were transduced with a GFP-luciferase retroviral
vector. CD4 cells were MACS isolated and analyzed by FACS for GFP expression, then cell suspensions with 1 106 GFP cells were transferred intravenously to PL/J recipients. 1 106 CD4 cells transduced to express GFP-luciferase were transferred to naïve (A) or MBP-immunized mice prior to clinical
signs of experimental allergic encephalitis (EAE) (B). Data are representative of 3 experiments with n 2–3/group. Although the patterns were initially the
same in both groups of animals, in the immunized animals the signal from the labeled lymphocytes persisted and appeared to localize to the lumbar region of
the spinal cord. In the naïve animals the signal initially appeared over the lymphoid organs and was eventually lost over time. Ventral and dorsal views are
shown as indicated at the 24-hour time point. This figure was adapted from Costa et al. [37].
Figure 3. Transgenic mice as a source of labeled cell populations. A GFPluciferase fusion gene was created by amplification of GFP (S65T) from plasmid
pCDM7-GFP [49], with a 19-amino acid linker [50] attached at the 3 end. This
was inserted into the plasmid pGL3 (Promega, Madison, WI, USA) upstream of
the luciferase gene such that the two open reading frames were fused in frame. The
fusion gene (diagrammed in the lower part of the figure) was cloned into the
expression vector pJW4304 [51]) downstream of a CMV immediate early promoter and intron, resulting in plasmid pJW-GyL [16]. The expression cassette was
excised from the plasmid DNA and introduced into pronuclei derived from the
FVB strain of mice via microinjection. Offspring were monitored for luminescence at age 4 weeks. The labeled animal (A) exhibits widespread luminescence
whereas its littermate (B) is negative. Transgenic mice with ubiquitous and constitutive reporter gene expression may serve as reliable sources of labeled cell populations for immune cell trafficking studies as well as a source of labeled tissues for
studies in transplantation [30].
J. Hardy et al./Experimental Hematology 29 (2001) 1353–1360
of this procedure to track any cell of interest in real time, especially cells of the immune system, will reveal new information about the physiological behavior of cells as well as their
function in different diseases. This method will provide new
insight into cellular roles in infectious disease, autoimmunity,
tumor immunology, and transplantation biology. The availability of a wide range of knockout and transgenic animals as
lymphocyte donors will allow the study of functional roles of
individual molecules in vivo, especially in regard to their influence on homing and proliferation. This method would be
very useful in the analysis of anti-tumor immunology by labeling both the tumor and the effector population. To enable
such studies, considerable efforts are now underway to discover and utilize novel luciferases that have different wavelengths of emission and/or use different bioavailable substrates. Should these possibilities be realized, a new
dimension in optical imaging will be available.
Imaging and hardware developments
Not to be underestimated is the potential for further technological advancement of CCD imaging, and the refinement of
imaging devices for these novel imaging strategies. The
CCD cameras currently in use for the majority of optical imaging are macroscopic. There is no reason why higher-powered lenses, such as those employed in dissecting scopes or
even microscopes, could not be mounted for use in imaging.
The feasibility of this approach has been demonstrated in
the analysis of bacterial interaction with mammalian cells in
culture [52]. Such tools may lead to finer levels of analysis
that can be readily coupled to the macroscopic imaging
strategies. There are limitations to microscopic analysis of
bioluminescent emission, in that the resolution is constrained by the scatter of light as it passes through tissue,
and so the extent of these potential improvements remains
to be elucidated. Currently, the images obtained by the CCD
camera are two-dimensional. Modifications of the instrumentation could change this situation, yielding three-dimensional, or possibly even tomographic, images by manipulating the camera or the subject to obtain a series of images
from around the animal. These images would then be combined by computer analysis into a three-dimensional representation of cell trafficking or gene expression patterns.
Summary and future directions
In conclusion, the field of imaging technology has advanced
to the point of soon revealing secrets of lymphocyte behavior that have previously been elusive. Medical imaging
techniques such as PET, SPECT, and MRI show great
promise in the detection of labeled lymphocyte populations
in vivo, and more improvements are certain. Optical imaging is especially promising because of the ability of luciferases to emit light without external excitation and their
utility as genetic tags. Tumor models employing luciferasebased imaging already reveal the interaction of lymphocytes
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and their targets, the location of the tumors, and the efficacy
of treatment regimens. Lymphocytes labeled in vitro have
been traced to their targets in models of autoimmunity. The
exploitation of these techniques in the search for the temporal movements of many lymphocyte subsets and antigenspecific clones is only a matter of time.
Acknowledgments
This work was supported in part by the National Institutes of Health,
grant number 1RO1 DK58664, the National Cancer Institute, grant
number 1 R33 CA88303, and unrestricted gifts from the Hess and
Mary L. Johnson Research Funds (CHC). We gratefully acknowledge Dr. Gary Glover for his contribution to the MRI section.
References
1. Altman JD, Moss PAH, Goulder PJR, et al. (1996) Phenotypic analysis
of antigen-specific T lymphocytes (published erratum appears in Science 1998 Jun 19;280(5371):1821). Science 274:94
2. Burrows SR, Kienzle N, Winterhalter A, Bharadwaj M, Altman JD,
Brooks A (2000) Peptide-MHC class I tetrameric complexes display
exquisite ligand specificity. J Immunol 165:6229
3. Reinhardt RL, Khoruts A, Merica R, Zell T, Jenkins MK (2001) Visualizing the generation of memory CD4 T cells in the whole body. Nature 410:101
4. Green MV, Seidel J, Vaquero JJ, Jagoda E, Lee I, Eckelman WC
(2001) High resolution PET, SPECT and projection imaging in small
animals. Comput Med Imaging Graph 25:79
5. Korf J, Veenma-van der Duin L, Brinkman-Medema R, Niemarkt A,
de Leij LF (1998) Divalent cobalt as a label to study lymphocyte distribution using PET and SPECT. J Nucl Med 39:836
6. Melder RJ, Brownell AL, Shoup TM, Brownell GL, Jain RK (1993)
Imaging of activated natural killer cells in mice by positron emission
tomography: preferential uptake in tumors. Cancer Res 53:5867
7. Beekman FJ, Kamphuis C, King MA, van Rijk PP, Viergever MA
(2001) Improvement of image resolution and quantitative accuracy in
clinical single photon emission computed tomography. Comput Med
Imaging Graph 25:135
8. Chatziioannou AF, Cherry SR, Shao Y, et al. (1999) Performance
evaluation of microPET: a high-resolution lutetium oxyorthosilicate
PET scanner for animal imaging. J Nucl Med 40:1164
9. Cutler PD, Cherry SR, Hoffman EJ, Digby WM, Phelps ME (1992)
Design features and performance of a PET system for animal research.
J Nucl Med 33:595
10. Ishizu K, Mukai T, Yonekura Y, et al. (1995) Ultra-high resolution
SPECT system using four pinhole collimators for small animal studies
(see comments). J Nucl Med 36:2282
11. Herschman HR, MacLaren DC, Iyer M, et al. (2000) Seeing is believing: non-invasive, quantitative and repetitive imaging of reporter gene
expression in living animals, using positron emission tomography. J
Neurosci Res 59:699
12. Kornblum HI, Araujo DM, Annala AJ, Tatsukawa KJ, Phelps ME,
Cherry SR (2000) In vivo imaging of neuronal activation and plasticity
in the rat brain by high resolution positron emission tomography (microPET). Nat Biotechnol 18:655
13. Gambhir SS, Barrio JR, Wu L, et al. (1998) Imaging of adenoviral-directed herpes simplex virus type 1 thymidine kinase reporter gene expression in mice with radiolabeled ganciclovir. J Nucl Med 39:2003
14. MacLaren DC, Gambhir SS, Satyamurthy N, et al. (1999) Repetitive,
non-invasive imaging of the dopamine D2 receptor as a reporter gene
in living animals. Gene Ther 6:785
15. Yu Y, Annala AJ, Barrio JR, et al. (2000) Quantification of target gene
1360
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
J. Hardy et al./Experimental Hematology 29 (2001) 1353–1360
expression by imaging reporter gene expression in living animals. Nat
Med 6:933
Lewin M, Carlesso N, Tung CH, et al. (2000) Tat peptide–derivatized
magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol 18:410
Contag CH, Contag PR, Mullins JI, Spilman SD, Stevenson DK, Benaron DA (1995) Photonic detection of bacterial pathogens in living
hosts. Mol Microbiol 18:593
Bugaj JE, Achilefu S, Dorshow RB, Rajagopalan R (2001) Novel fluorescent contrast agents for optical imaging of in vivo tumors based on a
receptor-targeted dye-peptide conjugate platform. J Biomed Opt 6:122
Ebert B, Sukowski U, Grosenick D, et al. (2001) Near-infrared fluorescent dyes for enhanced contrast in optical mammography: phantom experiments. J Biomed Opt 6:134
Hoffman R (1999) Orthotopic transplant mouse models with green fluorescent protein-expressing cancer cells to visualize metastasis and angiogenesis. Cancer Met Rev 17:271
Weissleder R, Tung CH, Mahmood U, Bogdanov A Jr (1999) In vivo
imaging of tumors with protease-activated near-infrared fluorescent
probes Nat Biotechnol 17:375
Hossain P, Liversidge J, Cree MJ, et al. (1998) In vivo cell tracking by
scanning laser ophthalmoscopy: quantification of leukocyte kinetics.
Invest Ophthalmol Vis Sci 39:1879
Cheong WF, Prahl SA, Welch AJ (1990) A review of the optical properties of biological tissues. IEEE Journal of Quantum Electronics 26: 2166
Jobsis FF (1977) Noninvasive, infrared monitoring of cerebral and myocardial oxygen sufficiency and circulatory parameters. Science 198:1264
Chance B, Luo Q, Nioka S, Alsop DC, Detre JA (1997) Optical investigations of physiology: a study of intrinsic and extrinsic biomedical
contrast. Philos Trans R Soc Lond B Biol Sci 352:707
Reynolds JS, Troy TL, Mayer RH, et al. (1999) Imaging of spontaneous canine mammary tumors using fluorescent contrast agents. Photochem Photobiol 70:87
Sevick-Muraca EM, Reynolds JS, Troy TL, Lopez G, Paithankar DY
(1998) Fluorescence lifetime spectroscopic imaging with measurements of photon migration. Ann N Y Acad Sci 838:46
Contag CH, Bachmann MH, Weissleder R, Fraser SE (2000) Applications of in vivo molecular imaging in biology and medicine. NeoReviews 1:233
Contag CH, Jenkins D, Contag PR, Negrin RS (2000) Use of reporter
genes for optical measurements of neoplastic disease in vivo. Neoplasia 2:41
Contag C, Spilman S, Contag P, et al. (1997) Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem
Photobiol 66:523
Edinger M, Sweeney TJ, Tucker AA, et al. (1999) Noninvasive assessment of tumor cell proliferation in animal models. Neoplasia 1:303
Contag PR, Olomu IN, Stevenson DK, Contag CH (1998) Bioluminescent indicators in living mammals. Nat Med 4:245
Lipshutz GS, Gruber CA, Cao Y, Hardy J, Contag CH, Gaensler KML
(2001) In utero delivery of adeno-associated viral vectors: intraperitoneal
gene transfer produces long-term expression. Molecular Therapy 3:284
Rehemtulla A, Stegman LD, Cardozo SJ, et al. (2000) Rapid and quan-
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
titative assessment of cancer treatment response using in vivo bioluminescence imaging. Neoplasia 2:491
Castano JP, Kineman RD, Frawley LS (1996) Dynamic monitoring
and quantification of gene expression in single, living cells: a molecular basis for secretory cell heterogeneity. Mol Endocrinol 10:599
White MR, Masuko M, Amet L, et al. (1995) Real-time analysis of the
transcriptional regulation of HIV and hCMV promoters in single mammalian cells. J Cell Sci 108:441
Costa GL, Sandora MR, Nakajima A, et al. (2001) Adoptive immunotherapy of experimental autoimmune encephalomyelitis via T cell delivery of the IL-12 p40 subunit. J Immunol 167:2379
Koransky ML, Ip TK, Wu S, et al. (2001) In vivo monitoring of myoblast
transplantation into rat myocardium. J Heart Lung Transplant 20:188
Zhang WS, Feng JQ, Harris SE, Contag PR, Stevenson DK, Contag CH
(2001) Rapid in vivo functional analysis of transgenes in mice using
whole body imaging of luciferase expression. Trangenic Research 10:423
Sweeney T, Mailander V, Tucker A, et al. (1999) Visualizing tumor
cell clearance in living animals. Proc Natl Acad Sci U S A 96:12044
Rice BW, Cable MD, Nelson MB (2001) Imaging of light-emitting
probes. Journal of Biomedical Optics (In Press)
Viviani VR, Bechara EJ, Ohmiya Y (1999) Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases:
relationship between bioluminescence spectra and primary structures.
Biochemistry 38:8271
Eames BF, Benaron DK, Stevenson DK, Contag CH (1999) Construction and in vivo testing of a red-emitting firefly luciferase. In: Contag
CH, Bornhop D, Sevick-Murac E (eds.) Reporters, dyes, and instrumentation. Pittsfield, MA: Laurin Publishing Company, pp. 36–39
Alvarnas JC, Linn YC, Hope EG, Negrin RS (2001) Expansion of cytotoxic CD3CD56 cells from peripheral blood progenitor cells of patients
undergoing autologous hematopoietic cell transplantation. Biol Blood
Marrow Transplant 7:216
Lu PH, Negrin RS (1994) A novel population of expanded human
CD3CD56 cells derived from T cells with potent in vivo antitumor
activity in mice with severe combined immunodeficiency. J Immunol
153:1687
Koehne G, Gallardo HF, Sadelain M, O’Reilly RJ (2000) Rapid selection of antigen-specific T lymphocytes by retroviral transduction. Blood
96:109
Nakajima A, Seroogy CM, Sandora MR, et al. (2001) Antigen-specific
T cell–mediated gene therapy in collagen-induced arthritis. J Clin Invest 107:1293
Day RN, Kawecki M, Berry D (1998) Dual-function reporter protein
for analysis of gene expression in living cells. Biotechniques 25:848
Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants
of the green fluorescent protein (GFP). Gene 173(1):33
McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552
Lu S, Arthos J, Montefiori DC, et al. (1996) Simian immunodeficiency
virus DNA vaccine trial in macaques. J Virol 70:3978
Pettersson J, Nordfelth R, Dubinina E, et al. (1996) Modulation of virulence factor expression by pathogen target cell contact. Science 273:1231