In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease

I N N OVAT I O N

In vivo imaging using bioluminescence: a tool for probing graft-versus-host diseaseRobert S. Negrin and Christopher H. Contag

Abstract | Immunological reactions have a key role in health and disease and are complex events characterized by coordinated cell trafficking to specific locations throughout the body. Clarification of these cell-trafficking events is crucial for improving our understanding of how immune reactions are initiated, controlled and recalled. As we discuss here, an emerging modality for revealing cell trafficking is bioluminescence imaging, which harnesses the light-emitting properties of enzymes such as luciferase for quantification of cells and uses low-light imaging systems. This strategy could be useful for the study of a wide range of biological processes, such as the pathophysiology of graft-versus-host and graft-versus-leukaemia reactions.

Normal immune function is crucial for maintaining health in a hostile environ-ment containing many potential pathogens. To protect against pathogens, immune responses must be both rapid and sustained. In addition, effector cells of the immune system provide immune surveillance against malignancy and promote tissue remodelling and repair. The importance of a normal immune response is further exemplified by the observation that patients with many diseases, including cancer and infectious and auto immune diseases, have dysfunctional immune responses. Therefore, a greater understanding of normal and pathological immune responses will not only provide insights into basic biological mechanisms but also will aid in the development of effective treatments for a range of diseases.

In this Innovation article, we discuss how bioluminescence imaging (BLI) can be used to analyse aspects of complex immune reactions in living animals. We use graft-versus-leukaemia (GVL) reactions and graft-versus-host disease (GVHD) as examples of what BLI can teach us about clinically relevant immune responses.

Haematopoietic-cell transplantationNowhere in clinical medicine is the impor-tance of effective immune responses clearer than after allogeneic haematopoietic-cell transplantation (HCT). Over the past several decades, HCT has emerged as an effective and often life-saving treatment for a broad array of haematological malignancies, as well

as for genetic and acquired immune deficien-cies1. HCT involves the transfer of the entire haematopoietic and immune systems from a donor to a recipient. The procedure involves pretreatment of the patient with high doses of chemotherapy, with or without irradiation, to eliminate malignant or defective haemato poietic cells. This is followed by the transfer to the patient (recipient) of donor-derived haematopoietic cells, which home to the bone marrow and re-establish haematopoiesis. After HCT, all of the haema-topoietic cells in the recipient, including professional antigen-presenting cells (APCs) and immune effector cells, are of donor origin. The functional consequences for the recipient of the new donor-derived immune system are dramatic and include the ability to reject the underlying malignancy or replace damaged haemato poietic-cell populations with normal cells. For example, the damaged red blood cells in patients with thalassaemia or sickle-cell disease can be replaced with healthy cells. The rejection of malignancy is known as the GVL effect. The antigens on the tumour cells that are recognized by the donor leukocytes are largely unknown but they include major and minor histocompatibility antigens (depending on the genetic disparity between the donor and the recipient) as well as potential tumour-specific antigens.

Despite its success in promoting the rejec-tion of malignancy, allogeneic HCT carries the significant risk that the donor-derived immune cells will recognize and respond to recipient tissues and result in a syndrome

known as GVHD2,3. Severe GVHD limits the overall effectiveness of HCT and precludes the application of this life-saving therapy to other clinical settings, such as for the treat-ment of severe autoimmune disorders4 or for the induction of tolerance to organ transplan-tation. The risks of GVHD are substantial, such that 20–60% of patients will develop this complication following allogeneic HCT, depending on disease-related factors, such as the stage of disease, the age of the recipient and the degree of genetic disparity between the donor and recipient. The reasons why some patients develop severe GVHD after HCT, whereas others do not, remain unclear. Given the substantial risk of GVHD, applica-tion of current HCT procedures requires that the donor is a fully matched histocompatible sibling or unrelated donor. Unfortunately, many patients that need HCT are unable to secure a well-matched donor, resulting in lethal consequences for the patient due to disease progression. Therefore, the study of GVHD and GVL reactions provides insight into both normal and pathological immune reactions and has significant implications for the development of new and more effective strategies for clinical management of disease.

Experimental models of HCTHCT has been widely studied in both rodent and canine models, and these studies have been crucially important in developing the theoretical basis of HCT. Initial studies in mice established the concept of allogeneic immune responses and GVHD. Mouse studies have also been crucial for studying allorecognition, and for exploring effector-cell populations and mechanisms, owing to the defined genetics and availability of strains that lack key effector molecules. Early stud-ies in dogs showed that in some litter-mates long-term engraftment occurred, whereas in others GVHD developed. The outbred canine model has been useful in the develop-ment of preparative regimens for transplan-tation, which were translated to the clinic5,6, and to study the use of drug prophylaxis for both acute and chronic GVHD in large animals. More recent studies have led to the development of non-myeloablative HCT, in which the intensity of the preparative regimen of the recipient is reduced and replaced with immuno suppressive medications to prevent graft rejection. This strategy of HCT is associated with reduced transplant-related morbidity and mortality7. Non-myeloablative HCT has been widely applied in the clinic; however, GVHD remains a serious complication of the therapy with an associated mortality rate

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of 10–20% (REF. 8). Recent studies in which the immune environment of the recipient is altered before transplantation using total lymphoid irradiation (TLI) and depleting antibodies that target T cells (anti-thymocyte globulin) — an approach pioneered in mouse model systems — have been trans-lated to the clinic with an apparent reduced risk of acute GVHD9.

Mouse studies have also been important in the development of a conceptual framework for GVHD reactions. As in humans, GVHD in animal models shows an unusual tissue tropism; it mainly affects the skin, gastrointes-tinal tract and liver. Tissue injury in the recip-ient owing to the preparative regimen results in the release of pro-inflammatory cytokines that fuel the GVHD reaction2,3. Alloreactive donor-derived T cells can become activated in the recipient, and can then infiltrate and sub-sequently cause damage to tissues of the skin, gut and liver, resulting in the pathophysiology of GVHD. In mice, this alloreactivity trans-lates as end-organ damage such as hair loss, ruffled fur, weight loss, diarrhoea and eventu-ally mortality. However, these experimental end-points are reflective of end-stage disease and provide little information about the spatial and temporal events in the induction of GVHD at time points when intervention could affect the ultimate outcome. To explore GVHD and GVL reactions in greater detail, imaging strategies have been used to visualize the spatial and temporal events in GVHD induction.

Visualizing immune responsesUntil recently, much of our understanding of immune-cell trafficking and the factors that control this process has been obtained by using culture systems, in which the influ-ence of intact organ structure, circulation, endothelial barriers and tissue effects have been removed. Insights into the specific locations and timing of immune-cell migra-tion and proliferation that can be gained using imaging methods in living animals hold promise for providing new informa-tion on physiology and pathophysiology.

Various imaging modalities are emerg-ing for the study of small animal models of human biology and disease, and several of these have been applied to the study of immune-cell migration. There are preclinical versions of clinical imaging systems, such as magnetic resonance imaging (MRI), positron emission tomography (PET) and single photon emission computed tomography (SPECT). For example, MRI has recently been used to track cardiac-graft rejection in a rat model10. The use of these tools in studies of small animals allows ready translation of new reagents and imaging approaches to the clinic. However, the use of optical markers, such as those that are bioluminescent or fluorescent, to assess cell fate and function in whole animals offers several advantages that can be used to refine and accelerate the study of mouse models of disease (BOX 1). The instru-mentation for acquiring whole-body images using optical reporters that are expressed in

small animals also has several advantages over other approaches: it can usually accommodate multiple animals in a single image at each time point; it is user-friendly so dedicated imaging technicians are not required; and it is less expensive than the instrumenta-tion required for SPECT, MRI and PET. The use of two-photon intravital microscopy offers high-resolution images of cell–cell interactions in tissues11, but is constrained by a limited field of view and can be severely hampered by motion artefacts. As a result of these limitations and constraints, only certain tissues can be observed using this approach.

In the field of optical imaging, there are several approaches that have been used to generate whole-body images of biological processes in rodents. These include the use of light scatter, absorbance, fluorescence and bioluminescence. In this Perspective article, we focus on the use of biolumin-escence to generate images of cell migra-tion and proliferation in vivo and on the

Box 1 | Molecular imaging in immunology: watching and waiting for an immune response

Imaging modalities. Two-photon intravital microscopy offers resolution of cells in vivo11, but it is constrained by small fields of view and motion artefacts. Non-invasive measures of immunological processes in vivo have been accomplished using positron emission tomography (PET)49–51, single photon emission computed tomography (SPECT)52, magnetic resonance imaging (MRI)53, bioluminescence imaging (BLI) and fluorescence imaging (FLI)49,54–56. Ultrasound and X-ray computed tomography (CT) provide anatomical information, and when used in combination with other modalities, this information improves localization of the signals obtained by PET, SPECT or optical imaging57. PET, SPECT, ultrasound MRI and CT have potential clinical uses, and therefore are useful in translational studies.

Optical methods. BLI and FLI can be used to refine and accelerate studies of animal models, but they have limited clinical application. Imaging times for optical imaging methods are generally short, which facilitates the analysis of greater numbers of animals. Optical methods also allow a range of image resolutions from microscopic to macroscopic, produce images without the use of ionizing radiation, offer the choice of many reporters and dyes, and benefit from user-friendly and inexpensive instrumentation58. Signal-to-noise ratios (SNRs) for BLI are excellent59,60, enabling detection of subtle changes non-invasively, thereby obviating the need to remove overlying tissue.

Reporter genes. The use of dyes and contrast agents allows visualization of the early events, but they are diluted by cell division. To prevent loss of labels during cell division, genes that encode reporter proteins can be integrated into the genome. Reporter genes are available for PET, SPECT, MRI and optical imaging49,57, each with strengths and weaknesses. The radiotracers used for PET and SPECT often produce signals from kidney, liver and bladder that can obscure the target tissue, and MRI is generally less sensitive than imaging of reporter-gene expression with PET or SPECT. In summary, optical imaging of reporter-gene expression in vivo offers the greatest versatility, sensitivity and SNR of all of the modalities used for small animals.

Figure 1 | Schematic representation of a bio-luminescence imaging strategy using cells from a transgenic donor mouse. Cells express-ing the transgene encoding a luciferase–GFP–green fluorescent protein (GFP) fusion protein are isolated from a transgenic donor animal and selected by cell-sorting technologies, using the GFP signal or fluorescent antibodies specific for selected cell-surface markers. Luciferase–GFP-positive cells are then transferred to recipient syngeneic or allogeneic animals. Recipient ani-mals are then injected with the luciferase sub-strate to allow serial imaging of the biolumines-cent signal in vivo. The tracking of effector cells involved in graft-versus-host disease or graft-versus-leukaemia reactions can be carried out in recipient animals that have been prepared using myelo ablative or non-myeloablative regimens.


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BLI strategiesThe use of in vivo bioluminescence was first shown in tracking bacterial pathogens12. In vivo BLI has since been applied to the study of gene-expression patterns13, as a measure of successful gene transfer14, to indicate the extent of tumour growth and response to therapy15, to assess the extent of protein–protein interactions in vivo16 and to determine the location and proliferation of stem cells17. These examples show the versa-tility of the approach and some of the basic principles of the method (FIG. 1).

BLI is based on the expression of a light-emitting enzyme (such as luciferase) in target cells and tissues. In the presence of its substrate (such as luciferin), an energy-dependent reaction releases photons that can pass through tissues and be detected using sensitive detection systems. As with all optical imaging approaches, BLI is sub-ject to the optical properties of tissues18. For example, in the visible region of the spec-trum, haemoglobin is the main absorber in

the body, and this has a marked influence on the transmission of light through tissues. The influence of tissue on the detection of bioluminescent reporters in vivo has been studied using four luciferases in three animal models19. The data generated were consist-ent with those reported using external light sources, and they showed that the longer wavelengths of bioluminescence (>600 nm), in the red and near-infrared regions of the spectrum, are transmitted through mam-malian tissues more efficiently than the shorter wavelengths of light, in the blue and green regions of the visible spectrum. This indicates that using luciferases that have a significant portion of their emission greater than 600 nm, such as luciferase derived from fireflies and click beetles (approximately 60% of the light emitted from these two enzymes has wavelengths greater than 600 nm), will lead to more-sensitive detection of the labelled cells in vivo. In each animal model, the sensitivity of detection should be measured by determining cell numbers (FIG. 2), assessing luciferase activity in excised tissues20 or using other imaging modalities21. Cells can be counted after recovery from animals (FIG. 2a–c) or before introduction

into animals (FIG. 2d,e). These measures have indicated that, in mice, a minimum of 100–1,000 cells can be detected at specific anatomical sites. Among the parameters that affect the sensitivity of detection are the wavelength of light emission, expression levels of the enzyme in the target cells, the location of the source of bioluminescence in the animal, the efficiency of the collection optics and the sensitivity of the detector.

Detection of internal biological sources of light requires sensitive detection systems with spectral sensitivity in the red region of the spectrum. Charge-coupled device (CCD) cameras (similar to a home camcorder) in general, have a spectral range that is appropri-ate for detecting biological light sources. But most CCD detectors are not sensitive enough to detect the light from inside the body that is needed for the study of cell migration or other biological processes. To increase their sensitivity, these detectors can be cooled. Alternatively, approaches can be used to amplify or intensify the signal, but many intensifiers used for amplifying optical signals are not sensitive in the red region of the spec-trum. So, CCD cameras, in which the CCD chip is thinned, back illuminated, placed in

Figure 2 | Sensitivity of detection in bioluminescence imaging studies. The detection of weak optical signals, such as those generated during bio-luminescence imaging (BLI), from inside small animals is influenced by sev-eral factors that include: the level of cell brightness (photon flux from source), the depth at which the bioluminescent source is located in the tis-sue, the wavelength of the emitted light, the quantum efficiency and noise of the detector, the nature of the collection optics and the background emission levels from the live animal. a | In this example, studies of the detec-tion sensitivity of BLI were carried out in a mouse B-cell lymphoma model, in which the tumour cells were labelled with luciferase and green fluor-escent protein (GFP), by imaging whole animals at various times during the

disease course. One time point is shown for two mice. b | After recovery from the animals, tumour cells from the liver and spleen of these animals can then be quantified by flow cytometry using the GFP signal and fluorescent anti-bodies specific for the B-cell marker CD19; the results for the two animals in part a are shown. c | The quantity of GFP-positive cells detected by flow cytometry correlated well with the bioluminescent signals detected in vivo, showing that BLI is a sensitive and reliable measure of cell number in vivo. d | Using the same detector, the detection sensitivity of BLI can also be ana-lysed by measuring the bioluminescent signal emitted from known numbers of cells in culture or following transfer in vivo (e). Images are adapted, with permission, from REF. 24 © (2003) the American Society of Hematology.




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a vacuum and cooled to temperatures as low as –105°C, are at present the most common cameras for imaging weak biological sources of light in the body.

For BLI, cells must be engineered to express the reporter luciferase and the sub-strate for the reaction must be injected into the animal for light to be emitted. Luciferases have been cloned from both marine (such as Renilla luciferase) and terrestrial (such as firefly and click-beetle luciferases) eukaryotic organisms. The substrates that these luciferases use seem to group with their origins — marine bioluminescent organ-isms use coelenterazine as a substrate and terrestrial organisms use d-luciferin. The biodistribution of these substrates has been studied in animals and differs significantly. d-luciferin has a longer circulation time than coelenterazine and there is little catalysis of d-luciferin by mammalian proteins22. These two differences determine the use of the enzyme–substrate pair for in vivo BLI in which luciferase–luciferin reactions provide a longer duration of signal at a longer wavelength that is less influenced by tissue absorption than enzyme–substrate reactions that emit blue light (for example, that are pro-duced by luciferases from marine organisms such as Renilla spp.). Coelenterazine-using enzymes can provide a short-lived signal that is useful when combining the assays with luciferin-using enzymes22,23. However,

all enzymes characterized so far that use co elenterazine emit blue light, which is highly absorbed by mammalian tissues. As more enzyme–substrate pairs are characterized and existing reactions are optimized for in vivo applications, more reagents will become avail-able for accelerating and refining animal stud-ies using BLI. At present, the luciferin-using enzymes from fireflies and click beetles that emit light greater than 600 nm offer the great-est sensitivity, and the coelenterazine-using enzymes can be used as secondary markers, despite their severe limitations as convenient and sensitive markers.

Using BLI to explore GVHDBLI is an effective means of evaluating com-plex biological processes such as stem-cell engraftment, GVHD and GVL reactions17,24,25. BLI is remarkably sensitive; it can detect as few as 10 cells in vitro and 100–1,000 cells in vivo (FIG. 2). The ability to track cell populations serially and non-invasively, so as to define key time points and locations for further analysis, has provided important new insights. Whole-body imaging of cell migra-tion guides investigators to specific times and organs for more labour-intensive assays.

A central limitation of all reporter-gene strategies is the need to introduce the reporter construct into the cell populations of interest. Gene transfer using viral vectors or non-viral strategies have been useful; however, they

suffer from variable efficiency, especially when attempting to introduce genes into certain primary cells. These techniques also often require cell activation and culturing for variable periods of time, and this might alter the biological activity of the cells. Transduction of haematopoietic stem cells (HSCs) has been carried out at high frequen-cies using viral vectors; these transduced cells can then be used directly in vivo to visualize engraftment26. In addition, transduced HSCs can be transplanted into immunodeficient mice, such as recombination-activating gene 2 (Rag2)–/– animals (which lack B and T cells), allowed to engraft and then effector cells can be isolated for secondary transfer27. This approach can be particularly useful for studying the migration of cells derived from particular strains of mice that lack key effec-tor molecules, as backcrossing these animals to reporter-gene-expressing mice is expensive and time consuming. However, if single HSCs or small numbers of HSCs are to be studied, then uniform integration of the reporter gene into a given genomic site17 is preferred to retroviral transduction, which results in each cell having the reporter gene integrated into different sites.

An alternative approach to gene transfer has been the generation of transgenic mice that express a luciferase–green fluorescent protein (GFP) fusion protein under the control of the chicken β-actin promoter and the cytomegalovirus enhancer in all haemato-poietic cells. Cells from these transgenic mice (known as L2G85 mice) can then provide a source of luciferase-positive donor cells for transplantation studies17. The presence of GFP facilitates the isolation of these cells from the donor animals and can also be used as a marker in fluorescence microscopy or flow-cytometry studies of transplanted cells in tissues from recipient animals. Although transgenic animals expressing GFP alone have been used to provide donor cells in studies of GVHD that show widespread infil-tration of tissues by donor-derived cells28, it is possible that the cells of interest lose expres-sion of the transgene or are recognized by the immune system and deleted29. Therefore, control experiments with wild-type cells are important to verify these results.

In studies with cells from L2G85 mice, syngeneic (genetically identical; FVB; H2q) or allogeneic (genetically different; BALB/c; H2d) recipient animals first received lethal irradiation (to delete the existing haematopo-ietic cells and simulate the clinical setting of HCT) followed by injection of T-cell-depleted bone-marrow cells from wild-type donor animals (to re-establish haematopoiesis) and

Figure 3 | Imaging of graft-versus-host disease. Bioluminescence imaging of luciferase-positive splenocytes transplanted to either irradiated syngeneic (top panels) or allogeneic (bottom panels) animals are shown. Serial images show markedly different patterns of lymphocyte trafficking, pro-liferation and tissue infiltration. At defined time points, tissue sites of interest, as determined by bioluminescence imaging, can then be further analysed. Images are reprinted, with permission, from REF. 25 © (2005) the American Society of Hematology.




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splenocytes from L2G85 animals (to induce GVHD)25. To visualize the donor cells, luci-ferin, the substrate for luciferase, was injected into the recipient just before imaging. Serial imaging showed striking differences between syngeneic and allogeneic recipients (FIG. 3). In the syngeneic animals, a waxing and waning BLI signal from the transplanted luciferase-positive cells was observed, which ultimately resulted in bone-marrow engraft-ment, probably from residual stem cells in the splenocyte preparations25. By marked contrast, the transplanted cells in allogeneic recipients showed early (in the first 24–48 hours) infiltration of cervical lymph nodes and structures in the gut. At 2–4 days after transplantation, marked proliferation of the donor cells was observed at these lymph node and gut sites, indicated by the increase in BLI signal. By day 6, infiltration of the skin (most obviously in the ears and tail) was readily apparent25. This study identified the key target structures and organs involved in the induction of GVHD, so further analyses, using ex vivo BLI-, immunofluorescence- and flow-cytometry-based approaches, can focus on these tissues.

Visualizing early migration of donor T cells. Before donor-cell infiltration of GVHD target organs, such as the skin, gut and liver, infiltra-tion of secondary lymphoid structures was seen. Histological analysis showed that, in the gut, these early sites of donor-cell prolifera-tion were the Peyer’s patches and mesenteric lymph nodes. These structures are extremely difficult to detect in irradiated animals but became easily visible by BLI following HCT25. In all of the lymphoid tissues analysed, CD4+ T cells infiltrated first (as early as 24 hours after transfer), followed (several days later) by CD8+ T cells.

Previous studies have indicated a key role for Peyer’s patches in the induction of GVHD as, under certain experimental conditions, animals that lack such structures do not develop disease30. Using a myeloablative-conditioning regimen, we have found that alloreactive T-cell activation and proliferation occurs in several sites, including Peyer’s patches, mesenteric lymph nodes and other nodal sites as well as the spleen (A. Beilhack, S. Schulz and R.S.N., unpublished observa-tions). This is consistent with results reported by others indicating that animals that lack Peyer’s patches still develop GVHD31. These studies indicate that priming (activation) of alloreactive T cells seems to occur in second-ary lymph nodes and the spleen. Following activation, alloreactive cells upregulate the expression of key molecules, such as α4β7-integrin, that are required for entry to GVHD target organs. A central question is whether cells are imprinted for entry to specific sites at their site of priming — for example, are cells that are activated in Peyer’s patches and mesenteric lymph nodes destined to enter the gut? And similarly, are other sites of activation required for cell infiltration of the skin? In vitro studies support this hypo-thesis and have indicated that APCs from particular sites, such as the Peyer’s patches, activate cells that can infiltrate the gut but not the skin32.

BLI is therefore extremely useful to define the time points and sites of donor-cell infiltration, and to direct further analyses using pheno typic and functional assays. Validation of these concepts in vivo with re-transplantation of luciferase-positive cells activated at specific sites and analysis by BLI will be invaluable for directly answering the remaining questions.

Using BLI to evaluate strategies to reduce GVHD. A major goal of these studies is to develop strategies that can reduce the risk of GVHD but do not interfere with GVL reac-tions. The studies described earlier indicate

that there are several possible approaches to control GVHD, which include blocking or limiting access of T cells to priming sites, controlling alloreactive T-cell proliferation and blocking entry to GVHD target organs.

Because access to secondary lymphoid structures was key to activation and prolif-eration of alloreactive T cells, several studies have indicated that different populations of T cells might have variable access to such sites and, therefore, differential abil-ity to induce GVHD25,33,34. For example, effector memory T cells (defined by a CD4+CD44hiCD62Llow phenotype) can provide increased immune reconstitution and GVL effects but have limited capacity to induce GVHD33,34. By contrast, naive T cells (defined by a CD4+CD44lowCD62Lhi pheno-type) readily induce GVHD. Using BLI to follow the trafficking and survival of these T-cell populations, it was shown that, unlike naive T cells, effector memory T cells did not infiltrate and proliferate in secondary lymph nodes25. Other studies have highlighted the importance of expression of the leukocyte-adhesion molecule CD62L by T cells in GVHD induction, also indicating that there is a need to access secondary lymphoid structures to initiate GVHD35.

Other populations of T cells and natural killer (NK) cells with cytolytic activity have also been studied in models of GVHD and GVL. The generation of T cells with defined reactivity — for example, against minor histo compatibility antigens that are expressed exclusively by malignant cells or against viral antigens in Epstein–Barr-virus-associated diseases — is currently being explored and holds significant promise for inducing GVL reactions36. Another T-cell population that can be expanded ex vivo, known as cytokine-induced killer (CIK) cells, has a limited capacity to induce GVHD in mouse models due, at least in part, to the production by CIK cells of interferon-γ (IFNγ)37. BLI studies of CIK cells have indicated that they might have reduced proliferative capacity compared with naive T cells, which is consistent with their attenuated capacity to induce GVHD. NK cells have also been observed to lack the capacity to induce GVHD but can have GVL effects38,39. Accordingly, infusion of NK cells from donors to HLA-mismatched recipients with relapsed malignancies results in very limited GVHD, but in some recipi-ents, especially those with acute myeloid leukaemia, beneficial GVL responses were observed40. The precise mechanisms under-lying the inability of NK cells to induce GVHD are unclear. BLI studies of NK cells

Figure 4 | Effect of transfer of conventional CD4+ and CD8+ T cells with and without CD4+

CD25+ regulatory T cells on tumour progres-sion. Leukaemia cells expressing a transgene encoding the fusion protein luciferase–yellow fluorescent protein were injected into recipient mice and, using bioluminescence imaging, can be observed infiltrating the bone marrow. Recipient mice that received irradiation and T-cell-depleted bone marrow only have progressive tumour growth at day 5 and 15 (left panels). Animals that received T-cell-depleted bone marrow and con-ventional T cells die rapidly due to acute lethal graft-versus-host disease (GVHD). By contrast, recipient mice that received both conventional T cells and CD4+CD25+ regulatory T cells (TReg cells) in equal proportions retain the ability to reject the tumour without significant GVHD. Images are reproduced, with permission, from Nature Medicine REF. 27 © (2003) Macmillan Publishers Ltd.




and have resulted in reduced levels of acute GVHD with retention of GVL activity9.

Another well-characterized population of regulatory T cells is the subset of naturally occurring CD4+ T cells that express the IL-2 receptor α-chain (also known as CD25)43 and the transcription factor forkhead box P3 (FOXP3): that is, CD4+CD25+ regula-tory T cells (TReg cells). Several groups have shown that the infusion of grafts containing an equal mix of TReg cells and conventional T cells results in the control of GVHD44–46; crucially, GVL reactions are retained in ani-mals treated in this way27,47. Using BLI, active rejection of the leukaemia could be observed following the infusion of equal numbers of luciferase-positive conventional T cells and TReg cells27 (FIG. 4). The mechanism of control of GVHD and retention of GVL seems to be due to the ability of TReg cells to suppress conventional T-cell proliferation, as shown using BLI to evaluate donor-derived conventional T-cell trafficking and numbers in the presence and absence of TReg cells27. By contrast, GVL reactions mainly required activation of CD8+ T cells, and this occurred even in the presence of TReg cells. However, important questions remain: where, how and for how long do TReg cells exert their immunological control? Clues have come from studies of TReg cells that are divided into subsets on the basis of CD62L expression. Both CD62L+ and CD62L– TReg cells sup-press cell proliferation in an in vitro T-cell

proliferation assay and express FOXP3; however, only CD62L+ TReg cells can suppress GVHD in vivo46,48, indicating that CD62L-mediated homing of TReg cells is required to control GVHD. The clinical application of TReg cells is under active development by several groups.

Concluding remarksBLI has provided new insights into complex biological processes that, until now, could not be evaluated. Future goals include improvement of the techniques that allow visualization of more than one population of cells simultaneously and improvement in quantification of cell numbers. Improved quantification might be possible through spectral imaging, in which the differential transmission of blue and green components of the luciferase emission spectra relative to the red components can be used to determine the depth of the signal in the body. Reconstruction of three-dimensional images from multiple views will also improve the quantification of biolumines-cence signals. Although the ability to carry out imaging studies using luciferase in humans will be limited to very superficial sites in which expression of a foreign gene is not problematic, lessons learned from using BLI in animal models will greatly affect the design of clinical trials for cellular transplantation. Perhaps the greatest benefit of BLI to clinical medicine will be through accelerating and refining preclinical mod-els. Evaluating the fate of transferred cell populations is likely to prove crucial to the evaluation of HCT and other cell-based therapeutics.Robert S. Negrin and Christopher H. Contag are at the Departments of Medicine, Center for Clinical Research

Building, 269 West Campus Drive and Pediatrics, Clark Center, 318 Campus Drive, Stanford University,

Stanford, California 94305, USA.Correspondence to R.S.N.

e-mail: [email protected]

doi:10.1038/nri1879

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in mouse models are ongoing, and it is hoped that they will provide further clues to why this cell population has such limited capacity for GVHD induction.

Monitoring regulatory T-cell function. An alternative approach to controlling GVHD has been to harness regulatory mechanisms in an effort to control alloimmune responses. Several populations of regulatory T cells that can control immune reactions, such as a mixed lymphocyte reactions in vitro and autoimmune diseases in vivo, have been described. One such population of regulatory T cells, known as natural killer T (NKT) cells, co-expresses both NK-cell and T-cell markers, recognizes CD1 through an invariant T-cell receptor and produces large amounts of interleukin-4 (IL-4) after activation41. Mouse recipients treated with TLI and anti-thymocyte serum (ATS) to delete the existing T cells have reduced numbers of conven-tional CD4+ and CD8+ T cells but increased numbers of NKT cells42. Interestingly, these animals are resistant to GVHD induction such that 1,000-fold more allogeneic donor cells can be transferred without GVHD developing42. The use of BLI to follow the fate of donor cells transplanted to animals treated with TLI and ATS compared with animals receiving total body irradiation is under active investigation. Recipient preparation strategies involving TLI and T-cell depletion before HCT have been translated to the clinic

Glossary

AllorecognitionAllorecognition occurs when the host immune system detects same-species, non-self antigens and triggers allograft rejection. It can occur by direct or indirect pathways: the direct pathway involves recognition of foreign MHC molecules on donor cells, and the indirect pathway involves processing and presentation of donor-derived MHC molecules by host antigen-presenting cells.

Graft-versus-host disease(GVHD). Tissue damage in a recipient of allogeneic tissue (usually a bone-marrow transplant) that results from the activity of donor cytotoxic T cells recognizing the tissues of the recipient as foreign. GVHD varies markedly in extent, but it can be life threatening in severe cases. Damage to the liver, skin and gut mucosae are common clinical manifestations.

Graft versus leukaemiaHosts with leukaemia who receive an allogeneic bone-marrow transplant have far fewer disease relapses than individuals who obtain autologous bone-marrow transplants. This results from the transplanted T cells recognizing alloantigens expressed by the leukaemia.

HaematopoiesisThe commitment and differentiation processes that lead from a haematopoietic stem cell to the production of

mature cells of all lineages: erythrocytes, myeloid cells (such as macrophages, mast cells, neutrophils and eosinophils), B and T cells, and natural killer cells.

Minor histocompatibility antigensPolymorphic peptides derived from normal cellular proteins that can be recognized in the context of MHC molecules. Immune responses to these polymorphic antigens can result in graft-versus-host reactions, graft rejection or beneficial antitumour responses.

Non-myeloablative haematopoietic-cell transplantationAn allogeneic haematopoietic-cell transplantation in a recipient who has received a conditioning regimen to achieve immunosuppression and prevent graft rejection without the complete ablation of host haematopoiesis. The recipient might develop (transient) mixed chimerism, owing to haematopoietic recovery of the host and engraftment of donor haematopoietic cells.

Two-photon intravital microscopyLaser-scanning microscopy that uses pulsed infrared laser light for the excitation of conventional fluorophores or fluorescent proteins. The main advantage is deep tissue penetration of the infrared light, owing to the low level of light scattering in the tissue.




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Competing interests statementThe authors declare competing financial interests: see web version for details.

DATABASESThe following terms in this article are linked online to:Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneCD4 | CD8 | CD25 | CD44 | CD62L | FOXP3 | interferon-γ | IL-4

FURTHER INFORMATIONChristopher H. Contag’s laboratory: http://sci3.stanford.edu/lab/Robert S. Negrin’s homepage: http://med.stanford.edu/profiles/robert_negrinAccess to this links box is available online.




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In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease