A clearer understanding of the cellular processes involved in rejection of transplanted organs is necessary to develop more specific therapeutic agents. However, the complexity and the highly dynamic nature of immune responses are difficult to capture using conventional approaches. Commonly used methods of cellular detection such as flow cytometry and histology, although extremely useful, only provide a static snapshot of the immune cells present at a given time. These approaches cannot differentiate between cells passing by one another or communicating with one another, and therefore cannot provide insight into the highly dynamic interactions of the immune cells.
Technological advances have provided various tools for performing intravital imaging in small animals. These include magnetic resonance imaging (MRI), ultrasound, positron emission tomography/computed tomography (PET/CT), single-photon emission computed tomography (SPECT), bioluminescence, and single-photon or two-photon laser-scanning intravital microscopy (LSIM) . With the exception of LSIM, the spatial resolution of these imaging tools does not provide single cell or subcellular levels of detection. In addition, only LSIM allows for fast time-lapse imaging (i.e. less than 1 min), a necessary feature to study the fast migratory rate of immune cells. For example, a single lymphocyte in lymphoid organs moves at a median velocity of 5–10 μm/s in three-dimensional space and thus can travel over several hundred micrometers in 1 h [2,3]. In addition, immune cells are influenced by the local milieu (including cytokines, chemokines and other mediators, anatomical location, and fluid forces). Examples of multiparameter, high-resolution, time-lapse imaging of cellular dynamics obtained by two-photon LSIM are shown in Fig. 1 and Supplementary Digital Content 1–2, http://links.lww.com/MOT/A5, http://links.lww.com/MOT/A6 (Supplementary Digital Content 1: Two-photon LSIM imaging of leukocyte adhesion in transplanted mouse kidney. Intravital time-lapse images of a transplanted kidney showing leukocyte crawling (white rectangle), adhering (white arrow), and transiently arresting (red arrow) in peritubular capillaries at 2 hours after graft reperfusion. Epithelial cell nuclei appear blue, capillary lumens green, and leukocytes yellow/green. Low MW dextran (orange/red) was injected i.v. 1 minute after the start of imaging. Movie reproduced from [4▪]; Supplementary Digital Content 2: Two-photon LSIM imaging of unexpected effector T-cell dynamics in the peri-islet area during rejection of islet allografts. An effector lymphocyte (red) is depicted crawling inside a blood vessel (light blue), stopping, extending a protrusion through the blood vessel wall, making a brief contact with another effector T cell infiltrating the parenchyma before retracting the protrusion and continuing its crawling motion inside the blood vessel. Yellow line shows the position of the effector T cell over time (tracking). The same time-lapse sequence is repeated twice, first without the blood vessel lumen and then with the blood vessel lumen. Three-dimensional rendering and cell tracking was generated using Imaris (Bitplane). Original images generated by G. Camirand). In the last decade, lymph node LSIM revealed several unexpected immune cell behaviors in steady state and in the context of inflammation in the presence of foreign antigens. For example, the complete activation of CD4 and CD8 T lymphocytes requires several initial short interactions, followed by prolonged interactions with dendritic cells bearing antigen [5,6]. In addition, activated B cells that are able to present antigens form stable interactions with activated CD4 T cells. Directed by B cells, which remained mobile, these pairs travel into B-cell follicles, while dragged T cells directly provide the necessary signals (e.g. CD40L and cytokines) to the B cells for the initiation of a germinal center reaction . Importantly, a reduction in these contact times inhibits lymphocyte activation [8–10]. In line with this, Foxp3+ CD4+ regulatory T cells (Tregs) were shown to suppress T-cell activation by reducing their contact duration with antigen-presenting cells (APCs), a process dependent of cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) [11–14]. Thus, visualizing immunological events as they occur can provide entirely new insights into rejection.
In this review, the technical principles of LSIM will be briefly explained, followed by an overview of the recent applications of LSIM in transplantation and their resulting findings in immune rejection. Finally, the future perspectives of LSIM in transplantation research will be discussed. This approach is now accessible to a greater number of researchers and promises to provide invaluable insights into allograft rejection, immunosuppression, tolerance, and organ function, which will greatly benefit the field.
PRINCIPLES OF LASER SCANNING INTRAVITAL MICROSCOPY AND ITS CURRENT APPLICATION IN TRANSPLANTATION
LSIM utilizes florescence to follow cells and detect their motility, location, and interaction with other cells in three-dimensional space over time. Two types of lasers are currently used in LSIM: conventional single-photon lasers (which are used in confocal imaging of explanted cells or tissues) and two-photon lasers. Imaging using two-photon lasers offers significant advantages over single-photon lasers. Two-photon lasers achieve greater tissue penetration (several hundred μm, depending on the tissue density and composition), cause less phototoxicity and photobleaching of fluorophores, and generate a greater signal to autofluorescence ratio. Two-photon lasers emit in the near-infrared range (around 700–1000 nm), which have less energy than photons at lower wavelengths. Thus, to excite commonly used fluorophores, the near-simultaneous absorption of two low-energy photons must occur. This is achieved by optical concentration of the low-energy photons at a single focal point . Also, an intrinsic signal called second-harmonic generation can be detected from various tissues using near-infrared emission. Photons interacting with spatially organized molecules (such as collagen) are scattered in ‘pairs’ (near simultaneous scattering of two photons), generating a signal that has twice the energy and half the wavelength of the original photons . Second-harmonic generation signals can define structures in organs, such as the external capsule of lymph nodes, without the use of fluorescent labeling .
One of the utmost challenges of LSIM is to maintain complete stability of the imaged tissue, while preserving physiological conditions (function, temperature, and blood perfusion). Motion artifacts are typically caused by breathing, heartbeats, even large pulsating blood vessels, and drastic changes in blood pressure. To circumvent this, a wide variety of custom-built immobilizing devices are used, which vary depending on the anatomical area of interest. We recently described a method to perform intravital imaging of transplanted kidneys in mice [4▪]. Our approach consists of harvesting kidney, renal vein and artery, as well as approximately 1 cm segments of the aorta and the inferior vena cava en bloc from the kidney donor mouse. The kidneys are then transplanted in the abdomen of recipient mice by end-to-side anastomosis. The relatively long native blood vessels allow immobilization of the transplanted kidney in a custom-built kidney holder (adapted from ). Our initial work described a significant reduction of kidney function and increased tissue damage within 3 h after transplantation, because of ischemia/reperfusion injury [4▪]. Using the same approach, we can now successfully image kidneys up to 7 days (and possibly longer) after transplantation (M. H. Oberbarnscheidt, F. G. Lakkis, unpublished observation). Interestingly, imaging of transplanted lungs in mice was the first documentation of LSIM in transplantation . A respirator was used to maintain breathing of the recipient mouse while the left lung was exposed and glued to a cover glass for imaging (using surgical tissue glue). More recently, this same group achieved LSIM of native and transplanted heart – which has been previously believed to be nearly impossible because of its constant contractility [20▪▪]. This remarkable achievement was made using an approach similar to that described for the transplanted lung. Because of the invasive nature of the imaging approaches described above and because of the use of glue used to mobilize the imaged tissues, the animals are sacrificed after completion of the imaging session. Thus, these approaches do not allow for sequential imaging of the same transplanted organ over several days. To circumvent this, other groups have either used a custom-made endoscope for performing single-photon LSIM of transplanted islets under the kidney capsule  or transplanted the islets in the anterior chamber of the eye of mice and imaged by two-photon LSIM through the cornea [22▪]. This allowed for repeated LSIM imaging of the same transplants over several weeks. In addition, imaging of allogeneic ear skin (epidermis and dermis) transplanted on the ventral side of mouse ears has been described [23▪▪]. Given the superficial location of this transplantation model, performing LSIM imaging spanning several days would be possible if one could avoid the use of tissue glue to stabilize the area.
RECENT DISCOVERIES IN TRANSPLANTATION REJECTION USING LASER SCANNING INTRAVITAL MICROSCOPY
The application of LSIM to transplantation was first reported in 2010. Since then, a handful of papers have been published. Because of their novel application, these publications were mostly descriptive in nature, with a main goal of demonstrating feasibility. However, several features of the immune system dynamics were revealed. For example, the immune responses between vascularized and nonvascularized transplanted organs can be distinguished from one another.
Innate immune responses in transplantation
Within minutes following transplantation, an intense inflammatory reaction occurs as a result of ischemia–reperfusion injury. This rapidly attracts innate immune cells, such as neutrophils, monocytes, and dendritic cells, which are the first line of defense against pathogens and play a crucial role in mounting adaptive immune responses to foreign antigens. Data from three LSIM studies show that neutrophils are the predominant cell type that infiltrates the graft early (1 h to 3 days), and this occurs independent of alloantigens [19,20▪▪,23▪▪]. In a nonvascularized skin graft model, neutrophils were scattered throughout the dermis [23▪▪]. In contrast, in vascularized grafts, neutrophils formed dynamic clusters in the coronary veins of transplanted hearts and within the alveolar tissue of transplanted lungs [19,20▪▪]. Interestingly, clusters of neutrophils in the transplanted lungs were partly generated by the recruitment of individual monocytes, possibly induced through chemokine secretion by those cells. These latter studies of vascularized grafts were however performed using syngeneic transplants. As monocytes have an innate allorecognition system [24,25], it would be interesting to visualize and compare the dynamics of neutrophils and monocytes in vascularized allogeneic vs. syngeneic transplants. However, comparing syngeneic and allogeneic skin transplantation has not revealed differences in innate immune cell infiltration [23▪▪].
Transplanted organs carry tissue-resident APCs that can activate recipient lymphocytes through the direct pathway of adaptive allorecognition. Concurrently, recipient APCs can indirectly present allogeneic antigens through MHC class II or cross-present alloantigens through MHC class I [26,27]. The relative contribution of direct versus indirect presentation in the adaptive immune response to an allograft is still the subject of debate. Because of the high precursor frequency (5–10%) of lymphocytes recognizing alloantigens directly by in-vitro assays , this pathway had been assumed to be dominant over indirect presentation – especially for acute alloresponses. Whereas the role of the indirect pathway would predominate over time as donor APCs decrease and indirectly alloreactive clones expand. However, this was directly challenged by a study showing that depletion of donor-derived APCs, using mice expressing diphtheria toxin receptor on their dendritic cells and pretransplant diphtheria toxin treatment of the donor, did not affect acute rejection of allogeneic hearts . In contrast, elimination of recipient dendritic cells significantly prolonged allograft survival. In agreement with several previously published studies, host natural killer cells rapidly killed donor APCs, limiting their ability to directly participate in the alloresponse [29–31]. These data are in line with a report which utilized LSIM showing that after skin intravital imaging in transplantation allografts, donor dendritic cells that migrated to the draining lymph node were immobile and had the morphology of dead cells, whereas donor dendritic cells that migrated from syngeneic skin grafts remained motile [23▪▪]. This study also showed that recipient innate immune cells that rapidly infiltrated skin allografts could then exit the graft and activate CD8 T cells in the draining lymph nodes, supporting an important role for indirect presentation during allograft rejection.
Adaptive immune responses in transplantation
Critical steps in rejection mediated by T lymphocytes include their initial activation and subsequent differentiation into effector T cells, and their migration to the allograft, where rejection occurs. Although the signals involved in T-cell activation and differentiation in lymphoid organs are well documented in different settings [32▪▪], the exact mechanisms by which T lymphocytes migrate into inflamed allografts have not been clearly elucidated. LSIM studies revealed differences between vascularized and nonvascularized allografts in terms of the anatomical location of T-cell extravasation from blood vessels into the tissues occurs. In nonvascularized transplants such as skin, effector T cells first infiltrated and accumulated in the recipient skin adjacent to the allograft (days 5–8), and then migrated to the allogeneic tissue (day 8) [23▪▪]. A similar scenario occurred when allogeneic islets were transplanted in the anterior chamber of the eye [22▪] or under the kidney capsule of diabetic mice (G. Camirand, unpublished observation). In both cases, the effector T cells accumulated in the peri-islet area before invading the transplanted islets. This occurred despite revascularization of the transplanted tissues by day 5, which potentially has the capacity to support lymphocyte entry. In contrast, in vascularized allografts (kidney transplantation), our data show that effector T cells directly migrate from the blood vessels (of donor origin) to the transplanted tissues. Interestingly, we found that extravasation from blood vessels into these kidney grafts by antigen-specific effector T cells occurred independently of chemokine receptor signaling, but required cognate antigen interactions (J. M. Walch, F. G. Lakkis, unpublished observation). This data is in line with other reports in which diabetogenic T-cell migration to islets in the pancreas was investigated [33–35]. Taken together, this suggests that neovascularization does not provide the necessary signals (i.e. antigenic presentation of donor antigens) for transendothelial migration of effector T cells. The exact mechanism behind this will require further investigation and can only be elucidated using LSIM.
CURRENT LIMITATIONS AND FUTURE PERSPECTIVES OF LASER SCANNING INTRAVITAL MICROSCOPY IN TRANSPLANTATION
LSIM is likely to become an invaluable tool for studying the different facets of organ function, immune rejection, and tolerance; however, this technology is not without important limitations. For example, in the absence of fluorophores, very little information is acquired (mainly originating from second-harmonic generation). Thus, the use of fluorescent transgenic mice and reagents to identify individual cell subsets and anatomical structures (e.g. blood vessels, organ compartments, etc.) is essential. Fluorophores compatibility (in terms of excitation and emission wavelength) and availability (transgenic mice) restrict the number of fluorescence parameters that can be distinguished simultaneously . This means that only a fraction of the biological events occurring in the imaged area can be observed. As in flow cytometry, the ability to simultaneously acquire more information will greatly enhance our understanding of any given environment. Ultimately, a multiparametric imaging setup may be able to allow simultaneous analysis of several cell subsets, anatomical structures (extracellular matrix, blood vessels, organ subcompartments, etc.), and biological or pharmacological modulators (antigen, cytokines, chemokines, enzymes, therapeutic drugs or reagents, etc.). Fortunately, new technologies are helping in achieving this goal. An increasing number of fluorescent transgenic mice are becoming available. In addition, new far-red and near-infrared emitting fluorescent proteins (such as mKate2 and tdKatushka2) coupled with an optical parametric oscillator (which allows emission of high wavelengths photons) will broaden the range of fluorophores available . Also, a recent publication reported the administration of fluorochrome-conjugated Fab fragments to label and identify lymphocyte subsets in vivo, without affecting their function [38▪▪]. If reproducible, this approach would be more flexible and allow a large variety of cell surface molecules to be targeted and imaged by LSIM. One of the most promising newly developed applications of LSIM is the investigation of therapeutic agent delivery and pharmacokinetics [39▪▪,40▪▪], and examination of their subsequent direct effect on the targeted cells in real time. The integration of these parameters will greatly benefit the timing and the efficacy of therapeutics aiming at inhibiting or enhancing specific biological processes in transplanted recipients. They may also identify new or unsuspected mechanisms of action.
Other limitations of LSIM include the limited depth and area that can be imaged in a single imaging session. For example, the volume imaged for transplanted kidneys is less than 0.01 mm3 at a maximum depth of 70 μm into the cortex [4▪]. The kidney is a highly vascularized and dense tissue with poor photon penetration. Thus, what is observed in this relatively small area of the cortex may not be generalizable to the whole kidney, where the infiltration of immune cells can differ. For example, LSIM of heart allografts revealed that neutrophil accumulation, clustering, and transendothelial migration occurred in coronary veins, but not in the coronary arteries [20▪▪], suggesting that not all anatomical compartments within organs offer the necessary signals and cell surface adhesion molecules that favor immune invasion. To circumvent some of these limitations, new technologies to reduce physiologic motion, achieve deeper tissue penetration, and large-scale assessment will be important.
Many unanswered immunological questions remain in transplantation rejection and tolerance. For example, it would be important to understand which host innate cell subset trigger alloimmune responses in lymphoid organs, how the indirect alloantigenic responses destroy the graft parenchyma and induce rejection, how Tregs regulate T effector responses in lymphoid organs and in the graft during tolerance induction, what signals mediate T effector and memory lymphocyte migration to allografts, and so on. In addition, it would be crucial to clearly elucidate the exact mechanisms of immunosuppressant and tolerogenic regimens on immune responses to allogeneic grafts. LSIM will be central in providing the answers to these questions.
With the recent addition of LSIM to study transplantation rejection and tolerance, the details of the spatiotemporal immune processes are starting to appear. However, further investigation in the biological relevance of the cell location and cell–cell interactions, and the precise mechanisms of cell migration to and from the allografts require additional work. LSIM promises to provide the key essential knowledge for the development of therapeutics specifically targeting precise immune processes leading to rejection and enhance regulation.
Many thanks to Drs David M. Rothstein, Fadi G. Lakkis, and Martin H. Oberbarnscheidt for their critical revisions of this article.
Conflicts of interest
Source of funding: G.C. is supported by the American Society of Nephrology (John Merrill Grant in Transplantation).
The author has no conflict of interest to disclose.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 112–113).
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