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- Materials and Methods
Over the past 25 years, lung transplantation has become the treatment of choice for patients with end-stage lung disease. Although outcomes have improved over this time period, long-term survival remains disappointing. According to the latest International Society for Heart and Lung Transplantation (ISHLT) Registry report, the median survival in the most recent era (2000–2006) was 5.5 years . Beyond the first year after transplantation, bronchiolitis obliterans syndrome (BOS), or chronic rejection, accounted for over 25% of deaths . Acute rejection episodes are a primary risk factor for the development of BOS; even a single episode increases the risk of developing BOS . Therefore, identifying acute rejection early is important to initiate treatment to reduce the risk of BOS.
Since mild and even moderate grade acute rejection can be clinically silent , surveillance transbronchial biopsies, the gold standard for diagnosing acute rejection, are performed at several centers. While these procedures are reasonably safe with experienced bronchoscopists, the number of biopsies required to reliably detect acute rejection can lead to increased risk of complications, including pneumothorax and bleeding . Sampling error also ultimately limits the diagnostic sensitivity of this approach. Therefore, techniques that can improve on the detection of acute rejection would be highly useful for improving management and outcomes in these patients.
Positron emission tomography (PET) imaging with [18F]fluorodeoxyglucose ([18F]FDG) has been used to quantify lung inflammation [5-8] and may be a useful approach for quantifying acute rejection. Evidence in the literature suggests that [18F]FDG is taken up by activated immune cells, including T cells, which are the key mediators of acute lung transplant rejection . T cells are known to take up glucose in response to activating stimuli to support the increased energy demands of the cell [10-12]. [18F]FDG uptake also increases with rejection in mouse models of acute rejection in lung, heart, kidney and liver transplantation [9, 13-15]. These data suggest that FDG-PET may be a useful approach for monitoring the efficacy of immunosuppressive therapy.
In this study, we characterized the time course of [18F]FDG uptake within the first 7 days after lung transplantation in an orthotopic left lung transplant mouse model. We hypothesized that in acutely rejecting lungs T cells are the major sinks for glucose uptake. Supporting our hypothesis, we demonstrated that recipients with ongoing acute lung allograft rejection have significant increases in [18F]FDG uptake driven primarily by the accumulation of T cells in the graft. In contrast, immunosuppression significantly reduced the overall sequestration of glucose tracer by the allogeneic lung graft T cell compartment, leading to decreased [18F]FDG uptake and thus allowing for clear PET-based discrimination of tolerant and rejecting lung grafts.
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- Materials and Methods
Our imaging data in a mouse model of orthotopic lung transplant suggest that FDG-PET may be useful non-invasive approach to detect acute rejection. When compared to mice that received syngeneic lung grafts, mice with acutely rejecting allogeneic lung grafts had markedly elevated [18F]FDG uptake that correlated with histological evidence of rejection. The syngeneic lung grafts also did not exhibit increased [18F]FDG uptake but instead had utilization that was similar to the native lungs. Our study thus agrees with published studies in other lung and solid organ transplant models that demonstrate increased [18F]FDG uptake with acute rejection [9, 13-15].
To determine if FDG-PET could be used to monitor tolerance in lung recipients, we immunosuppressed allograft recipients. Notably, DCB treatment nearly eliminated the [18F]FDG uptake that normally would have occurred as a result of rejection, suggesting that T cells in rejecting grafts play a significant role in driving glucose utilization under these conditions. Although T lymphocytes are necessary for acute lung rejection , these grafts also accumulate large numbers of myeloid cells , which are likewise potential sources of glucose sequestration. To better characterize [18F]FDG allograft signals we employed 2-NBDG , a fluorescent 2-deoxyglucose probe that has been used as a sensitive surrogate measure of glucose uptake in T, B  and myeloid cells  in vivo. We adapted the use of this probe in a flow cytometric–based assay to forward the study of differential glucose utilization by several graft–infiltrating myeloid and lymphoid cell populations. We observed that patterns of total 2-NBDG uptake by graft–infiltrating cells was comparable to [18F]FDG uptake by microPET under these same conditions.
Both CD4+ and CD8+ T cells are dependent on co-stimulation signals that drive lymphocyte proliferation leading to solid organ rejection . Notably, the CD28 co-stimulation pathway is a critical regulator of T cell proliferation leading to the expression of glucose transporters such as Glut1 and the subsequent upregulation of glucose uptake . Under glucose limiting conditions, proliferating T cells rapidly undergo apoptosis , which is in line with previous observations that CD28 costimulation blockade promotes the activation–induced T cell apoptosis known to enable graft tolerance. [33, 34]. To study the impact of this approach on glucose uptake in T cells specifically under tolerant conditions, we used a DCB immunosuppressive regimen, which is dependent on CD28 blockade to promote lung allograft acceptance . DCB treatment not only significantly lowered glucose uptake in individual T cells but also attenuated levels of intragraft T cell abundance to levels comparable to that observed in syngeneic grafts. Additionally, we could detect changes in intragraft T cell glucose uptake by modulating the amount of immunosuppression administered to lung recipients. Both allograft [18F] FDG and T cell 2-NBDG uptake was significantly lower in high dose CsA and MP treated recipients when compared to low dose treated recipients. Finally, because the number of activated T cells were still more numerous than the other cell types present in rejecting lungs, the combination of both higher per cell glucose uptake and increased accumulation suggests that T cells are the primary source of [18F]FDG uptake during an acute rejection episode, regardless of the severity of inflammation.
Our detailed FACS analysis of 2-NBDG uptake in rejecting lungs also revealed novel insights about the glucose uptake responses of non–T cell hematopoietic cell types during acute rejection–associated inflammation. While other parenchymal cell types within the lungs are known to take up glucose under various inflammatory conditions [35, 36], the collagenase digestion method used to release resident hematopoietic cells destroys parenchymal tissue; therefore, analysis of these cell types was not possible with this method. We clearly observed 2-NBDG uptake in neutrophils and APCs on a per cell basis in addition to that observed in the T cells. Surprisingly, neutrophils took up similar amounts of 2-NBDG per cell irrespective of whether they were infiltrating syngeneic, allogeneic or DCB–treated allografts and regardless of the presence or absence of steroids. We have shown that glucose uptake increases in activated neutrophils entering extravascular spaces in human lungs  and that increase glucose uptake can be detected by PET even in the initial steps of activation, before neutrophils enter the airspace . As neutrophils must be activated to enter extravascular spaces , the observations from the current study could be the result of constitutive surveillance mechanisms that drive small numbers of neutrophils into syngeneic and tolerant allografts, in line with our previously published data with intravital 2-photon microscopy demonstrating low levels of constitutive neutrophil trafficking into non–inflamed lungs . Additionally, our results clearly demonstrate that neutrophil glucose uptake on a per cell basis is resistant to combination CsA and MP treatment. In particular, corticosteroids may have diametric effects on neutrophil metabolism since these agents are known to attenuate reactive oxygen intermediate production and degranulation but also halt apoptosis , which would suggest some overall demand to maintain glucose utilization. Although glucose metabolism by individual neutrophils was unaffected by immunosuppression levels, high dose CsA and MP had a dramatic impact on preventing lung allograft neutrophil infiltration. This may be the result of two complementary effects on neutrophil recruitment. CsA prevents naïve CD4+ T cells from differentiating into Th17 cells , a T cell subset found in acutely rejecting lungs  that promotes neutrophil accumulation by stimulating granulocyte production and mobilization , while corticosteroids work further downstream, acting as transcriptional regulators that inhibit the expression of chemokines and adhesion molecules that promote extravascular accumulation [43, 44]. In contrast, we observed that B cells took up little 2-NBDG. The reasons for this are unclear as B cells express several glucose transporters . However, as opposed to T cell and neutrophil activation, which can be initiated in the lung [20, 46, 47], naïve B cell activation may mostly be restricted to draining secondary lymphoid organs and thus contributes little to intragraft glucose utilization during acute rejection.
We also evaluated the effect of administering low dose CsA at the time of transplant on [18F]FDG uptake in allogeneic lung grafts as well as low and high doses of CsA in combination with low and high dose MP treatment. The absolute [18F]FDG uptake decreased relative to untreated recipients but not as markedly as that seen with DCB treatment. Interestingly, the addition of low dose MP to CsA therapy did not improve the level of [18F]FDG uptake suppression; however, high dose CsA in combination with high dose MP more effectively reduced [18F]FDG uptake. Blunted antigen presentation mediated by primarily MP may partially explain the superior effectiveness of high dose CsA and MP treatment. Unlike CsA, high dose corticosteroids have been reported to prevent the upregulation of MHC molecules and costimulatory signals on dendritic cells known to drive IL-2 expression and proliferation of naïve T lymphocytes . Accordingly, a recent report suggests that CsA treatment alone is not sufficient to prevent lung graft rejection in mice .
To further test the ability of FDG-PET as such a biomarker, we treated allogeneic lung allograft recipients with anti–thymocyte globulin antibodies at Day 6, a time point at which acute rejection is already established, and obtained baseline pretreatment and immediate (approximately 24 h) posttreatment FDG-PET imaging. We observed a consistent reduction in the FDG-PET signal in animals treated with active antibody compared to those treated with the isotype control that again correlated with histological evidence of reduced T cell activation. These results support the potential for using FDG-PET to monitor responses to immunosuppressive interventions for acute rejection. Such an application for FDG-PET will most likely be most useful in patients who do not have other evidence of infection. Given our data demonstrating that neutrophil glucose utilization is not affected by immunosuppression, infectious processes would need to be excluded to improve the confidence of interpreting a lack of change in [18F]FDG uptake as a failure of immunosuppression therapy as opposed to superimposed infection. Thus, interpretation of the FDG-PET study for clinical applications should still occur within the context of other clinical information, including microbiology, biopsy and BAL results. Despite this limitation, FDG-PET may still provide useful information about the extent of rejection (e.g. confirming other areas potentially involved in the acute rejection process when biopsy and BAL results confirm the presence of acute rejection without superimposed infection). FDG-PET may even be potentially useful in directing which segments to interrogate by bronchoscopy, thus improving the diagnostic yield of bronchoscopic procedures. Finally, the development of novel tracers that can target different immune cells more specifically could be very helpful in differentiating infection from rejection.
While such clinical applications for FDG-PET are promising, the potential sensitivity of FDG-PET for detecting acute rejection must be determined to establish how FDG-PET might be used in the clinical management of lung transplant recipients. The only human study evaluating FDG-PET for imaging lung transplant recipients quantified [18F]FDG uptake using the Patlak graphical analysis, a model–free approach for quantifying kinetic PET data [49-51]. In that study, the rate of [18F]FDG uptake in the one subject with evidence of A2 rejection was at the upper range of values reported in healthy volunteers to date [5, 8, 52]. In our experience, such kinetic analyses in human studies appear to be more sensitive for quantifying differences in low levels of lung inflammation , unlike the %ID/cc used in this study or related SUV that is used clinically. However, dynamic imaging is used more frequently for research applications and rarely used for clinical FDG-PET scans. Thus, evaluating whether more clinic–friendly image acquisition protocols can provide the same information would also be helpful in establishing the potential utility of this technique for clinical practice.
In conclusion, we have demonstrated that FDG-PET may be useful biomarker of acute lung transplant rejection in a mouse model of orthotopic lung transplant. Our data support further assessing the utility of this approach for quantifying acute rejection in human lung transplant recipients.