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Ultrasound Molecular Imaging of Lymphocyte-endothelium Adhesion Cascade in Acute Cellular Rejection of Cardiac Allografts

Xie, Yu PhD1,2; Chen, Yihan MD1,2; Zhang, Li PhD1,2; Wu, Meiying PhD3; Deng, Zhiting MD3; Yang, Yali PhD1,2; Wang, Jing PhD1,2; Lv, Qing PhD1,2; Zheng, Hairong PhD3; Xie, Mingxing PhD1,2; Yan, Fei PhD3

doi: 10.1097/TP.0000000000002698
Original Basic Science—General
Open
SDC

Background. Acute cellular rejection is one of the main reasons for graft failure after heart transplantation. A precise diagnosis at the early stage of acute cellular rejection is a big challenge for clinicians. Given the importance of the interaction between T cells and graft endothelia in initiating rejection, we developed T cell-microbubble complexes (cell-MBs) as ultrasound molecular imaging probes to monitor the lymphocyte–endothelium adhesion cascade in cardiac acute cellular rejection.

Methods. Cell-MBs were fabricated by incubating lymphocytes with anti-CD4 antibody-conjugated MBs (MBCD4). The potential of cell-MBs as probes for detecting acute cardiac rejection was examined. Donor hearts from Brown Norway or Lewis rats were transplanted into Lewis recipients. Ultrasound molecular imaging was performed on allografts of untreated or cyclosporin A (CsA)-treated recipients, and isografts on posttransplantation day 3. Histology was used to assess rejection grades.

Results. We detected a significantly stronger ultrasound molecular imaging signal of cell-MBs than that of MBCD4 or plain MBs in allografts of untreated and CsA-treated recipients. No signal enhancement was observed in isografts with cell-MBs. The signal of cell-MBs in allografts of the untreated group was significantly higher than that in the CsA-treated group, and the signal in the CsA-treated group was higher than that in isografts. Histology confirmed grade 3R rejection in the untreated group, grade 2R rejection in CsA-treated group, and no rejection in isografts.

Conclusions. Our study suggests that cell-MBs can function as a promising probe to image the dynamic lymphocyte–endothelium adhesion cascade for noninvasive diagnosis of cardiac acute cellular rejection.

1 Department of Ultrasound, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

2 Hubei Province Key Laboratory of Molecular Imaging, Wuhan, China.

3 Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.

Received 9 September 2018. Revision received 20 February 2019.

Accepted 21 February 2019.

This work was supported by National Natural Science Foundation of China (Grant No. 81530056, 81727805, 81771851, 81671705, 81801715, 81571701, 11325420, 11534013, 81527901), Natural Science Foundation of Guangdong Province (Grant No. 2014A030312006), and Shenzhen Science and Technology Innovation Committee (Grant No. JCYJ20170413100222613, JCYJ20170307165254568).

The authors declare no conflicts of interest.

Y.X. participated in research design, writing of the article, the performance of research, and data analysis. Y.C., L.Z., M.W., Z.D., Y.Y., J.W., Q.L., and H.Z. participated in research design and data analysis. M.X. and F.Y. participated in research design, writing of the article, and data analysis.

Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com).

(a) Correspondence: Mingxing Xie, PhD, 1277 Jeifang Avenue, Wuhan 430022, China. (xiemx@hust.edu.cn).

(b) Correspondence: Fei Yan, PhD, 1068 Xueyuan Avenue, Shenzhen University Town, Shenzhen 518055, China. (fei.yan@siat.ac.cn).

This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

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INTRODUCTION

Heart transplantation remains the standard treatment for patients with end-stage heart failure, with median survival exceeding 10 years for most patients after surgery.1,2 Acute rejection is still the main reason for graft failure and early mortality after heart transplantation.1 It is a big challenge for clinicians to make a precise diagnosis at the early-stage of acute rejection. Currently, endomyocardial biopsy (EMB) is the gold standard approach for diagnosis of rejection.3 However, the invasive method bears a series of serious disadvantages, including sample error, cardiac tamponade, permanent cardiac block, procedure-related deaths, etc.3 Besides, stress related to the invasive EMB procedure may further activate immune responses and exacerbate acute rejection.4 Therefore, development of novel alternative, noninvasive diagnostic methods is urgently needed.

Pathologically, cellular rejection and antibody-mediated rejection are recognized to be the two main mechanisms leading to graft injury during acute rejection.5,6 Classically, acute cellular rejection is the most frequent type and is characterized by the presence of inflammatory cells and tissue damage in graft myocardium. Recently, several noninvasive approaches have been developed for diagnosing acute cellular rejection. The Allomap assay and cell-free donor-derived DNA (cfdDNA) in peripheral blood have been suggested to be biomarkers for detecting heart transplant rejection.7,8 But the Allomap assay has a relatively low positive predictive value, and the quality of the method for quantifying cfdDNA varies with the age of patients and the time after transplant.7,8 Neither Allomap assay nor cfdDNA test can diagnose transplant rejection alone, and a confirmatory test is needed further.9 Apart from blood biomarkers, electrocardiography (ECG), echocardiography, and cardiac magnetic resonance (CMR) are the widely used clinical techniques for surveillance of cardiac dysfunction. Abnormalities in these examinations, for instance, low values of T slew in ventricular-evoked responses, reduced global longitudinal peak systolic strain (GLS) in advanced echocardiography, and increased T2 relaxation time in CMR, are found to be associated with acute rejection.3,10 But these abnormalities are not specific for acute rejection and the conclusions are controversial.3,10 Noninvasive monitoring of acute rejection at the molecular level may be an alternative for precise diagnosis.

Molecular imaging provides a promising approach to address this issue. Ultrasound molecular imaging possesses many advantages, including high sensitivity, portability, relatively low cost, and good safety profile. Several studies concerning ultrasound molecular imaging diagnosis of acute cardiac rejection have been reported. Weller et al11 demonstrated the effectiveness of ultrasound imaging of acute cardiac allograft rejection with microbubbles (MBs) targeted to intercellular adhesion molecule-1 (ICAM-1). Kondo et al12 showed that leukocyte-targeted myocardial contrast echocardiography could assess the degree of acute allograft rejection in rats. But neither ICAM-1 upregulation on graft endothelia nor leukocyte infiltration was specific for acute rejection. Both processes also occur in ischemic/reperfusion injury,13,14 which is an unavoidable process in transplantation. More recently, T-lymphocyte-targeted nanobubbles were developed to diagnose acute cardiac rejection in rat models and showed that T lymphocytes could function as good target cells for monitoring acute cellular rejection.15,16 Nevertheless, nanobubbles, having smaller dimensions, produce lower acoustic signals as the backscatter coefficient is inversely proportional to the radius of scatterers.17,18 Nanobubbles need more targets than MBs to scatter resolved echo signal.

Pathologically, acute cellular rejection is a typical T-lymphocyte-mediated adaptive immune response characterized by lymphocyte infiltration and cardiomyocyte damage.5 Lymphocytes transmit through vascular endothelial barriers before they infiltrate into tissue interstitium. The lymphocyte–endothelium recognition process is a programmed cascade involving reversible rolling mediated by selectins, chemoattractant-induced activation, and stable adhesion via adhesion molecules.19 If it were possible to detect that lymphocytes are attracted to and adhere on cardiac endothelia, early diagnosis of cardiac acute cellular rejection might be realized.

In this study, we tried to monitor the dynamic lymphocyte–endothelium adhesion by ultrasound molecular imaging. We developed lymphocyte-MB complexes (cell-MBs) by linking lymphocytes with anti-CD4 antibody-conjugated MBs (MBCD4) (Figure 1). In an acute cellular rejection setting, cell-MBs would be chemoattracted to the endothelia of sites undergoing cardiac rejection and involved in the adhesion cascade (Figure 1). By imaging of the lymphocyte–endothelium adhesion cascade, cardiac acute cellular rejection may be assessed.

FIGURE 1

FIGURE 1

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MATERIALS AND METHODS

Materials

Lipid components, DSPC, and DSPE-PEG2000 were purchased from Avanti Polar Lipids (Alabaster, AL), while DSPE-PEG2000-biotin was from Nanocs (Boston, MA). Streptavidin was obtained from Solarbio (Beijing, China) and fluorescein isothiocyanate (FITC)-avidin was from Sigma-Aldrich (St. Louis, MO). Biotinylated anti-CD4 antibodies were purchased from Invitrogen (Waltham, MA). Cyclosporin A (CsA) was acquired from Abcam (ab120114). Lymphocyte separation kits were obtained from Solarbio (P8850). DiI and DiR are fluorescent lipophilic dyes for labeling cell membranes. DiI and DiR were purchased from Beyotime (Haimen, China) and KeyGene Biotech (Nanjing, China), respectively. Anti-rat CD31 and anti-mouse IgG (FITC) antibodies were acquired from Santa Cruz Biotechnology (Dallas, TX) and Cell Signaling Technology (Danvers, MA), respectively.

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Isolation of Rat Lymphocytes

Single cell suspensions of rat spleens were prepared by grinding the tissues through a 70-μm nylon filter. Lymphocytes were isolated through density gradient centrifugation with lymphocyte separation kits (P8850, Solarbio). Cells from Lewis recipient origin were used for all in vivo and ex vivo experiments, and cells from SD rats were used for in vitro assays.

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Fabrication of Cell-MBs

Cell-MBs were prepared by linking lymphocytes to MBCD4. In brief, biotinylated MBs were prepared as previously described,20 followed by incubation with streptavidin (3 μg/107 MBs) for 30 minutes and biotinylated anti-CD4 antibodies (25 μg antibodies/109 MBs) for 30 minutes. Unbound streptavidin or antibodies were separated by centrifugation and the MBCD4 were obtained. MBCD4 were further incubated with lymphocytes for 30 minutes to form cell-MBs. The ratio of MBs to cells in the experiment was 50:1. Unbound cells were removed by centrifugation. As a control, plain MBs without antibodies and cells were also obtained.

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Characterization of Cell-MBs

Size distributions of the plain MBs, MBCD4, lymphocytes, and cell-MBs were determined by using Accusizer 780 Optical Particle Sizer (Particle Sizing Systems, Santa Barbara, CA). For confocal laser scanning microscopic images, lymphocytes were labeled with DiI and MBs were labeled by FITC-avidin. The dual fluorescence-labeled cell-MBs were examined under a confocal laser scanning microscope (CLSM) (Leica TCS SP5).

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Heart Transplantation Model

Brown Norway (BN) (200–220 g) and Lewis (200–220 g) rats (Vital River Laboratory, Beijing, China) were used to establish heart transplantation models. All animal experiments were performed in compliance with the Animal Ethics Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. Heterotopic heart transplantation surgery was conducted according to the method depicted by Ono-Lindsey (Video S1, SDC1, http://links.lww.com/TP/B714).21 During the surgery, 4% isoflurane in 1 L/min oxygen was used for anesthesia induction and 2.5% for maintenance. For ultrasound molecular imaging, 8 BN hearts into Lewis rats were as allografts in the rejecting group, and 4 Lewis hearts into Lewis rats were as isografts. Other 5 allotransplant Lewis recipients were treated with 7.5 mg/kg/d CsA subcutaneously.

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Ex Vivo Fluorescent Imaging

Lymphocytes were incubated with DiR at 5 μM for 20 minutes at 37°C. At day 5 after transplantation, DiR-labeled lymphocytes (108 cells/rat) were administrated into allogeneic or syngeneic transplant recipients intravenously. At 2 hours after injection, rat main organs including transplanted hearts (heartTX), native hearts, livers, spleens, lungs, and kidneys were excised for ex vivo fluorescent imaging at 745 nm of excitation and 800 nm of emission (Caliper IVIS Spectrum, PerkinElmer, CT, USA).

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Fluorescence Microscopy

To further prove that T cells bind with endothelia in allografts, DiI-labeled lymphocytes (7 × 107 cells/rat) or phosphate buffered saline (0.5 mL/rat) were intravenously injected into allogeneic transplant recipients on day 3 after transplantation. At 15 minutes after injection, transplanted hearts were removed and examined for immunofluorescence. Microvessels were stained with anti-CD31 antibodies overnight and anti-mouse IgG (FITC) for 2 hours. Nuclei were labeled with DAPI. Fluorescence images were captured by IX71 inverted fluorescence microscope (Olympus, Japan).

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Ultrasound Molecular Imaging

Ultrasound molecular imaging was performed with Resona7 (Mindray, Shenzhen, China), using the L11–3 linear array transducer. Parameters were set as follows: frequency 5.6 MHz, depth 2.0 cm, gain 60 dB, frame rate 10 Hz, dynamic range 100 dB, and mechanical index (MI) 0.085. All imaging parameters were kept consistent during the whole experiment. Our approach to differentiate adhered contrast agents from freely circulating ones was according to the destruction-replenishment principle.22 In our preliminary study with plain MBs (1 × 108 MBs/rat), myocardial contrast signal could be barely detected at 5 minutes after injection (Figure S1, SDC, http://links.lww.com/TP/B715). Based on this observation, the ultrasound contrast signal at 5 minutes after MB or complex administration was mainly from adhered MBs and few remaining freely circulating MBs. Therefore, at 5 minutes after injection, we transmitted a high-power ultrasound pulse with a MI of 0.734 for 1025 ms to destruct all adhered and freely circulating contrast agents in the field. The signal after burst derived from circulating MBs replenishing the beam. Ten seconds were allowed for complete replenishment. The signal of adhered contrast agents, which was considered as ultrasound molecular imaging signal, could be calculated through subtracting mean contrast signal 10 s after burst from mean contrast signal before burst.

At posttransplantation day 3, rats were anesthetized with 2.5% isoflurane in 1 L/min oxygen and were positioned supine and kept immobile during the examination. The left-ventricular short-axis view was acquired and left-ventricular myocardium was the region of interest. An intravenous bolus of 1 × 108 MBs, or MBCD4, or cell-MBs in 0.2 mL saline was administrated. MBs and MBCD4 were given in a random order in each rat, and cell-MBs were administrated last to avoid cells’ effects on MBCD4. There was a 30-minute interval between administrations of different contrast agents. Ultrasound transmission was suspended until 30 seconds before burst. An ultrasound video from 30 seconds before burst to 30 seconds after burst was recorded for each infusion. Ultrasound molecular imaging signal was calculated as described above, that is, by subtracting mean contrast signal 10 seconds after burst from mean contrast signal before burst.

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Histological Analysis

Grafts were retrieved after ultrasound molecular imaging for hemotoxylin and eosin staining. Rejection grades were assessed by the 2004 International Society for Heart and Lung Transplantation grading system.5

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Statistical Analysis

Data were presented as mean ± standard deviation. Multiple comparison was analyzed with one way analysis of variance and post Bonferroni test. Data in two groups were compared by 2-tailed Student ttest. Statistical significance was defined as P < 0.05.

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RESULTS

Fabrication and Characterization of Cell-MBs

Cell-MBs were prepared by conjugating lymphocytes with MBCD4 (Figure 2;Figure S2, SDC, http://links.lww.com/TP/B715). Figure 2A displayed a representative image of cell-MBs under a light microscope. Each cell could bind with one or several MBs while there were still some unbound free MBs, indicating that all cells were in the cell-MB complexes. The mean particle size of cell-MBs was larger than the sizes of MBCD4 and plain MBs, with 3.16 ± 0.20 μm versus 1.67 ± 0.57 μm and 1.38 ± 0.07 μm, respectively, (P < 0.05) (Figure 2B). There was no significant difference in the mean particle sizes between MBCD4 and plain MBs (P > 0.05). The diameters of lymphocytes detected by Accusizer 780 Optical Particle Sizer were mainly 4–5 μm. Furthermore, lymphocytes were labeled with DiI (Figure S3, SDC, http://links.lww.com/TP/B715) and MBs were labeled with FITC-avidin. The results from CLSM confirmed the binding of these MBs with cells (Figure 2C).

FIGURE 2

FIGURE 2

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Ex Vivo Fluorescent Imaging

To determine whether exogenous lymphocytes could be chemoattracted to allografts, DiR-labeled lymphocytes were injected intravenously into syngeneic or allogeneic rat heart transplantation models. Figure 3A showed that DiR-labeled lymphocytes mainly distributed to the liver and spleen at 2 hours after injection. Much stronger signal intensity was found in the cardiac allograft than in the isograft. Quantitative analysis showed about 30% higher fluorescent signal intensity in allografts than in isografts (Figure 3B) (P < 0.01). There was no noticeable signal intensity difference in native hearts.

FIGURE 3

FIGURE 3

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Binding of Lymphocytes onto Allograft Endothelia

To examine whether lymphocytes could attach onto the endothelia in grafts during acute cellular rejection, phosphate buffered saline (0.5 mL/rat) or DiI-labeled lymphocytes (7 × 107 cells/rat) were intravenously injected into allogeneic rats on post-transplantation day 3. At 15 minutes after injection, the transplanted hearts were removed and cut into sections for immunofluorescence staining. As shown in Figure 3C, DiI-labeled lymphocytes could be found on graft endothelia, which were stained with FITC-labeled anti-CD31 antibodies. Nevertheless, some DiI-labeled T cells had already infiltrated into cardiac interstitium at that time (data not shown).

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Ultrasound Molecular Imaging

We performed ultrasound molecular imaging on untreated allogeneic recipients with MBs, MBCD4, or cell-MBs. The data were presented in Figure 4 and Videos S2–S4 (SDC, http://links.lww.com/TP/B716, http://links.lww.com/TP/B717, and http://links.lww.com/TP/B718). Figure 4A showed that ultrasound contrast signal of cell-MBs at 5 minutes after injection (before burst) was much stronger than the signal of MBCD4 or plain MBs, indicating the presence of more MBs in cardiac myocardium with cell-MBs at this time than with MBCD4 or plain MBs. No apparent ultrasound contrast signal could be observed at the first frame after burst, suggesting that almost all MBs were destroyed in the field. The ultrasound signals of all three contrast agents were still low even at 10 seconds replenishment after burst, confirming that there were few freely circulating MBs. As signal before burst represented the sum signal of vasculature-adhered contrast agents and circulating contrast agents, it could be speculated that most of the ultrasound signal before burst resulted from vasculature-attached MBs. Furthermore, after deducting the postburst signal intensity from the preburst signal intensity, the quantitative mean ultrasound molecular imaging signal intensity was presented in Figure 4B. The signal intensity of cell-MBs was significantly stronger than that of MBCD4 (13.59 ± 1.58 vs 8.65 ± 2.85; P < 0.05) or plain MBs (13.59 ± 1.58 vs 3.45 ± 1.32; P < 0.05) in this group. Besides, the signal intensity of MBCD4 was also significantly stronger than that of plain MBs (8.65 ± 2.85 vs 3.45 ± 1.32; P < 0.05).

FIGURE 4

FIGURE 4

We further performed ultrasound molecular imaging on cardiac isografts with these contrast agents. The ultrasound signals both before and after burst were substantially low with all the three contrast agents (Figure 4C). Consequently, their ultrasound molecular imaging signals, that is, the differences of the contrast signals before burst minus the contrast signals after burst, were low as well (Figure 4D). Molecular imaging signals of these three contrast agents were not significantly different from each other in isografts.

Furthermore, we performed ultrasound molecular imaging on allotransplant recipients treated with 7.5 mg/kg/d CsA (Figure 5A and B). The ultrasound molecular imaging signal of cell-MBs was significantly higher than the signals of MBCD4 (7.996 ± 2.57 vs 2.01 ± 0.69; P < 0.05) and plain MBs (7.996 ± 2.57 vs 2.12 ± 1.36; P < 0.05). However, the molecular imaging signal of MBCD4 was comparable with that of MBs in this group. Figure 5C exhibited that the ultrasound molecular imaging signal of MBCD4 in CsA-treated group was significantly lower than that in the untreated group but was comparable with that in isografts. Nevertheless, the ultrasound molecular imaging signal of cell-MBs in CsA-treated group was significantly lower than that in the untreated group, and was still significantly higher than that in isografts (Figure 5D).

FIGURE 5

FIGURE 5

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Histological Analysis

From the hemotoxylin and eosin staining images, we could see severe cell infiltration in allografts of untreated recipients (grade 3R rejection), moderate cell infiltration in allografts of 7.5 mg/kg/d CsA-treated recipients (grade 2R rejection), and no infiltration in isografts (grade 0R) (Figure 6).

FIGURE 6

FIGURE 6

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DISCUSSION

In this study, we designed cell-MBs as ultrasound molecular imaging probes to detect cardiac acute cellular rejection when T cells were attracted to the graft vasculature. Currently available literature suggests that both T cells and graft endothelial cells play essential roles in allograft rejection.23 Adaptive immune response to allogeneic cells is initiated through recognition of polymorphic proteins by T lymphocytes. Endothelial cells are prime targets of alloreactivity and also key players in the recruitment and extravasation of immune cells.23 Subsequent activation of proinflammatory allospecific T cells initiates a cascade of reactions leading to rejection of transplanted allogeneic tissues and organs.24-26 Therefore, it is possible to monitor early acute cellular rejection through ultrasound molecular imaging of dynamic lymphocyte–endothelium adhesion.

In this study, we used cell-MBs by conjugating MBCD4 onto rat lymphocytes as the molecular probe for the following reasons. First, T cells recognize allografts by direct or indirect recognition of alloantigens.27,28 Various T cell subsets are involved in the process and their recognition of alloantigens is initially dominant in the cellular rejection.29 Second, CD4+ T cells are essential for initiating allograft rejection. In vivo treatment with anti-CD4 antibodies or CD4 gene knockout significantly reduced both CD4+ helper T lymphocytes (HTL) and CD8+ cytotoxic T lymphocytes (CTL) in cardiac allografts, with ameliorated myocardial damage. However, in recipients treated with anti-CD8 antibodies or CD8 knockout mice, HTL infiltrated in cardiac allografts and resulted in extensive myocardial damage and loss of cardiac function.30,31 Moreover, it was reported that CD4+ HTL are required for both CTL accumulation in cardiac tissues and CTL activation in lymphoid tissues.32 Because of the tight association between CD4+ T cells and cardiac allograft rejection, we chose CD4+ cells in our model to detect acute cardiac rejection.

Early monocyte maturation and neutrophil infiltration have also been reported to promote acute rejection in cardiac allografts.33,34 However, in the early chemokine cascade following surgery trauma and ischemia/reperfusion injury, the neutrophil and monocyte/macrophage chemoattractants are comparable in cardiac isografts and allografts.35 Therefore, neutrophils or monocytes may not be able to differentiate acute rejection from other inflammation types.

As is evident from Figure 2B, cell-MBs had a significantly larger mean particle size than MBCD4, but were smaller than the sizes of rat lymphocytes. This could be due to the presence of some free MBs that did not bind to T cells (as depicted in Figure 2A) and could not be separated by centrifugation. They would reduce the mean value of particle diameters.

Although cells were preferentially recruited to the reticuloendothelial system as was previously confirmed,36Figure 3A and B showed that exogenous lymphocytes were more attracted to the rejected cardiac allografts than the nonrejected isografts at 2 hours after transfusion. This result was consistent with the report by Grabner et al in which exogenous xenogeneic T cells could target the kidney with acute rejection in 2 hours.36 Using fluorescence microscopy, we detected binding of exogenous T cells with allograft endothelia in 15 minutes (Figure 3C). Grabner et al37 even successfully detected xenogeneic lymphocytes in rejected kidneys by ultrasound molecular imaging at 15 minutes after cell transfusion. The binding of T cells onto allograft endothelia in a short time affords the superior modality of ultrasound molecular imaging over other molecular imaging technologies since MBs are cleared in several minutes.

Ultrasound molecular imaging signal of cell-MBs was higher than the signals of MBCD4 and plain MBs in allografts undergoing grade 3R rejection (Figures 4 and 6). MBs are confined in vasculature due to their micrometer sizes.38 Ultrasound molecular imaging only analyzes vasculature-adhered MBs, and freely circulating ones are eliminated by pre and postburst signal subtraction.22 For cell-MBs, lymphocytes were attracted to rejected sites following a chemokine gradient and adhered on graft vasculature (Figures S4–S6; Table S1, SDC, http://links.lww.com/TP/B715).37 The MBs in cell-MBs adhered on vessel luminal side through these attracted and vasculature-adhered cells. MBCD4 adhered on vasculature only through a few endogenous CD4+ lymphocytes that stayed on the luminal side. When comparing cell-MBs and MBCD4, the higher ultrasound molecular imaging signal of cell-MBs may be attributed to the attracted and vasculature-adhered cells that acted as important mediators linking MBs and allograft endothelia. In this way, the adhesion interaction of lymphocytes with endothelial cells in allografts was detected by ultrasound molecular imaging. Also, the molecular imaging signal of MBCD4 was higher than that of plain MBs in allografts undergoing grade 3R rejection. As MBs were confined in vasculature due to their micrometer sizes,38 the result indicated that some endogenous CD4+ T cells those stayed on and had not transmitted through vascular wall were detected by ultrasound molecular imaging with MBCD4. The results suggested that both cell-MBs and MBCD4 could target grade 3R rejected allografts, but the targeting efficiency of cell-MBs was higher than that of MBCD4. Besides, the signals of cell-MBs and MBCD4 in grade 3R rejected allografts were significantly higher than those in isografts (Figure 5), suggesting that ultrasound molecular imaging with either cell-MBs or MBCD4 could differentiate grade 3R rejection episodes from nonrejection ones. However, in the 7.5 mg/kg/d CsA-treated group undergoing grade 2R rejection (Figure 6), the molecular imaging signals of MBCD4 and plain MBs were comparable. But the molecular imaging signal of cell-MBs was still significantly higher than that of MBCD4 or plain MBs (Figure 5). This indicated that cell-MBs still significantly targeted grade 2R rejected allografts, but MBCD4 did not. In addition, the molecular imaging signal of cell-MBs in the CsA-treated group was still higher than that in isografts, but the molecular imaging signals of MBCD4 in the two groups were comparable. Thus, for grade 2R rejection, cell-MBs still had the ability to identify the weaker signal but MBCD4 did not. According to International Society for Heart and Lung Transplantation guidelines for the care of heart transplant recipients, asymptomatic patients with grade 2R acute rejection should be seriously treated with adjusted immunosuppressive strategies.39 Ultrasound molecular imaging with cell-MBs may be more advisable for clinicians than imaging with MBCD4.

MBs have a short circulating lifetime. As depicted in Figure 1, MBs are spherical gas cores with lipid monolayer shells.40 They were mainly metabolized by splenic macrophages and hepatic Kupffer cells, and the gas in MBs were diffused into the blood and exhaled by the lung.41,42 Figure S1 (SDC, http://links.lww.com/TP/B715) showed that myocardial contrast signals of all three contrast agents were very low at 8 minutes after a bolus of injection. There was nearly no signal on the screen at 30 minutes after injection (data not shown). It was reported that about 50% of vascular endothelial growth factor receptor 2-targeted MBs were cleared from the circulation at approximately 3.5 minutes after administration.41 Rapid elimination of MBs makes them a desirable choice for clinical contrast imaging.

Lumason (also known as SonoVue outside the United States), phospholipid shell-coated sulfur hexafluoride-filled MBs already approved for clinical use, was recommended for myocardial perfusion imaging at a dose of 0.3–0.5 mL in clinic.43 According to the manufacturer’s instructions for Lumason, the concentration is 1.5–5.6 × 108 MBs/mL after reconstitution. So, every injection of Lumason in clinic is about 2 × 108 MBs. At the MB/cell ratio of 50:1, about 4 × 106 cells, lymphocytes in 3–4 mL human peripheral blood, are needed if our model is to be used in clinic. Blood withdrawal of this volume is not harmful to patients. In a rat kidney transplantation model, Grabner et al37 had used xenogeneic human buffy coats for ultrasound molecular imaging of acute renal rejection with a 15-minutes waiting period after cell infusion. Our method with cell-MBs may also be applied in renal rejection even without a 15-minutes waiting period. However, because a large number of exogenous cells accumulate in the liver (Figure 3), the strong baseline signal may be an obstacle for its application in liver transplantation.

Overall, ultrasound molecular imaging is safe. The technology has been performed in a clinical trial on patients with ovarian or breast lesions.44 No serious adverse effects were observed. Our method of ultrasound molecular imaging with cell-MBs may be an appropriate choice for regular surveillance in the follow up of heart transplant patients. However, considering different transplant species and ultrasound devices, the ultrasound parameters, the doses of MBs and cells, the time of burst, and other parameters should be further investigated and adjusted in future clinical application.

In conclusion, we successfully fabricated cell-MBs by linking rat lymphocytes with MBCD4. As ultrasound molecular imaging probes, cell-MBs were used to detect acute cellular rejection in cardiac allografts. We observed the highest ultrasound molecular imaging signal of cell-MBs in untreated allogeneic group (grade 3R rejection), the second highest in 7.5 mg/kg/d CsA-treated allogeneic group (grade 2R rejection), and the lowest in isografts (no rejection). Our results suggested that cell-MBs could function as a promising probe to image the dynamic lymphocyte-endothelium adhesion cascade in acute cardiac rejection. Ultrasound molecular imaging with cell-MBs may provide a novel noninvasive imaging approach for early diagnosis of cardiac acute cellular rejection.

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REFERENCES

1. Stehlik J, Kobashigawa J, Hunt SA, et al. Honoring 50 years of clinical heart transplantation in circulation: in-depth state-of-the-art review. Circulation. 2018;137:71–87.
2. Lund LH, Khush KK, Cherikh WS, et al; International Society for Heart and Lung Transplantation. The registry of the international society for heart and lung transplantation: thirty-fourth adult heart transplantation report-2017; focus theme: allograft ischemic time. J Heart Lung Transplant. 2017;36:1037–1046.
3. Badano LP, Miglioranza MH, Edvardsen T, et al; Document reviewers. European association of cardiovascular imaging/cardiovascular imaging department of the Brazilian society of cardiology recommendations for the use of cardiac imaging to assess and follow patients after heart transplantation. Eur Heart J Cardiovasc Imaging. 2015;16:919–948.
4. Dhabhar FS. Effects of stress on immune function: the good, the bad, and the beautiful. Immunol Res. 2014;58:193–210.
5. Stewart S, Winters GL, Fishbein MC, et al. Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection. J Heart Lung Transplant. 2005;24:1710–1720.
6. Berry GJ, Burke MM, Andersen C, et al. The 2013 international society for heart and lung transplantation working formulation for the standardization of nomenclature in the pathologic diagnosis of antibody-mediated rejection in heart transplantation. J Heart Lung Transplant. 2013;32:1147–1162.
7. Pham MX, Teuteberg JJ, Kfoury AG, et al; IMAGE Study Group. Gene-expression profiling for rejection surveillance after cardiac transplantation. N Engl J Med. 2010;362:1890–1900.
8. De Vlaminck I, Valantine HA, Snyder TM, et al. Circulating cell-free DNA enables noninvasive diagnosis of heart transplant rejection. Sci Transl Med. 2014;6:241ra77.
9. Snyder TM, Khush KK, Valantine HA, et al. Universal noninvasive detection of solid organ transplant rejection. Proc Natl Acad Sci U S A. 2011;108:6229–6234.
10. Miller CA, Fildes JE, Ray SG, et al. Non-invasive approaches for the diagnosis of acute cardiac allograft rejection. Heart. 2013;99:445–453.
11. Weller GE, Lu E, Csikari MM, et al. Ultrasound imaging of acute cardiac transplant rejection with microbubbles targeted to intercellular adhesion molecule-1. Circulation. 2003;108:218–224.
12. Kondo I, Ohmori K, Oshita A, et al. Leukocyte-targeted myocardial contrast echocardiography can assess the degree of acute allograft rejection in a rat cardiac transplantation model. Circulation. 2004;109:1056–1061.
13. Lange V, Renner A, Sagstetter MR, et al. Heterotopic rat heart transplantation (lewis to F344): early ICAM-1 expression after 8 hours of cold ischemia. J Heart Lung Transplant. 2008;27:1031–1035.
14. Lindner JR, Song J, Xu F, et al. Noninvasive ultrasound imaging of inflammation using microbubbles targeted to activated leukocytes. Circulation. 2000;102:2745–2750.
15. Wu W, Zhang Z, Zhuo L, et al. Ultrasound molecular imaging of acute cellular cardiac allograft rejection in rat with T-cell-specific nanobubbles. Transplantation. 2013;96:543–549.
16. Liu J, Chen Y, Wang G, et al. Ultrasound molecular imaging of acute cardiac transplantation rejection using nanobubbles targeted to T lymphocytes. Biomaterials. 2018;162:200–207.
17. Phillips D, Chen X, Baggs R, et al. Acoustic backscatter properties of the particle/bubble ultrasound contrast agent. Ultrasonics. 1998;36:883–892.
18. Wang Y, Li X, Zhou Y, et al. Preparation of nanobubbles for ultrasound imaging and intracelluar drug delivery. Int J Pharm. 2010;384:148–153.
19. Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell. 1991;67:1033–1036.
20. Borden MA, Martinez GV, Ricker J, et al. Lateral phase separation in lipid-coated microbubbles. Langmuir. 2006;22:4291–4297.
21. Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg. 1969;57:225–229.
22. Abou-Elkacem L, Bachawal SV, Willmann JK. Ultrasound molecular imaging: moving toward clinical translation. Eur J Radiol. 2015;84:1685–1693.
23. Valujskikh A, Heeger PS. Emerging roles of endothelial cells in transplant rejection. Curr Opin Immunol. 2003;15:493–498.
24. Pietra BA, Wiseman A, Bolwerk A, et al. CD4 T cell-mediated cardiac allograft rejection requires donor but not host MHC class II. J Clin Invest. 2000;106:1003–1010.
25. Yamada A, Laufer TM, Gerth AJ, et al. Further analysis of the T-cell subsets and pathways of murine cardiac allograft rejection. Am J Transplant. 2003;3:23–27.
26. Gill RG. T-cell-T-cell collaboration in allograft responses. Curr Opin Immunol. 1993;5:782–787.
27. Jiang S, Herrera O, Lechler RI. New spectrum of allorecognition pathways: implications for graft rejection and transplantation tolerance. Curr Opin Immunol. 2004;16:550–557.
28. Auchincloss H Jr, Lee R, Shea S, et al. The role of “indirect” recognition in initiating rejection of skin grafts from major histocompatibility complex class II-deficient mice. Proc Natl Acad Sci U S A. 1993;90:3373–3377.
29. Al-Lamki RS, Bradley JR, Pober JS. Endothelial cells in allograft rejection. Transplantation. 2008;86:1340–1348.
30. Bishop DK, Chan S, Li W, et al. CD4-positive helper T lymphocytes mediate mouse cardiac allograft rejection independent of donor alloantigen specific cytotoxic T lymphocytes. Transplantation. 1993;56:892–897.
31. Krieger NR, Yin DP, Fathman CG. CD4+ but not CD8+ cells are essential for allorejection. J Exp Med. 1996;184:2013–2018.
32. Bishop DK, Shelby J, Eichwald EJ. Mobilization of T lymphocytes following cardiac transplantation. Evidence that CD4-positive cells are required for cytotoxic T lymphocyte activation, inflammatory endothelial development, graft infiltration, and acute allograft rejection. Transplantation. 1992;53:849–857.
33. Oberbarnscheidt MH, Zeng Q, Li Q, et al. Non-self recognition by monocytes initiates allograft rejection. J Clin Invest. 2014;124:3579–3589.
34. El-Sawy T, Belperio JA, Strieter RM, et al. Inhibition of polymorphonuclear leukocyte-mediated graft damage synergizes with short-term costimulatory blockade to prevent cardiac allograft rejection. Circulation. 2005;112:320–331.
35. Morita K, Miura M, Paolone DR, et al. Early chemokine cascades in murine cardiac grafts regulate T cell recruitment and progression of acute allograft rejection. J Immunol. 2001;167:2979–2984.
36. Grabner A, Kentrup D, Edemir B, et al. PET with 18F-FDG-labeled T lymphocytes for diagnosis of acute rat renal allograft rejection. J Nucl Med. 2013;54:1147–1153.
37. Grabner A, Kentrup D, Mühlmeister M, et al. Noninvasive imaging of acute renal allograft rejection by ultrasound detection of microbubbles targeted to T-lymphocytes in rats. Ultraschall Med. 2016;37:82–91.
38. Lindner JR. Microbubbles in medical imaging: current applications and future directions. Nat Rev Drug Discov. 2004;3:527–532.
39. Costanzo MR, Dipchand A, Starling R, et al; International Society of Heart and Lung Transplantation Guidelines. The international society of heart and lung transplantation guidelines for the care of heart transplant recipients. J Heart Lung Transplant. 2010;29:914–956.
40. Garg S, Thomas AA, Borden MA. The effect of lipid monolayer in-plane rigidity on in vivo microbubble circulation persistence. Biomaterials. 2013;34:6862–6870.
41. Willmann JK, Cheng Z, Davis C, et al. Targeted microbubbles for imaging tumor angiogenesis: assessment of whole-body biodistribution with dynamic micro-PET in mice. Radiology. 2008;249:212–219.
42. Toft KG, Hustvedt SO, Hals PA, et al. Disposition of perfluorobutane in rats after intravenous injection of sonazoid. Ultrasound Med Biol. 2006;32:107–114.
43. Porter TR, Mulvagh SL, Abdelmoneim SS, et al. Clinical applications of ultrasonic enhancing agents in echocardiography: 2018 American society of echocardiography guidelines update. J Am Soc Echocardiogr. 2018;31:241–274.
44. Willmann JK, Bonomo L, Carla Testa A, et al. Ultrasound molecular imaging with BR55 in patients with breast and ovarian lesions: first-in-human results. J Clin Oncol. 2017;35:2133–2140.

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