Development and Evaluation of a Novel Radiotracer 125 I-rIL-27 to Monitor Allotransplant Rejection by Specifically Targeting IL-27R α

Abstract

Noninvasive monitoring of allograft rejection is beneficial for the prognosis of patients with organ transplantation. Recently, IL-27/IL-27Rα was proved in close relation with inflammatory diseases, and ¹²⁵I-anti-IL-27Rα mAb our group developed demonstrated high accumulation in the rejection of the allograft. However, antibody imaging has limitations in the imaging background due to its large molecular weight. Therefore, we developed a novel radiotracer (iodine-125-labeled recombinant IL-27) to evaluate the advantage in the targeting and imaging of allograft rejection. In vitro specific binding of ¹²⁵I-rIL-27 was determined by saturation and competitive assay. Blood clearance, biodistribution, phosphor autoradioimaging, and IL-27Rα expression were studied on day 10 after transplantation (top period of allorejection). Our results indicated that ¹²⁵I-rIL-27 could bind with IL-27Rα specifically and selectively in vitro. The blood clearance assay demonstrated fast blood clearance with 13.20 μl/h of ¹²⁵I-rIL-27 staying in the blood after 24 h. The whole-body phosphor autoradiography and biodistribution assay indicated a higher specific uptake of ¹²⁵I-rIL-27 and a clear radioimage in allograft than in syngraft at 24 h, while a similar result was obtained at 48 h in the group of ¹²⁵I-anti-IL-27Rα mAb injection. Meanwhile, a higher expression of IL-27Rα was found in the allograft by Western blot. The accumulation of radioactivity of ¹²⁵I-rIL-27 was highly correlated with the expression of IL-27Rα in the allograft. In conclusion, ¹²⁵I-rIL-27 could be a promising probe for acutely monitoring allograft rejection with high specific binding towards IL-27Rα on allograft and low imaging background.

1. Introduction

Solid organ allotransplantation has been the most effective therapeutic strategy for patients with end-stage organ failure [1, 2]. However, the appearance of acute rejection is strongly related to the loss of allograft and a poor prognosis [3]. Therefore, early detection of acute rejection with the noninvasive method could greatly improve the prognosis after organ transplantation [4].

Recently, IL-27Rα (interleukin-27 receptor α), along with its ligand (IL-27), has been shown to trigger the immune response, including cancer, abdominal aortic aneurysm, Sjögren syndrome, virus infection, and transplantation [5–9]. IL-27Rα is the specific subunit of the IL-27 receptor and is restricted primarily to lymphocytes and monocytes [10]. The IL-27 pathway has been shown to inhibit tumor growth by enhancing the response of T cells and decreasing the proportion of Treg cells (T regulator cells) [11]. Furthermore, IL-27 showed a proinflammatory effect by enhancing the IL-1β (interleukin-1 β) secretion from monocytes and macrophages [12]. Moreover, IL-27 could also promote the function of NK cells (natural killer cells) by secreting more IFN-γ (interferon-γ) during influenza infection [13]. All of this suggested that IL-27 could activate IL-27Rα and enhance the proinflammation response.

Acute rejection of the allograft was a severe proinflammatory response participated by T cells and macrophages, and IL-27 has been shown to be closely related to allograft rejection [8, 14, 15]. IL-27Rα (IL-27 receptor α) expression in T cells exacerbated GVHD (graft-versus-host disease) by improving the effector function of Th1 cells (T helper 1 cells) and inhibiting subsets of Th2 and Treg cells [8], while IL-27Rα was apparently upregulated in alloreactive splenetic CD4⁺T cells, T cells, and macrophage when acute rejection occurred [16–18]. In our previous study with the allografted mouse model, we found that a large amount of IL-27Rα-positive T cells and macrophage infiltrated in the rejection of the allograft and anti-IL-27Rα mAb labeled with iodine-125 could obviously accumulate in the allograft noninvasively when rejection occurred [19].

Target tissue could be accurately and noninvasively diagnosed by molecular nuclear imaging with a specific probe, which was much more favorable than histopathological biopsies and traditional imaging examination [20–24]. Although histopathological biopsies were the “gold standard” of acute graft rejection, they still were an invasive examination and can induce complications, including pain, bleeding, and death [25, 26]. Noninvasive examinations, such as magnetic resonance imaging and ultrasound, reflected decreased graft function and were limited in targeting allograft [22, 24]. Targeted molecular imaging has advantages in tracking specific cells and monitoring the function of the target organ with probes that have detection signals [27–29]. Among them, radionuclide imaging was a noninvasive method by which a disease could be diagnosed effectively and quickly, and the therapeutic effect could be monitored with the help of a radio probe. Radionuclide imaging with radiolabeled macromolecular objects such as proteins and antibodies usually had the disadvantages of long time to reach the target tissue and the high background, resulting in poor image quality. However, small molecules could quickly accumulate in the target tissue and thus improve imaging. Therefore, the small-sized radio probe is a much more promising radiotracer in radionuclide imaging than the full-length antibody. Radiolabeled cytokines have been applied to track targeted immunocytes due to high contrast imaging, fast clearance, low background, and weak inflammation response [30–32]. Hartimath et al. developed [¹⁸F] FB-IL-2 to monitor activated T lymphocyte infiltration induced by cancer therapy in tumor [33]. Di Gialleonardo et al. demonstrated that ¹⁸F-FB-IL-2 could trace IL-2 receptor-positive cells [34]. [¹²⁴I] I-F8-IL-10 could accumulate in a patient with arthritic joints in rheumatoid arthritis. Meanwhile, fast clearance of [¹²⁴I] I-F8-IL10 and [¹³¹I] I-F8-IL-10 in nonspecific target tissues was found in a 24-hour span [35]. Consequently, imaging with radiolabeled cytokines had the advantage of specific recognition of target tissue with a low background and could be a promising strategy for allorejection detection.

In this study, we prepared a novel radio probe (¹²⁵I-labeled recombinant IL-27, ¹²⁵I-rIL-27) to specifically target IL-27Rα and evaluated its potential application in monitoring acute allograft rejection.

2. Materials and Methods

2.1. Chemicals, Reagents, and Equipment

IL-27Rα mAb was obtained from the R&D system (Minnesota, USA). Recombination IL-27 (rIL-27) was purchased from PeproTech (New Jersey, USA). Na¹²⁵I was provided by the China Institute of Atomic Energy (Beijing, China). The Sephadex G-25M PD10 column was purchased from GE Healthcare (Pennsylvania, USA). RPMI-1640 medium was obtained from Biological Industries (Kibbutz Beit Haemek, Israel). Bromophenol orchid, HEPES buffer, BSA (bovine albumin) and red blood cell lysis buffer were obtained from Solarbio (China, Beijing). SDS (sodium dodecyl sulfate) loading buffer, antibody dilution buffer, and blocking buffer were obtained from Beyotime (Shanghai, China). PBS (phosphate-buffered saline), TBST (tris-buffered saline and Tween) buffer, H&E (hematoxylin and eosin) staining, and immunofluorescence (IF) staining reagent were purchased from Servicebio (Wuhan, China). The GAPDH solution was obtained from Bioss (Beijing, China) and Bioworld (Illinois, USA). The HRP- (horseradish peroxidase-) labeled goat anti-Rat IgG solution and the HRP-labeled goat anti-rabbit IgG solution were obtained from Epizyme (Shanghai, China). The ECL (enhanced chemiluminescence) substrate and PVDF (polyvinylidene fluoride) membrane were purchased from Merck Millipore (Darmstadt, Germany). The 0.05 M PB (phosphate buffer) solution (250 ml) consisted of 0.6555 g NaH2PO4·2H2O and 7.2495 g Na2HPO4·12H2O dissolved in distilled water, with a constant volume of 250 ml. NaH2PO4·2H2O and Na2HPO4·12H2O were obtained from Solarbio (China, Beijing).

Radioactive counts were measured using the Gamma counter from Capintec Inc. (USA). The phosphor autoradiography images were captured and analyzed using Cyclone Plus Scanner (PerkinElmer, Life Sciences, USA). The membrane was scanned with the Tanon 5200 imaging system scanner (Tanon, Shanghai, Beijing).

2.2. Radiochemistry

2.2.1. Preparation of the Radio Probe

The preparation of the ¹²⁵I-labeled probe was performed according to reference (36). Briefly, 0.05 M PB solution (100 μl), IL-27Rα mAb (12 μg) or rIL-27 (8 μg), and Na¹²⁵I (11.9 MBq) were mixed in the tube with iodogen at room temperature for 15 minutes. The tube was gently shaken every 3-4 minutes. 150 μl 0.05 M PB was added to the tube and allowed to stand at room temperature for 10 minutes. 200 μl 5% BSA reagent (containing bromophenol blue) was added to the tube and gently shaken. The mixture was then added to the Sephadex G-25M PD10 column, followed by elution with 0.01 M PB solution. The eluent was collected in a tube (0.5 ml for each tube), and the radioactive count of 10 μl eluent of each tube was measured by the Gamma counter. $\begin{matrix} (1) & Radiochemical yield = (radioactivity of the first peak) / (summed radioactivity counts for each tube) . \end{matrix}$

Radiochemical purity was detected following the protocol [19]. Briefly, 2 μl of the radio probe was added to the filter paper (2 cm to the bottom). The bottom of the paper was then immersed in 0.9% saline and methanol solution (1 : 2, $v / v$ ). After 40 min, the paper was cut into slices (1 cm) and the radioactive count was measured using a Gamma counter. $Radiochemical purity = (radioactivity near the sample) / (total radioactivity of every slice)$ .

2.2.2. In Vitro Stability Study

The radio probe (12.5 μl) was dissolved in saline (100 μl) or mouse serum (100 μl), and the mixture was kept at 37°C for a period of time. At 1, 12, and 24 h, 2 μl of the sample was taken and analyzed to observe the change in radiochemical purity.

2.2.3. Determination of Lipophilicity

¹²⁵I-rIL-27 (0.2 μl, $4.08 \times 10^{- 4}$ MBq) was diluted in 1 M HEPES buffer (500 μl) and mixed with n-octanol (500 μl) for 30 min, followed by centrifugation for 10 min with $14000 \times g$ . Subsequently, aliquots of the n-octanol and water phases (400 μl) were taken out and then centrifuged again. Finally, the radioactive count of each phase (100 μl) was measured by the Gamma counter and the octanol/water partition coefficient (log $D_{o / w}$ ) was calculated.

2.3. Cell Assays

Cell assays were performed using isolated mouse model spleen cells on day 10 after transplantation. Briefly, the mouse model spleen was isolated and pressed on mesh 200. The cells were then treated with red blood cell lysis buffer, washed with PBS, and finally suspended in RPMI-1640 medium. Cells were cultured in 48-well plates for 2 h with each well $1 \times 10^{6}$ cells in 200 μl RPMI 1640 medium and used for further studies after attachment.

2.3.1. Competition Study

For competition binding assay, nonlabeled anti-IL-27Rα mAb (0 to 71.4 μM) was incubated with alloreactive spleen cells for 1 h before adding 147.09 nM ¹²⁵I-rIL-27. Cells were washed with cold PBS buffer twice, and the supernatant was discarded. The activity bound to the cells was measured by a Gamma counter. $B / B_{0}$ was described as the ratio of radioactive counts with nonlabeled anti-IL-27Rα mAb to radioactive counts without nonlabeled anti-IL-27Rα mAb. The inhibition constant ( $K_{i}$ value) was calculated in GraphPad Prism software.

2.3.2. Saturation Study

¹²⁵I-rIL-27 (3.68 to 147.09 nM) was incubated with spleen cells for 2 h at 37°C to obtain the total binding activity of ¹²⁵I-rIL-27. To test nonspecific binding, cells were pretreated for 1 hour with 10.46 μM nonlabeled rIL-27.

After incubation with ¹²⁵I-rIL-27, cells were washed twice with cold PBS buffer and radioactivity was measured with the Gamma counter. Maximum binding capacity ( $B_{\max}$ ) and dissociation constant [37] were calculated in the GraphPad Prism software. Specific binding was the value of total binding minus nonspecific binding.

2.4. Animal Experiments In Vivo

All animal experiments were performed according to the ARRIVE guidelines. The protocol was approved by the Shandong University Animal Care and Use Committee with the corresponding ethical approval code (LL-201602040, 2016-2022). Female BALB/c mice (H-2^d) and C57BL/6 mice (H-2^b) were purchased from Vital River Laboratory Animal Technology (Beijing, China) and housed in standard conditions with free access to water and standard food.

2.4.1. Animal Models

To establish the skin transplantation model, C57BL/6 mice and BALB/c mice were used as allogeneic and syngeneic transplant skin graft donors, respectively. BALB/c mice were recipients. Briefly, surgery was performed under anesthesia with 0.6% pentobarbital sodium (0.1 ml/10 g body weight) under sterility conditions. The mucous membrane and blood vessel of the graft were removed, and then, the graft was cut into a circle with 1 cm diameter. Then, remove the skin of the recipients on the right shoulder and transfer the graft to the recipients. Finally, petrolatum gauze was placed on the graft and covered with bandage. Acute rejection occurred on day 7 after transplantation when the bandage with the escharotic area was removed greater than 50%.

2.4.2. Blood Clearance Assay

At 1 h, 2 h, 6 h, 12 h, and 24 h after radio probe injection, mice were anesthetized with 0.6% pentobarbital sodium solution. Then, 5 μl blood was drawn from the tail vein. The activity of the radio probe that remained in the blood was counted by the Gamma counter. Each mouse was weighted, and the radio probe concentration in the blood (ng/μl) was calculated using 78 ml/kg as a blood factor. The AUCs (area under the curves) of ¹²⁵I-rIL-27 in 24 h and ¹²⁵I-anti-IL-27Rα mAb in 48 h were obtained using the GraphPad Prism software. Blood clearance (CL, μl/h) was calculated as the dose/AUC with the study referred to [38].

2.4.3. Dynamic Phosphor Autoradiography

Mice were divided into an allogroup, a syngroup, and a blocking group according to the allogeneic, syngeneic, and allogeneic transplantation model with specific antibody blocking. After being fed 3% NaI solution for 24 h, 60 μg nonlabeled IL-27Rα mAb was injected into the blocking group. One hour later, all mice were injected with ¹²⁵I-rIL-27 (0.37 MBq) and ¹²⁵I-anti-IL-27Rα mAb (0.37 MBq) on day 9 after transplantation, respectively. Mice were anesthetized and scanned with the Cyclone Plus Scanner. ROIs (regions of interest) were quantified using OptiQuant Image Analysis software and presented as digital light units per square millimeter (DLU/mm²).

2.4.4. Ex Vivo Biodistribution

Three groups of mice (allogroup, syngroup, and blocking group, $n = 3$ for each group) were sacrificed 24 h after intravenous injection of a radio probe (0.08 MBq in 200 μl of 0.01 M PB). Organs or tissues of interest, including blood, liver, lung, kidney, spleen, control skin, and graft, were excised and weighed. The activity was measured by a Gamma counter, and the uptake of the radio probe was expressed as the percentage of injected dose per gram (%ID/g). The T/NT (target/nontarget) ratio was calculated by dividing the %ID/g of the target graft to that of the control skin (opposite site), while T/B (target/blood) was %ID/g of the target graft to that of the blood.

2.4.5. H&E Staining and Immunofluorescence Staining

On day 10 after transplantation, the grafts were collected and histological sections were prepared. H&E staining and immunofluorescence staining were performed following the staining kit protocols. The image was obtained under an optical microscope. Briefly, in H&E staining, sections were covered with hematoxylin for 5 min. After applying 1% acid ethanol reagent for 5 seconds, sections were covered with a blue promotor solution for 5 seconds. The sections were then covered with eosin solution for 10 minutes. Between each step, distilled water was used to wash out excess buffer. In IF staining, sections were treated with EDTA antigen repair buffer (pH 9.0) and blocked with BSA for 30 min. Anti-IL-27Rα Ab was then diluted in PBS (1 : 200) and added to the section at 4°C, overnight. Sections were washed with PBS and covered with a second antibody for 1 hour. Later, sections were washed with PBS and the FITC regent (green) was added to the sections. The sections were then washed with TBST and covered with tissue autofluorescence quencher regent for 5 min. The excess regent was then washed with distilled water for 10 min. The sections were discarded excess liquid and incubated with DAPI regent (blue) for 10 min at room temperature. Finally, the sections were washed with PBS and then enclosed with antifade mounting medium.

2.4.6. Western Blot

After 10 d of transplantation, the grafts were separated, lysed, and reacted with SDS loading buffer. Electrophoresis was performed, and the protein was transferred to the PVDF membrane. The target membrane was then treated with blocking buffer and then covered with anti-IL-27Rα mAb solution and a GAPDH solution overnight. The membrane was then washed with TBST buffer and covered with HRP-labeled goat anti-rat IgG solution and HRP-labeled goat anti-rabbit IgG solution, respectively. Finally, the membrane was washed with TBST buffer, followed by ECL substrate covering. The band was scanned with the Tanon 5200 imaging system scanner and analyzed with ImageJ software.

2.5. Statistical Analysis

All data were quoted as $mean \pm standard deviation$ ( $mean \pm SD$ ), and each data point emerged from 3 independent experiments. Comparisons between two groups were analyzed using the unpaired Student $t$ -test. The correlation between DLU/mm² of ¹²⁵I-rIL-27 and the expression of IL-27Rα was calculated using a correlation assay. The statistically significant level was established at $p < 0.05$ .

3. Results

3.1. Radiochemistry

The labeling yields of ¹²⁵I-rIL-27 and ¹²⁵I-anti-IL-27Rα mAb were 84.4% and 99.0%, respectively. The radiochemistry purity of these radio probes was 93.3% and 95.3%. The stabilities of the ¹²⁵I-rIL-27 and ¹²⁵I-anti-IL-27Rα mAb were more than 90% in saline and mouse serum even after 24 h (supplement Figure 1), respectively. The results showed that the ¹²⁵I-rIL-27 and ¹²⁵I-anti-IL-27Rα mAb were quite stable. The log $D_{o / w}$ values for ¹²⁵I-rIL-27 were $- 1.18 \pm 0.23$ , which means that ¹²⁵I-rIL-27 has a hydrophilic character.

3.2. Cell Binding Assays

3.2.1. Saturation

Typical saturation graphs obtained after incubation of $1 \times 10^{6}$ cells with ¹²⁵I-rIL-27 are shown in Figures 1(a) and 1(b). The $B_{\max}$ values of ¹²⁵I-rIL-27 in alloreactive and synreactive splenocytes were 2545 cpm/10⁶ cells and 1607 cpm/10⁶ cells, respectively. Furthermore, $K_{d}$ values were found to be 48.59 nM and 49.04 nM for allo- and synreactive splenocytes, respectively.

Figure 1

Binding assay of 125I-rIL-27 in vitro.

(b)

(c)

3.2.2. Competition

Figure 1(c) shows that the binding of ¹²⁵I-rIL-27 decreased as anti-IL-27Rα mAb increased. Using the $K_{d}$ value of ¹²⁵I-rIL-27 from the saturation assay, the determination of the $K_{i}$ value was 769.9 nM using the Cheng-Prusoff equation.

3.3. Small Animal In Vivo Experiments

3.3.1. Blood Clearance Assay

To understand how fast ¹²⁵I-rIL-27 cleared in vivo, a blood clearance assay was performed. Blood clearance was represented as a clearance value. The allogroup showed a significantly lower concentration of ¹²⁵I-rIL-27 than ¹²⁵I-anti-IL-27Rα mAb in the blood ( $0.450 \pm 0.095$ vs. $0.808 \pm 0.089$ ng/μl, $p < 0.01$ ), and the syngroup showed the same pattern ( $0.342 \pm 0.281$ vs. $0.8967 \pm 0.0753$ ng/μl, $p < 0.05$ ) at 24 h after injection. The AUC was shown in Figure 2, and the retention of ¹²⁵I-rIL-27 in the blood was shorter than that of ¹²⁵I-anti-IL-27Rα mAb in the allogroup (35.52 vs. 53.17 h·ng/μl) at 24 h. The syngroup showed shorter retention of ¹²⁵I-rIL-27 than ¹²⁵I-anti-IL-27Rα mAb (31.53 vs. 52.65 h·ng/μl) at 24 h. ¹²⁵I-rIL-27 demonstrated faster blood clearance compared with ¹²⁵I-anti-IL-27Rα mAb in the allogroup (13.20 vs. 9.43 μl/h) and syngroup (14.87 vs. 9.53 μl/h) at 24 h. All these suggested a significantly faster blood clearance of ¹²⁵I-rIL-27 than ¹²⁵I-anti-IL-27Rα mAb in allograft rejection model mice.

Figure 2

Blood clearance assay.

3.3.2. Dynamic Whole-Body Phosphor Autoradiography Imaging

To investigate ¹²⁵I-rIL-27 imaging in vivo, we performed dynamic whole-body phosphor autoradiography imaging. As shown in Figure 3(a), high uptake of ¹²⁵I-rIL-27 was obtained in the allogeneic graft (DLU/mm²: $434241.58 \pm 53524.20$ ) at 6 h after intravenous injection and then reached a plateau up to 24 h (DLU/mm²: $163603.46 \pm 56677.03$ ), while lower uptake was obtained in the syngeneic graft (DLU/mm²: $66401.60 \pm 29698.30$ ) ( $p < 0.01$ ). The in vivo specificity of ¹²⁵I-rIL-27 was confirmed by blocking studies using excess unlabeled anti-IL-27Rα mAb (DLU/mm²: $68252.033 \pm 38373.75$ ). Ex vivo autoradiography apparently showed a high accumulation of activity in the allograft. A similar result of ¹²⁵I-anti-IL-27Rα mAb was observed at 48 h, and the uptake of ¹²⁵I-anti-IL-27Rα mAb in the allogeneic graft was also higher than in the syngeneic graft (Figure 3(b)). However, the image using ¹²⁵I-rIL-27 in the allogeneic graft exhibited a lower background compared to the image with that using ¹²⁵I-anti-IL-27Rα mAb. These indicated that ¹²⁵I-rIL-27 could target the allograft specifically and produce better images with high contrast and low background.

Figure 3

Dynamic whole-body phosphor autoradiography imaging assay.

(b)

3.3.3. Biodistribution Assay

To gain first insight into the potential relevance of ¹²⁵I-rIL-27 for transplantation imaging, a biodistribution assay was performed using skin transplantation mice. Biodistribution data for ¹²⁵I-rIL-27 was shown in Figure 4(a). Higher uptake was observed in the allogeneic skin graft compared to that in the syngeneic group (Figure 4(b)). The uptake of the activity of ¹²⁵I-rIL-27 in the allograft was higher than that in the syngraft (%ID/g: $5.648 \pm 1.735$ vs. $1.751 \pm 0.967$ , $p < 0.01$ ). The T/NT ratio and the T/B ratio increased significantly in the allogroup compared to the syngroup in Figures 4(b) and 4(c).

Figure 4

Biodistribution study on day 10 after transplantation.

(b)

(c)

(d)

More interestingly, compared to ¹²⁵I-anti-IL-27Rα mAb, fewer ¹²⁵I-rIL-27 in the blood were obtained 24 h after injection not only in the allogroup (%ID/g: $6.960 \pm 0.754$ vs. $4.083 \pm 0.710$ , $p < 0.01$ ) but also in the syngroup (%ID/g: $6.090 \pm 0.508$ vs. $3.230 \pm 1.835$ , $p < 0.05$ ). Furthermore, the activity uptake of ¹²⁵I-rIL-27 was also lower than that of ¹²⁵I-anti-IL-27Rα mAb in the liver, lung, kidney, and spleen. These indicated that ¹²⁵I-rIL-27 could specifically recognize IL-27Rα overexpressed in the allograft and have favorable imaging with low background.

3.3.4. IL-27Rα Expression in the Rejection of Allograft

To study the correlation between the accumulation of activity of ¹²⁵I-rIL-27 and the expression of IL-27Rα in the rejection of the allograft, IF staining was performed on day 10 after transplantation to determine the IL-27Rα expression.

HE staining in Figure 5(a) confirmed that a severe rejection response occurred in allogeneic graft, while mild inflammation occurred in syngeneic graft. IL-27Rα expression was obviously higher in the allograft (Figure 5(b)). The accumulation of activity (DLU/mm²) in the graft had a positive correlation with IL-27Rα expression (Figure 5(c)). Fluorescence imaging also confirmed the higher expression of IL-27Rα on the surface of infiltrated cells of rejecting the allograft (Figure 5(d)). All of these suggested that ¹²⁵I-rIL-27 could specifically bind the IL-27Rα in the allograft, monitoring acute rejection.

Figure 5

H&E staining and IL-27Rα expression.

(b)

(c)

(d)

4. Discussion

Early acute allorejection is usually more responsive to allograft transplant therapy, and therefore, timely detection of acute rejection could benefit the prognosis [39]. Up to now, molecular imaging with specific radio probes was a promising method responsible for the detection of allograft rejection [23]. Recently, IL-27, a pleiotropic cytokine with proinflammatory properties, was reported with enhanced antitumor and antivirus activities and participated in the rejection response [40–42]. IL-27 could promote the infiltration of CD4⁺T cells and CD8⁺T cells in the tumor and upregulate IFN-γ, granzyme B, and perforin production, resulting in an improved antitumor effect of T cells [11]. Moreover, IL-27 could also boost NK cells proliferation and cytotoxic activity synergistically with IL-15/IL-18 [43]. All of these indicated that IL-27/IL-27R was a promising target in the proinflammatory immune response. IL-27Rα, the subunit of the IL-27 receptor, which was also expressed in T cells and macrophages, had the highest expression during the acute rejection period in the allograft [44–46]. In our previous study, ¹²⁵I-anti-IL-27Rα mAb has been found with high specificity towards IL-27Rα [19]. However, it had limitations in nonspecific binding to Fc recognition, slow metabolism, and clearance, compared to a small antibody fragment or ligand [31, 47]. Therefore, a small radio probe could provide better imaging with a low background.

The cytokine was a small ligand of the cytokine receptor that was expressed on the surface of effector cells [47]. Many radio cytokine probes have already been applied in target imaging [48–51]. Radiolabeled IL-2 probes were used in clinics for the targeted detection of lymphocytic infiltration in transplantation and atherosclerotic plaque [48, 49]. Glaudemans et al. found that symptomatic plaques with high CD3⁺ cell infiltration had a significant uptake of ^99mTc-HYNIC-IL-2 and the lung of the rejection patient had increased ^99mTc-HYNIC-IL-2 uptake. In their researches, no side effects were found with the administration of ^99mTc-HYNIC-IL-2. We also developed the ¹²⁵I-rIL-27 targeted radio probe with high radiochemical purity. Because the Sephadex G-25M PD10 column allowed for rapid group separation of high-molecular-weight substances form low-molecular-weight substances, the radio probe which has high molecular weight would be eluted first. The radioactive probes were easier to observe because they contain proteins that could be stained blue with bromophenol blue. Meanwhile, the radio count assay of the elution would help to confirm the radio probe. We found that ¹²⁵I-rIL-27 would keep stable for 24 hours after synthesis using the paper chromatography method. This is a traditional method to determine radiochemical purity and has reproducible and accurate radio counts [52]. When the radio compound is not dissociated, the radio compound swims slowly in the medium because of its large molecular weight. Therefore, the place where the sample was had the highest radioactivity count. And when the radio compound dissociated, free iodine 125 would produce a higher radioactivity count on the upper paper. This method will help to demonstrate the radiochemical purity and stability of the radio probe. Also, we also found that this radio probe had no side effects in the mouse model.

The in vitro experiment showed that our ¹²⁵I-rIL-27 had a specific binding to IL-27Rα on the spleen cells. However, the binding ability and affinity of ¹²⁵I-rIL-27 were lower than those of ¹²⁵I-anti-IL-27Rα mAb. This could be due to the fact that ¹²⁵I-anti-IL-27Rα mAb has nonspecific binding of the Fc fragment. Matsushima et al. developed ¹²⁵I-labeled IL 1β in a human large granular lymphocyte cell line (YT cells), and this radio probe showed a higher affinity of 0.1 nM ( $K_{d}$ value) compared to our probe [53]. It may be due to the different receptor expressions of the cells. Furthermore, the isolation process of spleen cells may also result in some loss of receptors [54].

In the imaging of [¹²⁴I] I-F8-IL10, it was suggested that the target area had the highest uptake and target-to-background ratios at 24 h after injection of the radio probe [35]. Therefore, we carried out biodistribution and blood clearance of ¹²⁵I-rIL-27 within 24 h after radio probe injection. In the blood clearance assay, ¹²⁵I-rIL-27 showed faster blood clearance than ¹²⁵I-anti-IL-27Rα, which could be due to different levels of cytokines and antibody glycosylation, which influence receptor recognition and blood clearance [55]. The blood clearance assay showed a shorter retention of ¹²⁵I-anti-IL-27Rα in the blood compared to monoclonal antibody, which could be due to recognition of Fc [56]. The whole-body phosphor autoradiography imaging demonstrated that the allograft had more activity accumulation than the syngeneic graft, and this accumulation could be blocked by excess of anti-IL-27Rα mAb. A lower background was also observed at 24 h in the ¹²⁵I-rIL-27 group compared with ¹²⁵I-anti-IL-27Rα. The tumor necrosis factor superfamily (TNFSF) contains CD40L, FasL, TRAIL (TNF-related apoptosis-inducing ligand), LiGHT, VEGF (vascular endothelial growth factor), lymphotoxin alpha, lymphotoxin beta, and lymphotoxin alpha1/beta2, which could be fused with the F8 antibody for tumor targeting. In biodistribution, it was suggested that the %ID/g of ¹²⁵I-rIL-27 in the allograft was similar to that of F8-TRAILtrunc, lower than that of F8-CD40L, and higher than that of other TNFSF in the tumor [57]. The reason may be the different expressions and affinity of different receptors for the receptors. The %ID/g of ¹²⁵I-rIL-27 (47.8 KDa) in the blood was higher than that of F8-TNFSF, F8-IL-10 (18.6 KDa), and ^99mTc-VEGF₁₆₅ (16 KDa), probably due to the lower molecular weight of other cytokines [50]. However, the activity of ¹²⁵I-rIL-27 in the blood was much lower compared to that of ¹²⁵I-anti-IL-27Rα (155KDa), possibly caused by the nonspecific Fc binding of ¹²⁵I-anti-IL-27Rα. The uptake of ¹²⁵I-rIL-27 in the lung was higher than that in other organs, except graft and blood. This could be due to the enrichment of immune cells overexpressed with IL-27Rα in the lung. Meanwhile, blood pollution should also be considered. The accumulation of activity in the kidney was found to be higher than in the liver, which may be due to the hydrophilic character of ¹²⁵I-rIL-27. Furthermore, it was also found that the accumulation of ¹²⁵I-rIL-27 had a close correlation with the expression of IL-27Rα of the graft. All of these indicated that ¹²⁵I-rIL-27 was a promising radiotracer that could specifically target IL-27Rα for the imaging of acute rejection allograft with faster blood clearance and low background, compared with ¹²⁵I-anti-IL-27Rα mAb.

5. Conclusions

In this study, the acute rejection of allograft could be detected by targeting IL-27Rα in allograft specifically with ¹²⁵I-rIL-27. The rejecting allograft had higher specific ¹²⁵I-rIL-27 uptake than nonrejecting syngeneic graft, and the accumulation of activity was in close correlation with the expression of IL-27Rα of the graft. More importantly, low background and rapid clearance were obtained for ¹²⁵I-rIL-27 compared with ¹²⁵I-anti-IL-27Rα mAb. Imaging with this small radio probe could be a promising strategy for noninvasive monitoring of IL-27Rα-overexpressed rejecting allograft.

Footnotes

Data Availability

All data included in this study are available upon request by contact with the corresponding author.

Additional Points

Statement. A preprint has previously been published [].

Conflicts of Interest

The authors have no conflicts of interest to disclose.

Authors’ Contributions

Shanshan Zhao and Guihua Hou designed the studies. Shanshan Zhao and Qian Liu carried out the study, including data collection and data analysis. Shanshan Zhao, Qian Liu, Feng Gao, and Guihua Hou wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The author thanks the Translational Medicine Core Facility of Shandong University for the consultation and availability of instruments that supported this work. This research was funded by grants from the Natural Science Foundation of Shandong Province (grant numbers ZR2019MH019).

Supplementary Materials

References

Loupy

Lefaucheur

Antibody-Mediated Rejection of Solid-Organ Allografts

New England Journal of Medicine 2018 379

4200142

26 2579 2582

10.1056/NEJMc1813976

2-s2.0-85059211918

Grenda

Kaliciński

Combined and sequential liver-kidney transplantation in children

Pediatric Nephrology 2018 33

4200142

12 2227 2237

10.1007/s00467-017-3880-4

2-s2.0-85040365092

29322327

Jalalzadeh

Mousavinasab

Peyrovi

Ghadiani

M. H.

The impact of acute rejection in kidney transplantation on long-term allograft and patient outcome

Nephro-Urology Monthly 2015 7

4200142

1, article e24439

10.5812/numonthly.24439

2-s2.0-84932105931

25738128

Tsai

M.-K.

F.-L. L.

Lai

Lee

C.-Y.

R.-H.

Lee

P.-H.

Decreased acute rejection and improved renal allograft survival using sirolimus and low-dose calcineurin inhibitors without induction therapy

International Journal of Artificial Organs 2009 32

4200142

6 371 380

10.1177/039139880903200608

2-s2.0-70149087539

19670189

Peshkova

I. O.

Aghayev

Fatkhullina

A. R.

Makhov

Titerina

E. K.

Eguchi

Tan

Y. F.

Kossenkov

A. V.

Khoreva

M. V.

Gankovskaya

L. V.

Sykes

S. M.

Koltsova

E. K.

IL-27 receptor-regulated stress myelopoiesis drives abdominal aortic aneurysm development

Nature Communications 2019 10

4200142

1 5046 5046

10.1038/s41467-019-13017-4

31695038

Yao

Liang

Shi

Chen

Tang

Wang

Chen

Shi

Hou

Sun

Mesenchymal stem cell transplantation alleviates experimental Sjögren's syndrome through IFN-β/IL-27 signaling axis

Theranostics 2019 9

4200142

26 8253 8265

10.7150/thno.37351

31754394

Huang

Zak

Pratumchai

Shaabani

Vartabedian

V. F.

Nguyen

Xiao

Teijaro

J. R.

IL-27 promotes the expansion of self-renewing CD8(+) T cells in persistent viral infection

The Journal of Experimental Medicine 2019 216

4200142

8 1791 1808

10.1084/jem.20190173

2-s2.0-85071063566

31164392

Bastian

Liu

Schutt

Nguyen

H. D.

Daenthanasanmak

Sofi

M. H.

Zhang

Iamsuwat

X. Z.

IL-27 receptor signaling on T cells augments GVHD severity through enhancing Th1 responses

Journal of Immunology Research and Therapy 2018 3

4200142

1 151 157

30906912

Wang

Ning

Peng

Wei

Hou

Hoft

D. F.

Liu

Tristetraprolin inhibits macrophage IL-27-induced activation of antitumour cytotoxic T cell responses

Nature Communications 2017 8

4200142

1 867 867

10.1038/s41467-017-00892-y

2-s2.0-85031125590

29021521

10.

Jones

G. W.

Hill

D. G.

Cardus

Jones

S. A.

IL-27: a double agent in the IL-6 family

Clinical and Experimental Immunology 2018 193

4200142

1 37 46

10.1111/cei.13116

2-s2.0-85049776333

29437229

11.

Zhu

Liu

J.-Q.

Shi

Cheng

Ding

Zhang

J. C.

Davis

J. P.

Varikuti

Satoskar

A. R.

Pan

Zheng

Liu

Bai

X.-F.

IL-27 gene therapy induces depletion of Tregs and enhances the efficacy of cancer immunotherapy

JCI insight 2018 3

4200142

7, article e98745

10.1172/jci.insight.98745

2-s2.0-85062245603

29618655

12.

Petes

Wynick

Guzzo

Mehta

Logan

Banfield

B. W.

Basta

Cooper

Gee

IL-27 enhances LPS-induced IL-1β in human monocytes and murine macrophages

Journal of Leukocyte Biology 2017 102

4200142

1 83 94

10.1189/jlb.3A0316-098R

2-s2.0-85021736494

28377398

13.

Kumar

Rajasekaran

Nanbakhsh

Gorski

Thakar

M. S.

Malarkannan

IL-27 promotes NK cell effector functions via Maf-Nrf2 pathway during influenza infection

Scientific Reports 2019 9

4200142

1, article 4984

10.1038/s41598-019-41478-6

2-s2.0-85063320720

30899058

14.

Benzimra

Calligaro

G. L.

Glanville

A. R.

Acute rejection

Journal of Thoracic Disease 2017 9

4200142

12 5440 5457

10.21037/jtd.2017.11.83

2-s2.0-85039797872

29312755

15.

Gaignage

Marillier

R. G.

Cochez

P. M.

Dumoutier

Uyttenhove

Coutelier

J.-P.

Van Snick

The TLR7 ligand R848 prevents mouse graft-versus-host disease and cooperates with anti-interleukin-27 antibody for maximal protection and regulatory T-cell upregulation

Haematologica 2019 104

4200142

2 392 402

10.3324/haematol.2018.195628

2-s2.0-85061028660

30213828

16.

Petes

Mariani

M. K.

Yang

Grandvaux

Gee

Interleukin (IL)-6 inhibits IL-27- and IL-30-mediated inflammatory responses in human monocytes

Frontiers in Immunology 2018 9

4200142

256

10.3389/fimmu.2018.00256

2-s2.0-85042066811

29497424

17.

Qiu

S.-L.

Duan

M.-C.

Liang

Tang

H.-J.

Liu

G.-N.

Zhang

L.-M.

Yang

C.-M.

Cigarette smoke induction of interleukin-27/WSX-1 regulates the differentiation of Th1 and Th17 cells in a smoking mouse model of emphysema

Frontiers in Immunology 2016 7

4200142

553 553

10.3389/fimmu.2016.00553

2-s2.0-85009351732

27994590

18.

Wang

Zhang

Song

Liang

Jin

Kim

Y. C.

Wang

S. M.

Hou

Alternatively expressed genes identified in the CD4+ T cells of allograft rejection mice

Cell Transplantation 2011 20

4200142

2 333 350

10.3727/096368910x552844

2-s2.0-79955434456

21294963

19.

Zhao

Shi

Jiang

Zhang

Liang

Hou

IL-27Rα: a novel molecular imaging marker for allograft rejection

International Journal of Molecular Sciences 2020 21

4200142

4, article 1315

10.3390/ijms21041315

32075272

20.

Sharfuddin

Renal Relevant Radiology: Imaging in Kidney Transplantation

Clinical Journal of the American Society of Nephrology 2014 9

4200142

2 416 429

10.2215/cjn.02960313

2-s2.0-84893626955

21.

Roden

Aisner

Allen

Aubry

Barrios

Beasley

Cagle

Capelozzi

Dacic

Hariri

Lantuejoul

Miller

Mino-Kenudson

Moreira

Raparia

Rekhtman

Sholl

Smith

Diagnosis of acute cellular rejection and antibody-mediated rejection on lung transplant biopsies: a perspective from members of the pulmonary pathology society

Archives of Pathology & Laboratory Medicine 2017 141

4200142

437 444

10.5858/arpa.2016-0459-SA

2-s2.0-85014450705

22.

Huang

Sadowski

E. A.

Artz

N. S.

Seo

Djamali

Grist

T. M.

Fain

S. B.

Measurement and comparison of T1 relaxation times in native and transplanted kidney cortex and medulla

Journal of magnetic resonance imaging: JMRI 2011 33

4200142

5 1241 1247

10.1002/jmri.22543

2-s2.0-79955374464

21509885

23.

Köhnke

Kentrup

Schütte-Nütgen

Schäfers

Schnöckel

Hoerr

Reuter

Update on imaging-based diagnosis of acute renal allograft rejection

American Journal of Nuclear Medicine and Molecular Imaging 2019 9

4200142

2 110 126

31139495

24.

Liao

Zhang

Ren

Zheng

Zhang

Liu

Yin

Sun

Noninvasive quantification of intrarenal allograft C4d deposition with targeted ultrasound imaging

American Journal of Transplantation 2019 19

4200142

1 259 268

10.1111/ajt.15105

2-s2.0-85054037227

30171802

25.

D. M.

Chen

Zhou

F. D.

Zhao

M. H.

Risk factors for severe bleeding complications in percutaneous renal biopsy

The American Journal of the Medical Sciences 2017 353

4200142

3 230 235

10.1016/j.amjms.2016.12.019

2-s2.0-85020353485

28262208

26.

Chi

Hansen

B. E.

Tang

W. Y.

Schouten

J. N.

Sprengers

Taimr

Janssen

H. L.

de Knegt

R. J.

Multiple biopsy passes and the risk of complications of percutaneous liver biopsy

European Journal of Gastroenterology & Hepatology 2017 29

4200142

1 36 41

10.1097/meg.0000000000000731

2-s2.0-84983233505

27556687

27.

Freise

A. C.

Zettlitz

K. A.

Salazar

F. B.

Tavaré

A. M.

ImmunoPET imaging of murine CD4+ T cells using anti-CD4 Cys-diabody: effects of protein dose on T cell function and imaging

Molecular Imaging and Biology 2017 19

4200142

4 599 609

10.1007/s11307-016-1032-z

2-s2.0-85003781903

28.

Mall

Yusufi

Wagner

Klar

Bianchi

Steiger

Straub

Audehm

Laitinen

Aichler

M. J. C. R.

Immuno-PET imaging of engineered human T cells in tumors

Cancer Research 2016 76

4200142

14 4113 4123

10.1158/0008-5472.can-15-2784

2-s2.0-84978654959

29.

Sun

Yang

Liang

Zhang

Song

Han

Hou

Non-invasive imaging of allogeneic transplanted skin graft by 131I-anti-TLR5 mAb

Journal of Cellular and Molecular Medicine 2014 18

4200142

12 2437 2444

10.1111/jcmm.12423

2-s2.0-84911991268

25283154

30.

Berraondo

Sanmamed

M. F.

Ochoa

M. C.

Etxeberria

Aznar

M. A.

Pérez-Gracia

J. L.

Rodríguez-Ruiz

M. E.

Ponz-Sarvise

Castañón

Melero

Cytokines in clinical cancer immunotherapy

British Journal of Cancer 2019 120

4200142

1 6 15

10.1038/s41416-018-0328-y

2-s2.0-85056350770

30413827

31.

Ehlerding

E. B.

Lee

H. J.

Jiang

Ferreira

C. A.

Zahm

C. D.

Huang

Engle

J. W.

McNeel

D. G.

Cai

Antibody and fragment-based PET imaging of CTLA-4+ T-cells in humanized mouse models

American Journal of Cancer Research 2019 9

4200142

1 53 63

30755811

32.

Ying

Feng

Wang

Chen

Dimitrov

D. S.

Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases

MAbs 2014 6

4200142

5 1201 1210

10.4161/mabs.29835

2-s2.0-84913543666

25517305

33.

Hartimath

S. V.

Draghiciu

van de Wall

Manuelli

Dierckx

R. A. J. O.

Nijman

H. W.

Daemen

de Vries

E. F. J.

Noninvasive monitoring of cancer therapy induced activated T cells using [¹⁸F]FB-IL-2 PET imaging

OncoImmunology 2017 6

4200142

1, article e1248014

10.1080/2162402X.2016.1248014

2-s2.0-85007390227

28197364

34.

Di Gialleonardo

Signore

Glaudemans

A. W.

Dierckx

R. A.

De Vries

E. F.

N-(4-¹⁸F-Fluorobenzoyl)interleukin-2 for PET of human-activated T lymphocytes

Journal of Nuclear Medicine 2012 53

4200142

5 679 686

10.2967/jnumed.111.091306

2-s2.0-84860761137

22499614

35.

Bruijnen

S. T. G.

Chandrupatla

Giovanonni

Neri

Vugts

D. J.

Huisman

M. C.

Hoekstra

O. S.

Musters

R. J. P.

Lammertsma

A. A.

van Dongen

Jansen

Molthoff

C. F. M.

van der Laken

C. J.

F8-IL10: a new potential antirheumatic drug evaluated by a PET-guided translational approach

Molecular Pharmaceutics 2019 16

4200142

1 273 281

10.1021/acs.molpharmaceut.8b00982

2-s2.0-85059648564

30550295

36.

Shi

Liu

Zhao

Zhang

Liang

Hou

TLR5 is a new reporter for triple-negative breast cancer indicated by radioimmunoimaging and fluorescent staining

Journal of Cellular and Molecular Medicine 2019 23

4200142

12 8305 8313

10.1111/jcmm.14707

2-s2.0-85073963466

31576678

37.

Ceylan

M. N.

Akdas

Yazihan

The effects of zinc supplementation on C-reactive protein and inflammatory cytokines: a meta-analysis and systematical review

Journal of Interferon & Cytokine Research 2021 41

4200142

3 81 101

10.1089/jir.2020.0209

33750215

38.

Ding

Carlton

Povoski

Milum

Kumar

Kothandaraman

Hinkle

Colcher

Brody

Davis

Pokora

Phelps

Martin

Tweedle

Site specific discrete PEGylation of ¹²⁴I-labeled mCC49 Fab’ fragments improves tumor microPET/CT imaging in mice

Bioconjugate Chemistry 2013 24

4200142

11 1945 1954

10.1021/bc400375f

2-s2.0-84888584556

24175669

39.

Zhang

Donor-specific antibodies in kidney transplant recipients

Clinical Journal of the American Society of Nephrology 2018 13

4200142

1 182 192

10.2215/CJN.00700117

2-s2.0-85040223052

28446536

40.

Belle

Agle

Zhou

Yin-Yuan

Komorowski

Eastwood

Logan

Sun

Ghilardi

Cua

Williams

C. B.

Gaignage

Marillier

van Snick

Drobyski

W. R.

Blockade of interleukin-27 signaling reduces GVHD in mice by augmenting Treg reconstitution and stabilizing Foxp3 expression

Blood 2016 128

4200142

16 2068 2082

10.1182/blood-2016-02-698241

2-s2.0-84992051377

27488350

41.

Jung

J.-Y.

Roberts

L. L.

Robinson

C. M.

The presence of interleukin-27 during monocyte-derived dendritic cell differentiation promotes improved antigen processing and stimulation of T cells

Immunology 2015 144

4200142

4 649 660

10.1111/imm.12417

2-s2.0-84924363518

25346485

42.

Petes

Mintsopoulos

Finnen

R. L.

Banfield

B. W.

Gee

The effects of CD14 and IL-27 on induction of endotoxin tolerance in human monocytes and macrophages

The Journal of Biological Chemistry 2018 293

4200142

45 17631 17645

10.1074/jbc.RA118.003501

2-s2.0-85056395233

30242126

43.

Choi

Y. H.

Lim

E. J.

Kim

S. W.

Moon

Y. W.

Park

K. S.

H.-J.

IL-27 enhances IL-15/IL-18-mediated activation of human natural killer cells

Journal for Immunotherapy of Cancer 2019 7

4200142

1 168 168

10.1186/s40425-019-0652-7

2-s2.0-85068685219

31277710

44.

Iwasaki

Fujio

Okamura

Yamamoto

Interleukin-27 in T cell immunity

International Journal of Molecular Sciences 2015 16

4200142

2 2851 2863

10.3390/ijms16022851

2-s2.0-84921897987

25633106

45.

Holz

Prinz

Brendecke

S. M.

Hölscher

Deng

Mitrücker

H. W.

Rose-John

Hölscher

Differing outcome of experimental autoimmune encephalitis in macrophage/neutrophil- and T cell-specific gp130-deficient mice

Frontiers in Immunology 2018 9

4200142

836

10.3389/fimmu.2018.00836

2-s2.0-85046254211

29770132

46.

Zhao

Liang

Zhang

Shi

Jiang

Hou

IL-27 Rα ⁺ cells promoted allorejection via enhancing STAT1/3/5 phosphorylation

Journal of Cellular and Molecular Medicine 2020 24

4200142

18 10756 10767

10.1111/jcmm.15700

32761753

47.

Calzada

García

M. F.

Alonso-Martínez

L. M.

Camachoc

Goicochea

Fernández

Castillo

A. X.

Díaz-Miqueli

Iznaga-Escobar

Montaña

R. L.

Alonso

Gambini

J. P.

Cabral

Fab(nimotuzumab)-HYNIC-99mTc: antibody fragmentation for molecular imaging agents

Anti-Cancer Agents in Medicinal Chemistry 2016 16

4200142

9 1184 1189

10.2174/1871520616666160310143828

2-s2.0-85021856621

26961312

48.

Glaudemans

Bonanno

Galli

Zeebregts

de Vries

Koole

Luurtsema

Boersma

Taurino

Slart

Signore

In vivo and in vitro evidence that ^99mTc-HYNIC-interleukin-2 is able to detect T lymphocytes in vulnerable atherosclerotic plaques of the carotid artery

European Journal of Nuclear Medicine and Molecular Imaging 2014 41

4200142

9 1710 1719

10.1007/s00259-014-2764-0

2-s2.0-84906062899

24737117

49.

Telenga

E. D.

van der Bij

de Vries

E. F. J.

Verschuuren

E. A. M.

Timens

Luurtsema

Slart

Signore

Glaudemans

^99mTc-HYNIC-IL-2 scintigraphy to detect acute rejection in lung transplantation patients: a proof-of-concept study

EJNMMI Research 2019 9

4200142

1 41

10.1186/s13550-019-0511-z

2-s2.0-85065706103

31076906

50.

Galli

Artico

Taurone

Manni

Bianchi

Piaggio

Weintraub

B. D.

Szkudlinski

M. W.

Agostinelli

Dierckx

R. A. J. O.

Signore

Radiolabeling of VEGF165 with 99mTc to evaluate VEGFR expression in tumor angiogenesis

International Journal of Oncology 2017 50

4200142

6 2171 2179

10.3892/ijo.2017.3989

2-s2.0-85019595027

28498441

51.

Gratz

Rennen

H. J.

Boerman

O. C.

Oyen

W. J.

Corstens

F. H.

Rapid imaging of experimental colitis with (99m)Tc-interleukin-8 in rabbits

Journal of Nuclear Medicine 2001 42

4200142

6 917 923

11390557

52.

Sedik

G. A.

Rizq

R. S. A.

Ibrahim

I. T.

Elzanfaly

E. S.

Motaleb

M. A.

Miniaturized chromatographic systems for radiochemical purity evaluation of ¹³¹I-ferulic acid as a new candidate in nuclear medicine applications

Applied Radiation and Isotopes 2021 167, article 109370

4200142

10.1016/j.apradiso.2020.109370

33065399

53.

Matsushima

Yodoi

Tagaya

Oppenheim

J. J.

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The Journal of Immunology 1986 137

4200142

10 3183 3188

10.4049/jimmunol.137.10.3183

2945861

54.

Nieto

J. C.

Cantó

Zamora

Ortiz

M. A.

Juárez

Vidal

Selective loss of chemokine receptor expression on leukocytes after cell isolation

PLoS One 2012 7

4200142

3, article e31297

10.1371/journal.pone.0031297

2-s2.0-84857756869

22403612

55.

Ohtsubo

Marth

J. D.

Glycosylation in cellular mechanisms of health and disease

Cell 2006 126

4200142

5 855 867

10.1016/j.cell.2006.08.019

2-s2.0-33748195979

56.

Matera

M. G.

Calzetta

Rogliani

Cazzola

Monoclonal antibodies for severe asthma: pharmacokinetic profiles

Respiratory Medicine 2019 153

4200142

3 13

10.1016/j.rmed.2019.05.005

2-s2.0-85065615860

31136930

57.

Hemmerle

Hess

Venetz

Neri

Tumor targeting properties of antibody fusion proteins based on different members of the murine tumor necrosis superfamily

Journal of Biotechnology 2014 172

4200142

73 76

10.1016/j.jbiotec.2013.12.010

2-s2.0-84892654619

24384233

58.

Gao

Zhao

Hou

Development and evaluation of a novel radiotracer 125I-rIL-27 to monitor allotransplantation rejection by specifically targeting IL-27Rα 2021

Research Square

10.21203/rs.3.rs-145989/v1