Development and Evaluation of a Novel Radiotracer 125I-rIL-27 to Monitor Allotransplantation Rejection by Specically Targeting IL-27Rα

Non-invasive monitoring of allograft rejection is benet for the prognosis of patients with organ transplantation. Recently, IL-27/IL-27Rα was proved in close relation with inammatory diseases, and 125 I-anti-IL-27Rα mAb our group developed demonstrated high accumulation in rejecting allograft. However, antibody imaging has limitation in the imaging background due to its large molecule weight. Therefore, we developed a novel radio tracer (Iodine-125 labeled recombinant IL-27) to evaluate the advantage on the targeting and imaging of allograft rejection. In vitro specic binding of 125 I-rIL-27 was determined by saturation and competitive assay. Blood clearance, biodistribution, autoradio-imaging and IL-27Rα expression were studied on day 10 post transplantation (top period of allorejection). Our results indicated that 125 I-rIL-27 could bind with IL-27Rα specically and selectively in vitro. Blood clearance assay demonstrated a fast blood clearance with 13.20 µl/h of 125 I-rIL-27 staying in blood after 24 h. Whole-body phosphor-autoradiography and biodistribution assay indicated that higher specic uptake of 125 I-rIL-27 and clear radio-image in allograft than syngraft at 24 h, while similar result was obtained at 48 h in group of 125 I-anti-IL-27Rα mAb injection. Meanwhile, higher IL-27Rα expression was found in allograft by the western blot study. The activity accumulation of 125 I-rIL-27 was highly correlated with IL-27Rα expression on allograft. In conclusion, 125 I-rIL-27 could be a promising probe to acutely monitor the allograft rejection with high specic binding towards IL-27Rα on allograft and low imaging background.


Introduction
Solid organ allotransplantation has been the most effective therapeutic strategy for the patient with endstage organ failure [1]. However, the occurrence of acute rejection is strongly related with the allograft loss and the poor prognosis [2]. Therefore, early detection of acute rejection with non-invasive method could greatly bene t for the prognosis after organ transplantation [3].
Acute allograft rejection is a severe pro-in ammation participated by T cells and macrophage [13] and IL-27 has been proved in close relation with allograft rejection [7] [14]. IL-27Rα (IL-27 receptor α) expression

on T cells exacerbated GVHD by enhancing effector function of Th1 cells (T helper 1 cells) and inhibiting
Th2 and Treg cell subsets [7], while IL-27Rα was found apparently up-regulated on alloreactive splenetic CD4 + T cells, T cell and macrophage when acute rejection happened [15][16] [17]. In our previous study with allografted mice model, we found a great amount of IL-27Rα positive T cells and macrophage in ltrated in rejecting allograft and iodine-125 labeled anti-IL-27Rα mAb could obviously accumulate in allograft non-invasively when rejection occurred [18].
Target tissue could be diagnosed precisely and non-invasively by nuclear molecular imaging with a speci c probe, which is much more favorable than histopathological biopsies and traditional imaging examination [19] [20][21] [22] [23]. Although histopathological biopsies was the "gold-standard" of acute graft rejection, it still was an invasive examine and may induce complication including pain, bleeding and death [24] [25]. The non-invasive examines such like MRI and ultrasound re ected the decrease graft function and were limited in targeting allograft [21] [23]. Targeted molecular imaging has advantages in tracking speci c cells and monitoring the function of the target organ with the probes which have the detection signals [26][27] [28]. Among them, radionuclide imaging is a non-invasive method by which the disease could be diagnosed effectively and timely, and the therapeutic effect could be monitored with the help of radio-probe. Radionuclide imaging with radiolabeled macromolecular such as protein, antibody and so on usually had the disadvantages in the long time to reach the target tissue and the high background, resulting in the poor imaging quality. However, small molecular could accumulate in the target tissue quickly, and thus make a better imaging. Therefore, radio-probe with small size is the much more promising radio tracer in radionuclide imaging compared with full length antibody. Radio-labeled cytokine has been applied to track targeted immune cells due to the high-contrast imaging, fast clearance, low background and the weak in ammation response. [29][30] [31]. Hartimath et al developed [18F]FB-IL-2 to monitor cancer therapy-induced activated T lymphocyte in ltration in tumor [32]. Di  [34]. Accordingly, imaging with radiolabeled cytokine had advantage in speci c recognition of target tissue with low background, and could be a promising strategy for allorejection detection.
In this study, we prepared a novel radio-probe ( 125 I labeled recombinant IL-27, 125 I-rIL-27) to speci cally target IL-27Rα, and evaluated its possibility for the potential application in acute allograft rejection monitoring. GAPDH solution was obtained from Bioss (Beijing, China) and Bioworld (Illinois, USA). HRP-labeled Goat Anti-Rat IgG solution and HRP-labeled Goat Anti-Rabbit IgG solution was get from EpiZyme (Shanghai, China). ECL substrate was purchased from Merck Millipore (Darmstadt, Germany).

Materials And
The radioactive counts were measured by Gamma Counter from Capintec Inc (USA). The phosphorautoradiography imaging was captured and analyzed by Cyclone Plus Scanner (PerkinElmer, Life Sciences, USA). The membrane was scanned by Tanon 5200 imaging system scanner (Tanno, Shanghai, Beijing).

Preparation of the Radio-probe
The preparation of 125 I labeled probe was performed according to the reference [35]. Brie y, 0.05M PB solution (100µL), IL-27Rα mAb (12µg) or rIL-27 (8µg) and Na 125 I (11.9 MBq) was mix in the tube with Iodogen. Then the mixture was added into the SephadexG-25M PD10 column, following by the elution with 0.01M PB solution. The eluent was collected in tube (0.5ml for each tube) and the radioactive count of 10µL eluent from each tube was measured by Gamma Counter.
The radiochemical purity were detected following the protocol. Brie y, 2µL of the radio-probe was added into the lter paper (2 cm to the bottom). Then bottom of the paper was immersed in the solution of 0.9% saline and methanol (1:2, v/v). After 40min, the paper was cut into slice (1cm) and radioactive count was measured by Gamma Counter..

In vitro stability study
Radio-probe (12.5µL) was dissolved in saline (100µL) or mouse serum (100µL), and the mixture was kept at 37℃ for a period of time. At 1, 12, 24h, 2µL of the sample was taken out and analyzed so as to observe the change of radiochemical purity 2.2.3 Determination of lipophilicity 125 I-rIL-27 (0.2μL, 4.08×10 -4 MBq) was diluted in 1M HEPES buffer (500μL) and mixed with n-octanol (500μL) for 30min, following by the centrifugation for 10min with 14000 × g. Subsequently, Aliquots of noctanol and water phases (400μL) was taken out and then centrifuged again. Finally, the radioactive count of each phase (100μL) was measured by Gamma Counter and the octanol/water partition coe cient (Log D o/w ) was calculated.

Cell Assays
Cell assays were performed using spleen cells isolated from the mouse model on day 10 post transplantation. Brie y, spleen of mouse model was isolated and pressed on mesh 200. Then, cells were treated with Red Blood Cell lysis buffer, washed with PBS and nal suspended in RPMI 1640 medium.
Cells were cultured in 48-well plates for 2h with each well 1 × 10 6 cells in 200 µL RPMI 1640 medium, and used for further studies after attachment.

Competition study
For competition binding assay, non-labeled anti-IL-27Rα mAb (0 to 71.4 µM) was incubated with alloreactive spleen cells for 1h before 147.09 nM 125 I-rIL-27 was added. Wash the cells with cold PBS buffer twice and discard the supernatant. The activity bound in the cells was measured by Gamma Counter. B/B 0 was described as the ratio of radioactive counts with non-labeled anti-IL-27Rα mAb to the radioactive counts without non-labeled anti-IL-27Rα mAb. The inhibition constant (K i value) was calculated in GraphPad Prism software.

Saturation study
125 I-rIL-27 (3.68 to 147.09 nM) was incubated with spleen cells for 2 h at 37 ℃ to obtain the total activity binding of 125 I-rIL-27.In order to test the non-speci c binding, cells were pre-treated with 10.46µM nonlabeled rIL-27 for 1 hour.
After incubation with 125 I-rIL-27, cells were washed with cold PBS buffer twice and radioactive counts were measured in Gamma Counter. The maximum binding capacity (B max ) and dissociation constant (K d ) were calculated in GraphPad Prism software. The speci c binding was the value of total binding minus non-speci c binding.

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

Animal models
To establish the skin transplantation model, C57BL/6 mice and BALB/c mice were employed as the skin graft donor of allogeneic and syngeneic transplantation, respectively. BALB/c mice were the recipients. Brie y, surgery was performed under anesthesia with 0.6% pentobarbital sodium (0.1mL/10g body weight) in sterility condition. The mucous membrane and blood vessel of graft was removed and then cut the graft into circle with 1cm in diameter. Then, remove the skin of recipients in right shoulder and transfer the graft to recipients. Finally, petrolatum gauze was put on the graft and covered with bandage. Acute rejection occurred on day 7 post transplantation when removing the bandage with escharotics area over 50%.

Blood clearance assay
At 1h, 2h, 6h, 12h and 24h post injection of radio-probe, mice were anaesthetized with 0.6% pentobarbital sodium solution. Then 5µL blood was collected from the tail vein. The activity of radio-probe stayed in the blood was counted by Gamma Counter. Each mouse was weighted and the concentration of radioprobe in blood (ng/µL) was calculated using 78 mL/kg as blood factor. AUCs (Area Under Curves) of 125 I-rIL-27 in 24h and 125 I-anti-IL-27Rα mAb in 48h were obtained using GraphPad Prism software. Blood clearance (CL, µl/h) was calculated as dose/AUC with the study referred [36].

Dynamic phosphor-autoradiography
Mice were divided into allo-group, syn-group and blocking group (n=5 for each group) according allogeneic, syngeneic transplantation and allogeneic transplantation model with speci c antibody blocking. After fed with 3% NaI solution for 24h, 60µg non-labeled IL-27Rα mAb was injected to the blocking group. One hour later, all mice were injected with 125 I-rIL-27 (0.37 MBq) and 125 I-anti-IL-27Rα mAb (0.37 MBq) on day 9 post transplantation, respectively. Mice were anesthetized and scanned by Cyclone Plus Scanner. Regions of interest (ROIs) were quanti ed using the OptiQuant Image Analysis Software and presented as Digital Light Units per square millimeter (DLU/mm 2 ).

Ex vivo Biodistribution
Three groups of mice (allo-group, syn-group, and blocking group, n = 3 for each group) were sacri ced with at 24 h after intravenous injection of radio-probe (0.08MBq in 200µL of 0.01M 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 Gamma counter and the uptake of radio-probe was expressed as the percentage of injected dose per gram (%ID/g). T/NT (Target/non-Target) 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.

H&E (hematoxylin and eosin) staining and Immuno uorescence staining
On day 10 post transplantation, grafts were collected and histological sections were prepared. H&E staining and immuno uorescence (IF) staining were performed following the protocols of staining kit. The image was obtained under the optical microscope. Brie y, in H&E staining, the sections were covered haematoxylin for 5min. After 1% acid ethanol regent for 5 second, sections were covered with blue promoting solution for 5 seconds. Next, the sections were covered with eosin solution for 10 minutes. Between every step, the distilled water was used to wash out the excess buffer. In IF staining, the sections were treated with EDTA antigen repair buffer (pH 9.0) and blocked with BSA for 30min. Then, anti-IL-27Rα Ab was diluted in PBS (1:200) and added on the section in 4℃, overnight. The sections were washed with PBS and covered with second antibody for 1 hour. Later, wash the sections with PBS and add the FITC regent (Green) on the sections. Next, the sections were washed with TBST and covered with tissue auto uorescence quencher regent for 5min. Then, excess regent was washed with distilled water for 10min. The sections were discarded excess liquid and incubated with DAPI regent (Blue) for 10min at room temperature. Finally, the sections were washed with PBS and then enclosed with antifade mounting medium.

Western Blot
After transplantation for 10 d, grafts were separated, lysed and reacted with SDS loading buffer. Electrophoresis was performed and protein was transferred to PVDF membrane. The target membrane was then treated with blocking buffer and then covered with anti-IL-27Rα mAb solution and GAPDH solution overnight. Later, membrane was washed with TBST buffer and covered with HRP-labeled Goat Anti-Rat IgG solution and HRP-labeled Goat Anti-Rabbit IgG solution, respectively. Finally, membrane was washed with TBST buffer, following by ECL substrate covering. Band was scanned using Tanon 5200 imaging system scanner and analyzed with Image J software.

Statistical analysis
All data were quoted as mean ± standard deviation (mean ± SD) and each data point arised from 3 independent experiments. Comparisons between two groups were analyzed using the unpaired student's t-test. Correlation between DLU/mm 2 of 125 I-rIL-27 and IL-27Rα expression was calculated by correlation assay. Statistically signi cant level was set at P<0.05.

Saturation
Typical saturation graphs obtained after incubation of 1 × 10 6 cells with 125 I-rIL-27 was shown in Figure   1A-B. B max values of 125 I-rIL-27 on allo-reactive and syn-reactive splenocytes were 2545 cpm/10 6 cells and 1607 cpm/10 6 cells, respectively. Moreover, K d values were found 48.59nM and 49.04nM for allo-and syn-reactive splenocytes, respectively. Figure 1C showed the binding of 125 I-rIL-27 decreased as anti-IL-27Rα mAb increased. Using the K d value of 125 I-rIL-27 from saturation assay, the determination the K i value was 769.9nM by Cheng-Prusoff equation.

Blood clearance assay.
To understand how fast 125 I-rIL-27 cleared in vivo, blood clearance assay was performed. The blood clearance was represented as Clearance Value ( 66401.60 ± 29698.30) (p<0.01). In vivo speci city of 125 I-rIL-27 was con rmed by blocking studies using excess unlabeled anti-IL-27Rα mAb (DLU/mm 2 : 68252.033 ± 38373.75). Ex vivo autoradiography showed apparently high activity accumulation in allograft. Similar result of 125 I-anti-IL-27Rα mAb was observed at 48h, and the uptake of 125 I-anti-IL-27Rα mAb in allogeneic graft was also higher than that in syngeneic graft ( Figure 3B). However, imaging using 125 I-rIL-27 in allogeneic graft exhibited lower background in comparison with that using 125 I-anti-IL-27Rα mAb. These indicated that 125 I-rIL-27 could target allograft speci cally and yield a better imaging with high contrast and low background.

Biodistribution assay
In order to have a rst insight into the potential relevance of 125 I-rIL-27 for transplantation imaging, biodistribution assay was performed using skin transplantation mice.The biodistribution data of 125 I-rIL-27 was shown in Figure 4A. Higher uptake was observed in allogeneic skin graft compared with that in syngeneic group ( Figure 4B). Activity uptake of 125 I-rIL-27 in allograft was higher than that in syngraft (%ID/g: 5.648 ± 1.735 vs 1.751 ± 0.967, p<0.01). T/NT ratio and T/B ratio signi cantly increased in allogroup compared with syn-group in Figure 4B-C.

IL-27Rα expression in rejecting allograft.
To study the correlation between activity accumulation of 125 I-rIL-27 and IL-27Rα expression in rejecting allograft, IF staining was performed on day 10 post transplantation, so as to determine the IL-27Rα expression.
The HE staining in Figure 5A con rmed that severe rejection response was occurred in allogeneic graft, while mild in ammation in syngeneic graft. IL-27Rα expression was obviously higher in allograft ( Figure   5B). The activity accumulation (DLU/mm 2 ) in graft had a positive correlation with IL-27Rα expression ( Figure 5C). Fluorescence imaging also con rmed the higher IL-27Rα expression on the surface of in ltrated cells in rejecting allograft ( Figure 5D). All these suggested that 125 I-rIL-27 could speci cally bind the IL-27Rαin the allograft, monitoring the acute rejection.

Discussion
Early acute allorejection is usually more responsive to the therapy of allograft transplantation, and thus detection of acute rejection detection timely could bene t for the prognosis [37]. To data, molecular imaging with speci c radio-probes was a promising method responsible for the detection of allograft rejection [22]. Recently, IL-27, a pleiotropic cytokine with pro-in ammation properties, was reported with enhanced antivirus and antitumor activities, and it participated in the rejection response [38] [39][40] [41].
IL-27 could promote in ltration of CD4 + T cell and CD8 + T cell in tumor, up-regulate IFN-γ, Granzyme B and Perforin production, resulting in improved antitumor effect of T cell [10]. Moreover, IL-27 could also boost NK cell proliferation and cytotoxic activity synergistically with IL-15/IL-18 [42]. All these indicated that IL-27/IL-27R was a promising target in pro-in ammation immune response. IL-27Rα, the subunit of IL-27 receptor which is also expressed on the T cell and macrophage, had highest expression on the top acute rejection period in the allograft [43][44] [45]. In our previous study, 125 I-anti-IL-27Rα mAb has been found with high speci city towards IL-27Rα [18]. However, it had limitations in non-speci c binding to Fc recognition, slow metabolism and clearance, as compared with small-sized antibody fragment or ligand [46] [30]. Therefore, small-sized radio-probe could provide a better imaging with low background.
Cytokine was a small-sized ligand of the cytokine receptor which is expressed on surface of effector cells [47]. Many radio-probes of cytokine have already been applied in targeting imaging [48] [49][50] [51]. Radiolabelled IL-2 probes were used in clinics for in targeted detection of the lymphocytic in ltration in transplantation and atherosclerotic plaque [48] [49]. Glaudemans et al found symptomatic plaques with high CD3 + cells in ltration had signi cant uptake of 99m Tc-HYNIC-IL-2 and the lung of rejection patient had increased 99m Tc-HYNIC-IL-2 uptake. However, tn their research, no side effect was found when administration of 99m Tc-HYNIC-IL-2. We also developed the targeted radio-probe 125 I-rIL-27 and also found no side effect in the mouse model.
In vitro experiment showed our 125 I-rIL-27 had a speci c binding to the IL-27Rα on the spleen cells.
However, binding ability and a nity of 125 I-rIL-27 was lower than that of 125 I-anti-IL-27Rα mAb. This might be due to that 125 I-anti-IL-27Rα mAb have non-speci c Fc fragment binding. Matsushima et al developed 125 I-labeled IL 1β in a human large granular lymphocyte cell line (YT cells) and this radio-probe showed higher a nity of 0.1 nM (K d value) compared with our probe [52]. It maybe was due to the different receptor expression of the cells. Besides, the isolation process of spleen cells may also result in some loss of receptors [53].
In the imaging of [ 124 I]I-F8-IL10, it was suggested that targeted area had highest uptake and target-tobackground ratios at 24 h post injection of the radio-probe [34]. Therefore, we carried out the biodistribution, blood clearance of 125 I-rIL-27 within 24 h after radio-probe injection. In the blood clearance assay, 125 I-rIL-27 showed faster blood clearance than 125 I-anti-IL-27Rα, which was might due to the different cytokine and antibody glycosylation level, in uencing the receptor recognition and blood clearance [54]. Blood clearance assay showed shorter retention of 125 I-anti-IL-27Rα in blood compared with monoclonal antibody, which might be due to Fc recognition [55]. Whole-body phosphorautoradiography imaging demonstrated that the allograft had more activity accumulation than syngeneic graft, and this accumulation could be blocked by the excess of anti-IL-27Rα mAb. Lower background was also observed at 24 h in 125 I-rIL-27 group compared with 125 I-anti-IL-27Rα. Tumor necrosis factor superfamily (TNFSF) contains CD40L, FasL, TRAIL, LiGHT, VEGI, lymphotoxin alpha, lymphotoxin beta and lymphotoxin alpha1/beta2, which could be fused with F8 antibody for tumor targeting. in the biodistribution suggested that the %ID/g of 125 I-rIL-27 in allograft was similar to that of F8-TRAILtrunc, lower than that of F8-CD40L and higher than that of other TNFSF in tumor [56]. The reason may be the different receptor expression and a nity of different cytokine to the receptors. The %ID/g of 125 I-rIL-27(47.8 KDa) in blood was higher than that of F8-TNFSF, F8-IL-10 (18.6 KDa), 99m Tc-VEGF 165 (16 KDa), which was probably due to the lower molecular weight of other cytokine [50]. However, the activity of 125 I-rIL-27 in blood was much lower compared with that of 125 I-anti-IL-27Rα (155KDa), perhaps caused by the non-speci c Fc binding of 125 I-anti-IL-27Rα. The uptake of 125 I-rIL-27 in lung was higher than that in other organs except graft and blood. This might result from the enrichment of IL-27Rα-overexpressed immune cells in lung. Meanwhile, blood pollution should also be considered. The activity accumulation in kidney was found higher than liver, which may be because of the hydrophilic character of 125 I-rIL-27. Moreover, these indicated that 125 I-rIL-27 was a promising radiotracer which could speci cally target IL-27Rα for the imaging of acute rejecting allograft with faster blood clearance and low background, compared with 125 Ianti-IL-27Rα mAb.

Conclusion
In this study, the acute allograft rejection could be detected by targeting IL-27Rα in allograft speci cally with 125 I-rIL-27. The rejecting allograft had higher speci c 125 I-rIL-27 uptake than non-rejecting syngeneic graft and the activity accumulation was in close correlation with IL-27Rα expression of the graft. More importantly, low background and fast clearance was obtained for 125 I-rIL-27 compared with 125 I-anti-IL-27Rα mAb. Imaging with this small-sized radio-probe might be a promising strategy for non-invasive  Blood clearance assay. Time-Radio probe concentration in blood curves after administration of 125I-IL-27 and 125I-anti-IL-27Rα mAb in allogeneic and syngeneic transplantation mice. The inset table was the AUC from1-24h of 125I-rIL-27 and 125I-anti-IL-27Rα mAb and blood clearance assay. Dynamic whole-body phosphor-autoradiography imaging assay. Allogeneic and syngeneic skin transplantation mice were established and represented as Allo and Syn group. Mice was injected with 125I-rIL-27 and 125I-anti-IL-27Rα mAb on day 9 post transplantation and scanned at different time. Graft and opposite control skin were isolated on day 10 (24h after radio probe injection). ARG means the autoradiography. A. Imaging at 1, 6, 12, 24h post 125I-rIL-27 injection and DLU/mm2 assay. The circle indicated the position of the graft. B. Imaging at 24, 48h post 125I-anti-IL-27Rα mAb injection. **p < 0.01 was used in Allo vs Syn group. #p < 0.01 was used in Allo vs Blocking group. Bio-distribution study on day 10 post transplantation. The organ was separated from allografted and syngrafted mouse model on day 10 and biodistribution assay at 24h post radio probe injection. T/NT and T/B assay was calculated. A. Biodistribution assay of 125I-rIL-27 injection. B-C. T/NT ratio (B) and T/B ratio (C) by 125I-rIL-27 injection. **p < 0.01 was used in Allo vs Syn group. #p < 0.05 was used in Allo vs Blocking group.