2.1 Chemicals, reagents, and equipment
IL-27Rα mAb was obtained from R&D system (Minnesota, USA). Recombination IL-27 (rIL-27) was purchased from peprotech (New Jersey, USA). Na125I was provided by China Institute of Atomic Energy (Beijing, China). SephadexG-25M PD10 column was purchased from GE Healthcare (Pennsylvania, USA). RPMI 1640 medium was get from Biological Industries (Kibbutz Beit Haemek, Israel). HEPES buffer and Red Blood Cell lysis buffer were got from Solarbio (China, Beijing). SDS loading buffer, antibody dilution buffer and blocking buffer were obtained from Beyotime (Shanghai, China). PBS TBST buffer, H&E staining and immunofluorescence (IF) staining regent were purchased from Servicebio (Wuhan, China). 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).
The radioactive counts were measured by Gamma Counter from Capintec Inc (USA). The phosphor-autoradiography 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).
2.2 Radiochemistry
2.2.1 Preparation of the Radio-probe
The preparation of 125I labeled probe was performed according to the reference[35]. Briefly, 0.05M PB solution (100µL), IL-27Rα mAb (12µg) or rIL-27 (8µg) and Na125I (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. Briefly, 2µL of the radio-probe was added into the filter 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..
2.2.2 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
125I-rIL-27 (0.2μL, 4.08×10-4MBq) 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 n-octanol 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 coefficient (Log Do/w) was calculated.
2.3 Cell Assays
Cell assays were performed using spleen cells isolated from the mouse model on day 10 post transplantation. Briefly, 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 final suspended in RPMI 1640 medium. Cells were cultured in 48-well plates for 2h with each well 1 × 106 cells in 200 µL RPMI 1640 medium, and used for further studies after attachment.
2.3.1 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 125I-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/B0 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 (Ki 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 ℃ to obtain the total activity binding of 125I-rIL-27.In order to test the non-specific binding, cells were pre-treated with 10.46µM non-labeled rIL-27 for 1 hour.
After incubation with 125I-rIL-27, cells were washed with cold PBS buffer twice and radioactive counts were measured in Gamma Counter. The maximum binding capacity (Bmax) and dissociation constant (Kd) were calculated in GraphPad Prism software. The specific binding was the value of total binding minus non-specific binding.
2.4 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.
2.4.1 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. Briefly, 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%.
2.4.2 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 radio-probe in blood (ng/µL) was calculated using 78 mL/kg as blood factor. AUCs (Area Under Curves) of 125I‐rIL-27 in 24h and 125I‐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].
2.4.3 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 specific 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 125I‐rIL-27 (0.37 MBq) and 125I-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 quantified using the 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 (allo-group, syn-group, and blocking group, n = 3 for each group) were sacrificed 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.
2.4.5 H&E (hematoxylin and eosin) staining and Immunofluorescence staining
On day 10 post transplantation, grafts were collected and histological sections were prepared. H&E staining and immunofluorescence (IF) staining were performed following the protocols of staining kit. The image was obtained under the optical microscope. Briefly, 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 autofluorescence 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.
2.4.6 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.
2.5 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/mm2 of 125I-rIL-27 and IL-27Rα expression was calculated by correlation assay. Statistically significant level was set at P<0.05.