At our center, we have maintained the Ewha IBD cohort since 2019, prospectively collecting patient data, colon biopsy specimen, blood, and stool sample. Between December 2020 and December 2021, we enrolled 14 patients with UC and 18 normal controls. After obtaining informed consent from patients under the approved human research protocol of the Institutional Review Board of Ewha Womans University Seoul Hospital (IRB number, SEUMC 2020-12-017), endoscopic colonic biopsies were performed at each five segments of the colon in patients with IBD and at 2–3 segments of the colon in normal controls. Additionally, we analyzed seven colon specimens collected from the Ewha IBD cohort (SEUMC 2019-04-019). Detailed demographic and clinical data were retrieved from medical record. Throughout the study, the authors did not have access to information that could identify individual participants.

The colon biopsy specimens were immediately placed into RNA later (Invitrogen, Waltham, MA, USA) and stored at -80°C for analysis. One milliliter of TRIzol Reagent (Ambion, Life Technologies, Carlsbad, CA, USA) was added to the colon specimen. After mincing, 200 μL of chloroform (Sigma, St. Louis, MO, USA) was added, followed by vortexing. The mixture was then centrifuged, and the supernatants were transferred to a new tube. An equal volume of isopropanol (Sigma) was added; after mixing by inversion, the sample was centrifuged again at 13,000 ×g (4°C) for 10 minutes. Subsequently, nuclease-free water was added, and the RNA concentration was measured using Nabi (NanoDrop) (MicroDigital, Seongnam, Korea), followed by the mixture of 2 μg of RNA and 0.5 μg of oligo dT primer, where the sample was left at 70°C for 10 minutes thereafter. Next, 200 units of Molony Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Promega, Fitchburg, WI, USA), 25 units of rRNasin Ribonuclease inhibitor (Promega), 5 × RT buffer, and 2 mM of dNTP were added, and the final volume was set to 25 μL by adding an appropriate amount of nuclease-free water. A Quantstudio 3 real-time polymerase chain reaction (PCR) system (Applied Biosystems, Waltham, MA, USA) was used for analysis using 0.1 μg of the synthesized cDNA as a template for the 2X Power SYBR Green PCR Master mix (Applied Biosystems) and each primer set for PD-1 and PDL1. Primers used in this study were obtained from Macrogen (Seoul, Korea) (Table 1). Each PCR was performed after 10 minutes of pre-denaturation at 95°C, 15 seconds at 95°C, and 1 minute at 60°C; this was repeated 40 times. After the PCR was completed, a melting curve was drawn to check the accuracy of gene amplification. For the internal compensation of gene expression level, the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also used, and relative gene expression levels were presented as 2-^ΔΔCt values.

Participants aged < 18 years were recruited for this study. Written informed consents were obtained from the legal guardians of the parents and/or patients for tonsil tissue studies. This study was approved by the Institutional Review Board of Ewha Womans University Medical Center (ECT 11-53-02). All experiments were performed following institutional ethical guidelines and the Declaration of Helsinki. All the tonsil tissues used in this study were obtained from a single donor. TMSCs were isolated and cultured as described in our previous studies [14]. Tonsil tissues were obtained during tonsillectomy from patients under 10 years of age. Minced and digested tonsillar tissues were washed using Dulbecco’s modified Eagle’s medium-high glucose (DMEM-HG; Welgene, Daegu, Korea) supplemented with 20% fetal bovine serum (FBS; Invitrogen) and washed again using DMEM-HG supplemented with 10% FBS. Mononuclear cells were isolated from the prepared tonsil tissues and cultured in cell culture plates. The adherent cells were further cultured for 2 weeks and passaged. The passaged cells (hereafter referred to as TMSCs) were stored in liquid nitrogen for future experiments. The MSC characteristics of TMSCs, according to the minimal criteria for defining multipotent mesenchymal stromal cells [16], were confirmed in our previous study [14] and other studies [17].

PD-1 and PD-L1 expression was measured in TMSCs. PD-1 was measured exclusively on the cell surface, while PD-L1 was secreted in soluble form in the conditioned medium and on the cell surface. PD-1 and PD-L1 expression on the surface of TMSC was also measured. The mRNA expression level of PD-1 and PD-L1 on the surface of TMSC were confirmed real-time PCR in the same procedure as previously described, and the soluble PD-L1 secreted from TMSC was confirmed by immunoassay. To measure PD-L1 (soluble form) secreted from TMSCs, TMSCs were sub-cultured and supernatants were collected and stored at -20°C or below before measurement. Extracted TMSC supernatants was used to measure PD-L1 levels using Quantikine Human PDL1 immunoassays (R&D Systems, Minneapolis, MN, USA). The diluted sample (supernatant, lysate protein) and standard solution were incubated in each 96-well plate coated with PD-L1 antibody for 2 hours at room temperature on a shaker at 500 rpm. After the reaction in each well, the remaining solution was washed with Wash Buffer and aspirated. A 200 μL PD-L1 conjugate was added to each well and incubated for 2 hours at room temperature. After repeating the washing process, 200 μL of substrate solution was added and incubated for 30 minutes at room temperature with light blocking. After the reaction was complete, the color was developed with 50 μL of stop solution, and the optical density was measured within 30 min using a microplate reader set to 450 nm.

The animal models used for the experiment were sevenweek-old C57BL/6 male mice (Orient Bio Co., Ltd., Sungnam, Korea) with an average weight of 20–22 g. The mice were acclimatized for seven days in a standardized environment at the facility of the Ewha Womans University Medical Research Institute before the experiment. Day and night conditions were provided at 12 hours intervals, and the temperature (23 ± 2°C) and humidity (45–55%) were set to appropriate levels. Experiments and procedures were approved and all experiments were performed following experimental research protocol approved by the Ethics Committee for Animal Research of Ewha Womans University (EUM19-0464, ESM18-0415). This study was performed following the standards described in ARRIVE guidelines. Acute colitis in mice was induced by oral administration of 2.5% DSS (MP Biochemical, Irvine, CA, USA) for seven days. Chronic colitis in mice was induced by oral administration of 1.5% DSS for five days, followed by an additional five days of tap water feeding; overall three such cycles (total 30 days) were performed. On the 7th day of the experiment after colitis induction in the acute colitis model and the 31st day of the experiment after colitis induction in the chronic colitis model, the mice were sacrificed by carbon dioxide inhalation, and the colon was dissected from the cecum to the anus for colon length measurement. Colon specimens of mice with colitis were also treated in the same manner as humans, and the levels of PD-1 and PD-L1 were measured using the method described above (Table 1).

In the acute colitis model, mice were randomly assigned to four groups: 1) normal control (n = 5), 2) TMSC control (n = 5), 3) DSS + phosphate-buffered saline (PBS) group (n = 5), and 4) TMSC-treated group (n = 5). TMSC was administered on day 3 following colitis induction in the TMSC-treated group, while in the TMSC control group, TMSC was administered on day 3 of the experiment without colitis induction. In the chronic colitis model, mice were randomly assigned to four groups: 1) normal control (n = 5), 2) TMSC control (n = 5), 3) DSS+PBS group (n = 5), and 4) TMSC-treated group (n = 10). In the TMSC-treated group, TMSC was administered on days 6, 9, 12, and 16 following colitis induction, while in the TMSC control group, TMSC was administered on days 6, 9, 12, and 16 of the experiment without colitis induction. For each group, weight change, stool consistency, and occult or grossly observed blood from stool or anus were checked daily and the DAI was assessed. The expression of PD-1, PD-L1, proinflammatory cytokine (IL-1β, IL-6, IL-17, and tumor necrosis factor-α [TNFα]) in colonic tissue were also measured as the method described previously (Table 1).