Clone ox 35

The tissue of transplanted area was minced with a scissors, after which the tissue fragments were incubated for 1 h at 37 ºC in Dulbecco's Modified Eagle Medium (DMEM: Invitrogen) in the presence of 0.2% collagenase (Wako Chemicals, Cell dissociation grade), and 25 μg/ml deoxyribonuclease (Sigma-Aldrich). The suspension was filtered through a cell strainer (Falcon 2350, 70 μm) to remove debris and tissue fragments, after which the cells were pelleted by centrifugation at 300 g for 5 min at room temperature. Next, the cells were resuspended in calcium- and magnesium-free Hank's Balanced Salt Solution (HBSS) (Fujifilm) supplemented with 2% FBS, 10 mM HEPES and 1% penicillin/streptomycin (P/S). The cells were stained with immune cell markers as described below; PE conjugated mouse anti-rat CD3 (BD Pharmingen, Clone G4.18, Catalog No:554833), FITC conjugated mouse anti-rat CD4 (BD Pharmingen, Clone OX-35, Catalog No:554837), Catalog No:561588), PE conjugated mouse anti-rat CD11b/c (BD Pharmingen, Clone OX-42, Catalog No:554862), and Alexa Fluor® 647 conjugated mouse anti-rat CD163 (Bio-Rad Laboratories, Inc., Clone ED2, Catalog No: MCA342A647). Flow-cytometric analysis was performed on FACSMelody (BD Biosciences), and the data were analyzed using FlowJo software (BD Biosciences).

For the performance of the phenotypic analyses, the cultured splenic T-cells were incubated with specific antibodies targeting T-cell surface markers. This included fluorescein (FITC)-conjugated anti-CD3 (1:200, clone G4.18, #554832, BD Bioscience, Franklin Lakes, NJ, USA) and phycoerythrin (PE)-conjugated anti-CD4 (1:200, clone OX-35, #554838, BD Bioscience). For the identification of Treg and Th17 cells, the cells were stained with peridinin-chlorophyll-protein (PerCP)–Cyanine5.5 conjugated anti-FoxP3 (1:100, clone FJK-16s, #45-5773-82, Thermo Fisher Scientific) for Treg cells or allophycocyanine (APC)-conjugated anti-retinoic-acid-receptor-related orphan nuclear receptor gamma (RORγt) (1:1000, clone 4G419, NBP2-24449, Novus Biologicals, Centennial, CO, USA) for Th17 cells, employing the FoxP3/Transcription Factor Staining Buffer Set (00-5523-00, eBioscience) [17 (link)]. The data were acquired using a 4-color flow cytometer (FACS Calibur; BD Bioscience, Franklin Lakes, NJ, USA) and were subsequently analyzed using FlowJo v10 software (BD Biosciences).

Single-cell suspensions of spleen were generated as has been described previously (Taylor et al., 2018 (link)). Blood was obtained from SS and SR rats, which had taken 20% fructose or tap water for 4 weeks. PBMCs were separated by Ficoll-Paque PLUS gradient centrifugation (GE Healthcare, Chicago, IL, USA). Briefly, phenotypic and intracellular analyses were performed by incubating cells with antibodies for T cell surface markers – fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat CD3 (1:100; clone G4.18, #554832; BD Biosciences, Franklin Lakes, NJ, USA) and PE-conjugated mouse anti-rat CD4 (1:100; clone OX-35, #554838; BD Biosciences) – for 30 min on ice in the dark. After washing, the cells were fixed and permeabilized using fix/perm concentrate (BD Biosciences) before incubation with antibodies for intracellular staining of APC rat anti-rat IL-17A (1:100; clone eBio17B7, #17-7177-81; Thermo Fisher Scientific) to identify Th17 cells and PerCP-cyanine5.5 rat anti-rat FOXP3 (1:100; clone FJK-16s, #14-5773-82; Thermo Fisher Scientific) to identify Treg cells. Cells were then washed and run through a four-color flow cytometer (FACSCalibur; BD Biosciences) and data were collected using CellQuest or FlowJo v10.0 software. An example of the flow cytometry gating strategy used is shown in Fig. S3.