Lymphocyte separation solution

Fresh peripheral blood (5 mL) was collected from CLL patients and healthy controls. Peripheral blood mononuclear cell (PBMC) was isolated using a lymphocyte separation solution (TBD Science). Peripheral blood lymphocytes were lysed in radioimmunoprecipitation assay buffer with protease; phosphatase inhibitors, as well as phenylmethanesulfonylfluoride and the supernatant, were collected after centrifugation. The protein concentration was determined by the bicinchoninic acid method. Sodium dodecyl sulfate (SDS) loading buffer was added to denature the proteins (all the reagents for protein extraction were purchased from Solarbio Company). Proteins were isolated by 12% SDS‐polyacrylamide gel electrophoresis and transferred to the polyvinylidene fluoride (PVDF) membrane (300 mA, 90 min). PVDF membrane was blocked for 1 h by 5% skimmed milk and then incubated with a polyclonal rabbit anti‐Tim‐3 (Proteintech Group) overnight at 4°C. Goat anti‐rabbit (Solarbio) horseradish peroxidase‐conjugated secondary antibody was incubated at room temperature for 1 h. β‐Actin (Cell Signaling Technology) in equal quantities was used as an internal reference. The enhanced chemiluminescence (Biogot Technology) method evaluated the protein expression level.

SF samples were pretreated in hyaluronidase at 37°C for 30 min. The lymphocyte suspension was isolated using lymphocyte separation solution (TBDscience, China). After centrifugation, 5 × 10⁵ cells were resuspended in Dulbecco's modified Eagle's medium (DMEM, HyClone, USA) containing 10% fetal calf serum (FCS, Biological Industries, Israel), inoculated into 24-well plates, and incubated with 10 μl of XBJ (CHASE SUN, China) for 48 h. The total cell suspension was collected, anti-human-CD4-FITC antibody (BioLegend, USA) and anti-human-CD25-PE antibody (BioLegend) were added, and the mixture was incubated at 4°C in the dark for 30 min. The proportion of Treg cells in the SF before and after XBJ stimulation was measured using a flow cytometer (ACEA, USA). Next, the cell suspension was centrifuged at 1000 ×g and 4°C for 15 min. The supernatant was used to measure changes in the secretion levels of IL-4 and IFN-γ using the human Th1/Th2 cytokine detection reagent kit (Cell-Genebio, China). The mean fluorescence intensity (MFI) was detected using a flow cytometer. Th1 cells are characterized by IFN-γ secretion, while Th2 cells mainly secrete IL-4. Based on the obtained MFI values of IFN-γ and IL-4, the Th1/Th2 ratio was calculated.

PBMCs were isolated from six patients with aGVHD using a lymphocyte separation solution (TBD Science). Subsequently, the Negative Selection CD4⁺ T‐cell sorting kit (STEMCELL Technologies) was used to enrich CD4⁺ T cells. The sorted CD4⁺T cells were re‐suspended to 5 × 10⁷/mL and incubated with Tim‐3‐PE antibody (BD, Clone:7D3, 2 µg/mL) in the dark for 15 min. Then, the incubation with 100 μL of EasySep® PE Selection Cocktail (STEMCELL Technologies) and 50 μL of EasySep® Magnetic Nanoparticles (STEMCELL Technologies) was performed. Finally, Tim‐3⁺CD4⁺ T cells were evaluated by flow cytometry, and their purity was over 90%.