Venous blood was collected from all participants. PBMCs were isolated from the whole blood using Human PBMC Separation Medium (TBD Science, China), centrifuged (Eppendorf, Germany) following the manufacturer’s instruction (TBD Science, China), and resuspended in PBS with 0.5% BSA.
For the isolation of lymphocytes from the lung and colon, the single-cell suspension was obtained according to a previous study.39 (link) Fresh lung tissues were minced and incubated with 1 mg/mL collagenase IV (Worthington, USA) and 50 μg/mL DNase I (Roche, Switzerland) in RPMI-1640 media (Biological Industries, Israel) before being mashed through 70 μm cell strainers. After removing adherent fat tissue and Peyer’s patches, the colon was washed twice with 20 mL HBSS medium containing 5 mM EDTA and 1 mM DTT to remove epithelial cells. Next, 2 mg/mL collagenase type III (Worthington, USA) and 50 μg/mL DNase I (Roche, Switzerland) in RPMI-1640 media were used to digest colon tissues. The digested tissues were filtered through 70 μm cell strainers to obtain cell suspension and enriched with a 40% Percoll gradient after red blood cells were lysed. PBMCs and single-cell suspensions from all tissues were used for subsequent flow cytometry staining.
For the isolation of lymphocytes from the lung and colon, the single-cell suspension was obtained according to a previous study.39 (link) Fresh lung tissues were minced and incubated with 1 mg/mL collagenase IV (Worthington, USA) and 50 μg/mL DNase I (Roche, Switzerland) in RPMI-1640 media (Biological Industries, Israel) before being mashed through 70 μm cell strainers. After removing adherent fat tissue and Peyer’s patches, the colon was washed twice with 20 mL HBSS medium containing 5 mM EDTA and 1 mM DTT to remove epithelial cells. Next, 2 mg/mL collagenase type III (Worthington, USA) and 50 μg/mL DNase I (Roche, Switzerland) in RPMI-1640 media were used to digest colon tissues. The digested tissues were filtered through 70 μm cell strainers to obtain cell suspension and enriched with a 40% Percoll gradient after red blood cells were lysed. PBMCs and single-cell suspensions from all tissues were used for subsequent flow cytometry staining.
