Collagenase d

LNs were digested as described above [39 (link), 40 (link)] or with Collagenase D (Worthington) and DNAse I (BD Biosciences) as described [2 (link), 41 ]. The cells from each mouse were plated into a 4-well chamber slide (Lab-tek, Nunc) in DME (Invitrogen) plus 10% fetal calf serum (Hyclone). After 24 h, non-adherent cells were removed and the remaining stromal cells were cultured in the presence of 30 μg/ml 10.1.1 Ab or control Hamster IgG for 5 days. Cells were stained with anti-Prox1 and anti-Ki67 Abs to identify proliferating LECs. Cells in at least 6 random 20x fields from each chamber were counted. Six mice were analyzed for each Ab treatment. Significance was determined using a Wilcoxon Ranked Sum test for paired samples using Prism (GraphPad Software).

To prepare the lung tissues for flow cytometry, they were first cut into small pieces and incubated with 1 mg/ml collagenase D (Worthington) and 0.5 mg/ml DNAse I (Roche) at 37 °C for 30 mins. Collagenase was inactivated with 1 ml sterile DMEM containing 10% FBS; the digested tissues were transferred to a 70-μM nylon cell strainer and disrupted using a syringe plunger to obtain single cell suspensions. Following lung tissue processing, the resulting single cell suspension, BAL, and peripheral blood samples were processed in 1 ml Red Blood Cell Lysing Buffer (Sigma). The cellular populations of the lung tissue, BAL, and peripheral blood from different groups were identified through the following markers: neutrophils were identified with anti-Ly6G (Biolegend) and CD11b (abcom) monoclonal antibody (mAb), macrophages/monocytes were identified with anti-F4/80 (eBiosciencce) and CD11b mAb, T cells were identified with anti-CD4 mAb (BD Pharmingen), eosinophils were identified with anti-siglecF mAb. The stained samples were analyzed with an Attune NxT Acoustic Focusing Cytometer (Thermo Fisher). Dead cells and debris were excluded from analysis via size exclusion.

Tumors were collected in ice-cold RPMI 1640 containing 2% FBS and minced into fine pieces, followed by digestion with 400 U/mL collagenase D (Worthington Biochemical Corporation, #LS004186) and 20 µg/mL DNase I (Sigma, #10104159001) at 37 °C for 40 min with periodic shaking. EDTA (Sigma, #1233508) was then added to the final concentration of 10 mM to stop digestion. Cell suspensions were filtered through 70 µM cell strainers, and TILs were obtained by collecting the cells in the interphase after Ficoll (MP Biomedicals, #091692254). Spleens were collected in ice-cold HBSS containing 2% FBS to prepare single-cell suspensions after lysis of red blood cells and filtering with 70 µM nylon mesh. Both TILs and splenocytes were resuspended in complete Click’s culture medium (Irvine Scientific, #9195-500 mL) for flow cytometric analyses. In some experiments, isolated TILs were cultured with 100 U/mL IL-2, with or without 1 μM Ruxo for 3 days and analyzed for FoxP3 expression and production of IFN-γ/TNF by flow cytometry, as described below.

Intestines were collected and incubated in pre-digestion buffer (1 mM DTT, 10 mM EDTA and 10 mM HEPES in PBS) at 37 °C for 30 min to remove epithelial cells. Remaining tissues were dissociated in digestion buffer containing 1 mg/mL collagenase D (Worthington), 20 μg/mL DNase I (Roche), and 10% FBS (Hyclone) with constant stirring at 37 °C for 30 min. Mononuclear cells were then collected at the interface of a 40–70% Percoll gradient (GE Healthcare).

The isolation of lamina propia (LP) lymphocytes was performed16 (link). The small intestine (SI) and large intestine (LI) were removed, opened longitudinally and cut into pieces. After vigorous shaking in HBSS containing EDTA, the supernatant containing epithelial cells and IELs was discarded. The remaining intestinal pieces were digested with Collagenase D (Worthington) and pelleted. The pellet was resuspended and placed in a Percoll gradient, and after centrifugation, the interface containing the LP lymphocytes was collected for further analysis.

Isolation of LPLs was performed as previously (Li et al., 2017 (link)). The small intestine and large intestine were removed, opened longitudinally, and cut into pieces. After vigorous shaking in HBSS containing EDTA, the supernatants containing epithelial cells and IELs was discarded. The remaining intestinal pieces were digested with collagenase D (Worthington) and pelleted. The pellet was resuspended and placed in a Percoll gradient as described above, and after centrifugation, the interface containing the LPLs was collected and prepared for further analysis.