Hyaluronidase

Cells from intestinal lamina propria, Peyer’s patches, and mesenteric lymph nodes were isolated as described previously (Rios et al., 2016 (link)). Briefly, Peyer’s patches and mesenteric lymph nodes were mechanically dissociated in ice-cold PBS. The resulting cell suspensions were passed through a 70-um mesh cell strainer. To prepare the intestinal lamina propria cells, associated fat and Peyer’s patches were removed, the intestinal tissue was washed in ice-cold PBS to remove the luminal contents and cut open longitudinally, and the tissue was cut into four equal-sized pieces. Epithelial cells were removed by shaking the tissues in PBS with 1 mM EDTA, 1 mM dithiothreitol, and 10% fetal calf serum for two rounds of 20 min at 37°C. Then, the pieces were washed three times with PBS to remove the EDTA, minced exactly 40 times in a microfuge tube, and incubated in 20 ml of RPMI-1640 supplemented with 1.5 mg ml^-1 Collagenase II (Biosharp), 2.5 mg ml^-1 hyaluronidase (Biosharp), and 0.25 mg ml^-1 DNase I (Solarbio) for 45 min at 37°C with constant shaking. Cell suspension was then extracted by passing the tissue and supernatant over a 70-µm mesh cell strainer. The cell suspension was then centrifuged, and the resuspended pellet was further purified from the interface of a 45/72% Percoll density gradient.

Cells from colonic lamina propria (LP), mesenteric lymph nodes (MLN) and spleen were isolated as described previously [22 (link), 27 (link)]. Briefly, MLN and spleen were mechanically dissociated in ice-cold PBS. The resulting cell suspensions were passed through a 70-μm mesh cell strainer, and spleen also needed erythrocyte lysis. To prepare colonic LP cells, associated fat and Peyer’s patches were removed, the colonic tissue was washed in cold PBS to remove fecal contents and cut open longitudinally, and the colon was cut into 2-cm pieces and washed twice in 2 mL PBS containing 1 mM dithiothreitol, 10% FCS and 1 mM EDTA with constant agitation for 40 min at 37 °C. And then the pieces were minced exactly 50 times to obtain 1 mm tissue fragments and incubated in 2 mL of RPMI-1640 supplemented with 2.5 mg/mL Hyaluronidase (Biosharp), 1.5 mg/mL Collagenase II (Biosharp), and 0.25 mg/mL DNase I (Solarbio) with constant agitation for 45 min at 37 °C. The cells suspension was extracted through filtration and centrifugation, and the resuspended pellet was further purified from the interface of a 45%/72% Percoll density gradient.

Cell isolation was performed according to a previous procedure^{60 (link)–62 (link)} with some adaptations. Briefly, after 4 mice were sacrificed, subcutaneous tumors were collected and cut into small pieces (<2 mm in diameter) and then digested with 10 ml of dissociation solution (RPMI 1640 medium containing 10% FBS, 1 mg/mL collagenase type IV (Biosharp, Hefei, China), 100 µg/mL DNase I (Biosharp, Hefei, China), and 2.5 µg/mL hyaluronidase (Biosharp, Hefei, China)) for 60 min on a 37 °C shaker. Next, 4 ml RPMI 1640 medium containing 10% FBS was added to dilute the suspensions. Then, the cell suspensions were passed through 70 µm cell strainers, and the lower layer was collected after centrifuging at 400 × g for 10 min. Subsequently, the erythrocytes were lysed with red blood cell lysis buffer (Biosharp, Hefei, China) according to the manufacturer’s instructions and then washed and resuspended the remaining single cells in PBS.