Isolation of intestinal and splenic lymphocytes

To isolate lymphocytes from the spleen and MLN, tissues were mashed through 70-µm cell strainers. Small intestine LP lymphocytes and IEL were isolated as previously described (Qiu and Sheridan, 2018 (link); Sheridan and Lefrançois, 2012 (link)). Briefly, small intestines were cut into 1-inch long pieces after removal of Peyer’s patches and luminal content. Intestinal tissues were treated twice with 1 mM dithioerythritol (Sigma-Aldrich) solution in a shaker at 220 rpm and 37°C for 20 min. Supernatants were collected, combined, and subjected to 44%/67% Percoll (GE Healthcare) gradient for the isolation of IEL. The remaining intestine tissues were treated twice with 1.3 mM ethylenediaminetetraacetic acid (Invitrogen) solution in a shaker at 220 rpm and 37°C for 30 min to remove intestinal epithelial cells, followed by digestion with 100 U/ml of collagenase (Invitrogen) in a shaker at 300 rpm and 37°C for 45 min. Supernatants were collected after collagenase digestion and the undigested tissues were mashed through 70-µm cell strainers into the collected supernatant, which was then subjected to 44%/67% Percoll gradient for the isolation of LP lymphocytes.