Isolation of Intestinal Epithelial and Lamina Propria Lymphocytes

Steady-state intestinal epithelial and lamina propria lymphocytes were harvested as previously described^{49 (link)}. To isolate IECs, intestinal tissues were first incubated in 5 mM EDTA (Invitrogen) in HBSS (Gibco) containing 1 mM DTT (Invitrogen) for 20 minutes at 37°C with 200rpm agitation, and then incubated in a second wash of 5 mM EDTA in HBSS without DTT for another 20 minutes at 37°C with agitation. Single-cell suspensions released into the EDTA solutions were pooled and confirmed to contain over 85% EpCAM⁺ IECs as reported previously^{39 (link)}. For the lamina propria, IEC-depleted tissues from post-EDTA washes were digested in a HBSS solution containing 10% Fetal Bovine Serum (Peak Serum), 1.0 mg/ml Collagenase D (Roche), 100 μg/ml DNase I (Sigma-Aldrich), and 50 U/ml dispase (Worthington Biochemical Corporation) at 37°C for 30–45 minutes. Lamina propria mononuclear lymphocytes were purified from the interphase of a 40:80% Percoll^™ (Cytiva; formerly GE Healthcare Life Sciences) gradient.