Tissue Processing

Small intestinal lamina propria lymphocyte preparations were prepared by opening longitudinally and removing the Peyer’s patches, associated fat and luminal content by gently shaking in cold PBS. Epithelial cells and intra-epithelial lymphocytes were removed by shaking tissues in stripping buffer (1 mM EDTA, 1 mM DTT and 5% FCS) for two rounds of 20 min at 37°C. Lamina propria lymphocytes were isolated by digesting the remaining tissue in 1 mg/mL collagenase D (Roche) and 20 µg/mL DNase I (Sigma-Aldrich) for 45 min at 37°C. Liberated cells were then extracted by passing the tissue and supernatant over a 70μm nylon filter and centrifuged to isolate lamina propria lymphocytes. Isolated Peyer’s patches were processed by passing them through a 70μm nylon filter. In a small number of cases Peyer’s patches were retained during intestinal tissue digest to facilitate concurrent analysis of tissue-resident plasma cells and B cell subsets.