Blood collected in EDTA tubes was processed by Ficoll density gradient to separate peripheral blood mononuclear cells (PBMCs), and 5 pinch biopsies from the rectal mucosa were processed by collagenase digestion to separate mucosal mononuclear cells (MMCs) as described previously.40 (link) A complete list of antibodies used for phenotyping is available in the supplementary materials. Mononuclear cells isolated from the blood and rectal biopsies were stained with LIVE/DEAD Near-IR Dead Cell stain (Life Technologies) at room temperature for 15 min in PBS to stain for dead cells. Cells were then washed with FACS wash and stained on the surface using antibodies specific to CD3, CD4, CD8, CCR7, CD45RO CD28, CCR5, α4β7, CD38, HLA-DR and then treated with 1x BD FACS Lysing solution for 10min at room temperature, permeabilized with 1x BD Permeablizing solution for 10 min at room temperature, washed with FACS wash, stained with anti-Ki67 antibodies, washed 2x with FACS wash, and acquired on the LSR-Fortessa platform and analyzed with Flowjo software (Treestar Inc. CA). Staining for HLA-DR was suboptimal during the study; therefore, further analyses of this antibody were not performed.