Rectal Immune Cell Characterization

Single-cell suspensions from rectal pinches were prepared as previously described^{30 (link)}. Rectal intraepithelial lymphocytes (IEL) and lamina propria(LP) were collected and subjected to flow cytometry analysis. The single-cell suspensions were first incubated with Fc Receptor blocking reagent (Miltenyi Biotec), followed by staining with viability dye (Invitrogen). The antibody mixtures were then incubated as previously described^{27 (link)}. For immune activation, the following antibodies were used: CD45-PerCP, CD3-PE-Cy7, CD4-BV605, CD8-APC-Cy7, CD14-V450, Ki67-APC, HLA-DR PE-Cy5, and CCR5-PE (BD Pharmingen); CD69-Alexa Fluor 700 (Biolegend); and CD38-FITC (STEMCELL Technologies). For detection of Treg and MDSCs, the following antibody mixture were used: CD45-PerCP/Cy5.5, CD3-PE-Cy7, CD4-BV605, CD8-BV785, lin 1-FITC (BD Pharmingen), FOXP3-APC (eBioscience), HLA-DR-APC-Cy7, CD11b-PE-Cy5, CD14-BV711, CD8-BV785, CD25-BV421, CD15-Alexa700 (Biolegend), CD33-PE (Milteny). An LSRII flow cytometer was used for data acquisition. FlowJo software (Tree Star Inc.) was used for data analyses.