For the collection of the immune cells in colon, mice were sacrificed by CO2 overdose and colon was removed and placed in ice-cold HBSS (GIBCO) with 5% FBS. Isolated cells were resuspended in 10 ml of the 40% fraction of a 40: 80 Percoll gradient and overlaid on 5 ml of the 80% fraction in a 15 ml Falcon tube. Percoll gradient separation was performed by centrifugation for 20 min at 1,800 rpm at room temperature. lymphocytes were collected at the interphase of the Percoll gradient, washed and resuspended in medium, and then stained for sorting and analyzed by flow cytometry. Dead cells were eliminated through 7-AAD staining. According to the manufacture's introduction, single-cell RNA-seq libraries were constructed using Single Cell 3' Library andGel Bead Kit V3. The libraries were finally sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing).