Freshly harvested spleens from GvHD B6 recipients of Balb/c T cells at 33-35 days post transplantation were dissociated mechanically using gentle force with iron strainer and 3ml syringe. Transfer the homogenized cell solution into the 50ml conical tube and spin down at 1500rpm for 5min at RT. Aspirate the supernatant, resuspend the pellet in 2ml ACK buffer, and incubate it for 5min at RT to lyse red blood cells. After the incubation, add 10ml of R2 (2%FBS in RPMI media) and pour the splenocytes into 70um cell strainer to filter the solution. After spinning down the tube at 1500rpm for 5min, cells were stained with CD8 APC, PD-1 BV421 antibodies, and Amcyan live/dead viability dye in FACS buffer (2% FBS and 2mM EDTA in PBS). Alive PD-1+ CD8 T cells were sorted using a FACS Aria II (BD Bioscience). Use LUNA-FL™ Automated Fluorescence Cell Counter (logos biosystems) to consult the 10x Genomics Single Cell Protocols Cell Preparation Guide and the Guidelines for Optimal Sample Preparation flowchart (Documents CG00053 and CG000126 respectively) for more information on preparing cells. Libraries were prepared using the Chromium controller according to the 10x chromium Next GEM Single Cell 3' v3.1 protocol(CG000315). Briefly, the cell suspensions were diluted in nuclease-free water to achieve a targeted cell count of 10,000. Cell suspension was mixed with master mix and loaded with Single Cell 3'V3.1 Gel Beads and Partitioning Oil into a chromium Next GEM chip G. RNA transcripts from single cells were uniquely barcoded and reverse-transcribed within droplets. cDNA molecules were pooled and the cDNA pool then go through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library. The purified libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide(KAPA) and qualified using the Agilent Technologies 4200 TapeStation(Agilent technologies). And then the libraries were sequenced using HiSeq platform(Illumina) according to the read length in the user guide.