Clonal expansion of T regulatory cells in rejecting versus tolerant mouse lung grafts

It has been previously shown that T regulatory cells play a key role in regulating local immune responses within lung grafts after transplantation to promote tolerance. However, it remains unknown whether T regulatory cells within the graft undergo clonal expansion after transplantation. In this study, we examine the T cell receptor repertoire of T regulatory cells within the local graft and also within the recipient spleen. Overall design: Transplanted left lungs and recipient spleens from four mice were collected 7 days following transplantation. Two samples were collected at a time and pooled for further processing. Lung tissue was digested, and single-cell suspensions were prepared as previously described (PMID: 28615357). Cells were stained using DAPI (BD Biosciences, #564907) and were sorted by flow cytometry for CD45+CD4+Foxp3+ Tregs into 250 µL cell resuspension buffer (0.04% BSA in PBS). Collected cells were centrifuged (300 rcf for 5 min at 4 °C) and resuspended in collection buffer to a target concentration of 1,000 cells/µL. Cells were counted on a hemocytometer before proceeding. Single-cell suspensions of sorted lung and spleen Tregs were submitted to the Genome Technology Access Center core facility (Washington University in St. Louis) for single-cell GEM construction and cDNA synthesis and library construction. Two sets of hashtagged samples were submitted as follows: tolerant lung CD4+, tolerant lung Treg, tolerant spleen Treg, rejecting lung Treg. Sorted cell types were labeled using a TotalSeq anti-mouse hashtag antibody (Biolegend, #155861, #155863, #155865, #155807) prior to pooling for sample submission. Samples were processed using the Chromium Single Cell 3' Library & Gel Bead Kit (10X Genomics, v3) and Chromium Single Cell V(D)J Reagent Kits (10x Genomics) following manufacturer's protocols. The libraries were sequenced on NovaSeq S4 (Illumina) targeting 50,000 reads per cell and 500 million read pairs per library. Cells were aligned to the mouse mm10-2020-A transcriptome using CellRanger (10× Genomics, v6.1.1) to generate feature-barcoded count matrices.