Irradiation and graft preparation

Irradiation and cell transplants were conducted as previously described [18–20]. This conditioning and cell transplantation model results in successful immune cell chimerism [19]. Briefly, 1 day prior to transplantation, recipient B6D2F1 mice received 1100 centigray (cGy) of total body irradiation (Gamma cell irradiator), split into two doses that were administered 3 h apart to minimise gastrointestinal toxicity. The following day, each recipient B6D2F1 mouse received a tail vein injection containing 5 × 10⁶ bone-marrow cells. Following transplantation, all mice were housed in sterilised cages and received autoclaved food and water.

The ‘syngeneic’ or the non-cGVHD control group received a cell transplant from donor B6D2F1 mice. For the allogeneic transplant (the ‘cGVHD’ group), B6D2F1 recipients received cells from donor C57BL/6 mice. In allotransplant recipients, the bone-marrow mixture was also supplemented with 0.5 × 10⁶ mature T cells, isolated from whole spleen and using magnetic bead-mediated depletion to remove non-T cells. Briefly, whole spleen was mashed, followed by red cell lysis. Next, the single-cell splenocyte suspension was incubated with a cocktail of mAb CD19 [HB305], CD11b [TIB128], anti-B220 [RQ36B2], anti-Gr1 [RB6-8C5], and anti-Ly76 [TER119]) for 20 min on ice. Cells were subsequently resuspended in goat anti-rat IgG BioMag beads (QIAGEN) and incubated again for 20 min on ice, followed by depletion of antibody-bound cells using magnetic separator. Non-bound T cells were stained with anti-CD3-BV421 (Biolegend) and confirmed to be 80–90% pure.