Three-way T-cell chimerism and alloreactivity using single cell profiling in a patient receiving a second intestinal transplant

Background: Three-way T-cell chimerism and alloreactivity following re-transplantation have not been explored.Methods: We monitored blood and intragraft chimerism by flow cytometry in a patient receiving a second intestinal transplant.Alloreactive graft1-vs-host (G1vH), graft1-vs-graft2 (G1vG2), graft2-vs-host (G2vH), graft2-vs-graft1 (G2vG1) clones were identified using bulk TCR-sequencing on proliferated cells in pre-transplant mixed lymphocyte reactions and tracked in post-transplant samples.Donor2 T-cells were analyzed using single-cell TCR+RNA sequencing.Results: The patient (20 months old: m/o) received a liverintestinal transplant from Donor1 (3 m/o).Severe rejection occurred on postoperative day 19 (POD19) with rapid intragraft recipient T cell replacement (> 95%) and low levels of blood Tcell chimerism (peaking at 2.36% on POD9).Intractable rejection was followed by graft explantation and multivisceral transplantation from Donor2 (19 m/o) on POD786.The patient then displayed much higher blood T-cell chimerism from Donor2 (peak 29.9%) and slower mucosal graft T-cell replacement.Surprisingly, blood T-cell chimerism from Donor1 reappeared, peaking at 8.98% and persisting >1000 days.G1vH-, G1vG2-, G2vH-and G2vG1-reactive clones were detected in post-transplant blood, implicating alloreactivity in long-term chimerism.G2vH-and G2vG1-crossreactive clones were found in the graft and blood, likely due to HLA sharing between Host and Donor1.ReTx introduced additional clones (e.g.nonG2vH but G2vG1) that expanded the alloreactive repertoire.Single-cell profiling identified Donor2 T-cell clones, including G2vH and G2vG1 CD8 clones with cytotoxic effector phenotypes, shared between baseline Donor2 mucosa and post-second transplant blood and bone marrow, reflecting lymphohematopoietic alloreactivity.Conclusions: Three-way chimerism and alloreactivity may play a significant role in re-transplantation outcomes.