Temporospatial profiling reveals three-way T cell chimerism and alloreactivity in a patient receiving a second intestinal transplant

Introduction: Retransplantation is a limited option for failed intestinal allografts, which face among the highest rejection rates in solid organ transplantation. Yet, the immune dynamics of retransplantation remain poorly understood. Here, we report a case of simultaneous multi-donor chimerism after intestinal retransplantation and dissect the multidirectional alloreactivity underlying long-term graft acceptance.
Methods: Using HLA allotype-specific antibodies, we traced chimerism in blood, bone marrow, and intragraft compartments via flow cytometry, distinguishing cells from Donor 1 (G1), Donor 2 (G2), and the Recipient (H). Alloreactive clones in all directions (G1vH, G1vG2, G2vH, G2vG1, HvG1, HvG2) were identified via bulk TCRβ sequencing of proliferated cells in pre-transplant mixed lymphocyte reactions and tracked longitudinally post-transplant. Donor 2 T cells were further analyzed using single-cell TCRαβ+RNA sequencing.
Results: A 20-month-old patient received a liver-intestinal transplant from Donor 1 (3 months old). Severe rejection occurred by postoperative day 19 (POD19), characterized by rapid intragraft recipient T-cell replacement (> 95%) and low-level peripheral blood T-cell chimerism (peak 2.36% on POD9). Intractable rejection was followed by graft explantation and multivisceral transplantation from Donor 2 (19 m/o) on POD786. Post-retransplant, the patient exhibited significant blood T-cell chimerism from Donor 2 (peak 29.9%) and slower mucosal graft T-cell replacement. Surprisingly, blood T-cell chimerism from Donor 1 reappeared, peaking at 8.98%, with T cells from both donors persisting for over 1000 days. G1vH-, G1vG2-, G2vH- and G2vG1-reactive clones were detected across blood, bone marrow, and the intestinal allograft, demonstrating sustained multidirectional alloreactivity. We identified alloreactive clones recognizing one or two individuals within the chimera, consistent with partial sharing of HLA alleles between each pairwise combination of the three individuals (G1, G2, H). Notably, Donor 1 T cells repopulated the Donor 2 graft, revealing a “pseudo HvG” response. Single-cell profiling of Donor 2 T cells identified two dominant mucosal trajectories: (1) G2vH and G2vG1 clones in the pre-transplant ileal graft and early post-transplant peripheral blood/bone marrow exhibited cytotoxic effector phenotypes consistent with three-way lymphohematopoietic GvH reactivity; (2) another population, including G2vH- and G2vG1-reactive cells, transitioned toward tissue-resident memory-like phenotypes during long-term graft acceptance.
Conclusion: Our study provides the first mechanistic dissection of multidirectional alloreactivity in a retransplant patient with multi-donor chimerism. These findings illustrate the stable co-existence of immune cells from multiple individuals and suggest that balanced alloreactivity may facilitate long-term graft stability.