Donor-Derived Cell-Free DNA (dd-cfDNA) as an Early Noninvasive Biomarker of Graft Injury in Pig-to-Monkey Islet Xenotransplantation

Background: In pig-to-nonhuman primate islet transplantation, reliable, sensitive biomarkers are needed to detect graft damage at an early stage before irreversible islet loss occurs. In our study, we investigated donor-derived cell-free DNA (dd-cfDNA) as an early, noninvasive biomarker of graft injury by analyzing its correlation with porcine C-peptide levels, complement activation markers, and donor-specific antibodies (DSAs). Methods: Streptozotocin-induced diabetic cynomolgus monkeys received 50 000–100 000 IEQ/kg of intraportal islets from quadruple-knockout (QKO; GGTA1, CMAH, B4GALNT2, and A3GALT2) pigs. Cohort 1 received antithymocyte globulin (ATG), tacrolimus, mycophenolate mofetil (MMF), and anti-inflammatory agents (i.e., anakinra, adalimumab, and tocilizumab), whereas Cohort 2 received the same regimen plus rituximab and crovalimab. Graft function and immune responses were assessed by measuring porcine C-peptide levels, complement activation markers, histology, and dd-cfDNA kinetics. Results: Cohort 1 showed transient porcine C-peptide secretion with marked dd-cfDNA elevation at 7 d postoperatively that coincided with complement activation (i.e., C5a and membrane attack complex (MAC)) and dense CD3⁺ T-cell and CD68⁺ macrophage infiltration, which resulted in early graft loss. Cohort 2 maintained stable C-peptide levels, lower dd-cfDNA levels, and reduced complement activation with improved graft preservation. Moreover, dd-cfDNA correlated negatively with C-peptide and positively with C5a but not with MAC. In both cohorts, DSA levels remained unchanged. Conclusions: Our study revealed that dd-cfDNA levels correlate with graft damage and C5a in QKO porcine islet xenografts, which corroborates dd-cfDNA utility as an early biomarker for predicting instant blood-mediated inflammatory reaction (IBMIR). These findings indicate that dd-cfDNA may be able to detect early islet xenograft damage.