Flow Cytometric Immune Profiling Enables Early Detection of Rejection in Islet Xenotransplantation

Islet xenotransplantation is a strategy for treatment of Type 1 Diabetes, but immune-mediated rejection limits graft survival. This study investigated a strategy with flow cytometry-based immune monitoring to evaluate immunosuppressive therapy and provide indicators of graft rejection.

Porcine neonatal islet cell clusters (NICC) from GTKO-hCD55-hCD59 transgenic pigs were transplanted into streptozotocin-induced diabetic baboons (n = 10). Recipients received anti-CD2 induction with anti-CD154, Belatacept, and full-dose (n = 7) or reduced/no dose Tacrolimus (n = 3). Leukocyte subsets and activation status were monitored using multiplex flow cytometry. Graft function was assessed by blood glucose measurements, porcine C-peptide ELISA, and histological analysis.

Porcine C-peptide was detected in all recipients. Seven animals receiving full-dose Tacrolimus maintained NICC function for up to 675 days, whereas three experienced acute rejection within two months. Flow cytometry generated absolute leukocyte counts equivalent to standard full blood counts (Deming regression slope 0.91-1.03, p<0.001), while requiring less blood (50 µl vs ∼500 µl) and processing time (50±15 vs 150±30 min). Anti-CD2 treatment significantly reduced unoccupied surface CD2 on circulating T cells (mean 73.21%, range 55.26-91.16%, p<0.001). Long-term graft survival was associated with greater first-month T-cell suppression (36.47%, 13.35-50.75%) than acute rejection (176.28%, 171.26-181.31%). Following immunosuppression withdrawal, lymphocyte counts increased by 40%-142% in all recipients. Multiplex immunofluorescence of endpoint grafts from these IS-free animals revealed cell-mediated destruction with increased lymphocyte infiltration and activated cytotoxic CD8⁺ T cells.

Multiplex flow cytometry provides a reliable method for immune monitoring in islet xenotransplantation and may help predict graft rejection.