Detailed knowledge on the preformed donor-specific T cell repertoire in transplant recipients prior to transplantation would allow guidance on individualized immunosuppressive drug treatment early after transplantation. As current tests for the detection of alloreactivity are not suitable for rapid and repeated use in a clinical setting, we now developed a simple flow-cytometric assay to simultaneously quantitate alloreactive CD4 and CD8 T cells directly from whole blood samples after an incubation period of only 6h. This assay is based on the intracellular accumulation of IFN-Γ and the induction of CD69 and was evaluated in a total of 376 alloreactivity tests (275 in dialysis patients awaiting renal transplantation and 101 in healthy controls) that were performed by pair wise combinations of whole blood from 22 patients and 9 controls. Respective autologous combinations served as negative controls. In addition, 114 pairs (73 from patients and 41 from controls) were re-tested after 3 months.

The detection limit was 0,0081% among CD4 and 0.0143% among CD8 T cells. Alloreactivity in the cytotoxic T-cell compartment was more common, as 27.7% of all pairs had detectable alloreactive CD8 T cells compared to 9.0% of pairs with respective CD4 T cells (p<0.0001). Interestingly, CD8 T-cell alloreactivity was significantly more frequent in dialysis patients than in controls (32.4% versus 14.9% of combinations, p<0.001). Of note, individual CD8 T-cell frequencies may reach 6.9%, and the magnitude of alloreactive CD8 T cells was significantly higher in dialysis patients (median 0.19%, IQR 0.05–1.67) compared to controls (0.04%, IQR 0.02–0.06, p=0.0013). Re-testing of 114 pairs after 3 months showed that 80% of transplant candidates with alloreactive CD8 T cells remained positive on follow-up. In addition, the frequency of alloreactive CD8 T cells observed at the two time points showed a strong correlation (r=0.82; 95% CI 0.72–0.88, p<0.0001).

Conclusion: Preformed cellular alloreactivity is dominated by CD8 T cells and can be detected from whole blood samples within one day. Together with published evidence based on more laborious assays, the substantially higher burden of T-cell alloimmunity among transplant candidates and the stability over time may indicate increased risk for graft rejection and feasibility to identify the risk arising from preformed alloreactivity for each individual recipient–donor pair in solid organ transplantation.

Sester, M.: Other, Patent Pending, Patent Pending. Sester, U.: Other, Patent Pending, Patent Pending.

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