The risk of rejection by cellular alloreactivity to the transplant donor is not routinely assessed. Here we analyzed alloreactive T cells in kidney transplant recipients and report how their detection may have helped to prevent rejection of a second kidney graft in a patient with a history of acute accelerated steroid-resistant nonhumoral rejection. Alloreactive CD4 and CD8 T cells were quantified using a flow-cytometric mixed lymphocyte reaction assay based on interferon-γ induction. A group of 16 nonrejecting transplant recipients did not show any alloreactive T-cell immunity to their respective donors, whereas alloreactivity to third-party controls was detectable. In the patient with rejection, HLA-specific antibodies were not detectable before and shortly after rejection, but after transplantation the patient showed exceptionally high frequencies of alloreactive T cells against 2 of 11 HLA-typed controls (0.604% and 0.791% alloreactive CD4 T cells and 0.792% and 0.978% alloreactive CD8 T cells) who shared HLA alleles (HLA-A*24, -B*44, -C*02, -DQB1*5) with the kidney donor. These HLA alleles were subsequently excluded for allocation of a second graft. No alloreactive T cells were observed toward the second kidney donor, and this transplantation was performed successfully. Thus, shared HLA alleles between the donor and third-party controls may suggest that alloreactive T cells had contributed to rejection of the first graft. The rejecting patient highlights that determination of cellular alloreactivity before transplantation may be applied to identify unacceptable mismatches and to reduce the risk for acute cellular rejection episodes.

The risk of allograft dysfunction increases with increasing numbers of human leukocyte antigen mismatches (HLA-MM). Although this seems to be mediated by preformed alloreactive T-cells, the association between cellular alloreactivity and HLA mismatches and potential differences in healthy controls and hemodialysis patients prior to transplantation are poorly characterised. Therefore, a 6h flow-cytometric assay based on a mixed lymphocyte reaction from whole blood was used to analyse paired combinations of cells from 113 individuals (52 dialysis patients and 61 healthy controls) in a total of 1502 alloreactivity tests (681 from dialysis patients and 821 from controls). Respective autologous combinations served as negative control. Alloreactive CD4 and CD8 T-cell frequencies were correlated with the number of HLA mismatches (MM), age, and gender. Frequencies of alloreactive T cells were low in most individuals and median levels were higher in CD8 T cells (0.002% (IQR 0-0.008%)) than in CD4 T cells (0.001% (IQR 0-0.003%). Among the 1502 tests, a significant association was found between the frequency of alloreactive CD8 T cells and the number of HLA-A/B/C-MM (p=0.030). Likewise, alloreactive CD4 T-cell frequencies strongly associated with the number of HLA-DR-MM (p<0.0001). Of note, peak levels of alloreactive CD8 T cells increased with increasing numbers of HLA-A/B/C-MM (0MM: 0%; 1MM: 0.041%; 2MM: 2.752%; 3MM: 4.118%; 4MM: 7.001%; 5MM: 8.549%; 6MM: 6.864%) and peak CD4 T cell frequencies increased with increasing numbers of HLA-DR-MM (0MM: 0.078%; 1MM: 0.578%; 2MM: 0.780%). Dialysis patients showed higher levels of alloreactive CD4 (p=0.015) and CD8 T-cell levels (p<0.0001) as compared to controls. No association was found between the level of alloreactivity and age or gender in neither controls nor dialysis patients. In conclusion, the 6h whole blood assay allows for a rapid quantitation of preformed alloreactivity in a pre-transplant setting. Alloreactivity correlated with HLA-MM in the whole population but not on an individual level. This technical approach may be used to explore whether the presence of alloreactive T cells can serve as a more specific parameter to assess the risk of graft dysfunction as compared to HLA typing. DISCLOSURES:Sester, U.: Stockholder, Red Flag Diagnostics, Other, patent pending, patent pending. Sester, M.: Stockholder, Red Flag Diagnostics, Other, patent pending, patent pending.