Intracellular interferon-γ staining analysis of donor-specific t-cell responses in liver transplant recipients

Objective: Biomarkers that accurately reflect, detect, and/or predict detrimental immune responses to grafts are important in organ transplantation. We established a new detection method for alloreactive T cells on the basis of intracellular staining for interferon (IFN)-γ, using CD40-activated B cells as stimulators, and assessed temporal changes in alloreactive T-cell frequencies in patients who received liver transplantation. Methods: Peripheral blood mononuclear cells and CD40-activated B cells were used as responder and stimulator cells, respectively. The responder cells were cultured with the stimulator cells for 7 days, restimulated for 5 hours, and flow cytometrically tested by intracellular staining for IFN-γ. Results: The relative postoperative-preoperative ratio of donor-specific CD8 ⁺ T cells in the nonrejection group was significantly lower than that in the rejection group and found to be <1 in most individuals of the group throughout the postoperative periods, indicating an induction of donor-specific suppression of the CD8 ⁺ T-cell responses. In contrast, such differences were not found in the donor-specific CD4 ⁺ T cells. These results suggest that the relative postoperativepreoperative ratio of the donor-specific CD8 ⁺ T cells is a good indicator of graft rejection. Conclusion: We established a new flow cytometric method for the detection of alloreactive T cells by intracellular staining for IFN-γ, using CD40-activated B cells as stimulator cells. Using this system, we found that the relative postoperativepreoperative ratio of the donor-specific CD8 ⁺ T cells is a possible evaluative indicator of the risk for graft rejection.