NIV Congres

Introduction CD4⁺CD25^highCD127^- are potent regulators of cytokine production and proliferation of alloreactive T-cells. Although, cytotoxic T-cells are the effector cells that destroy the allograft. The influence of regulatory T-cells (Tregs) on the donor-specific cytotoxic capacity of cytotoxic T-lymphocytes (CTL) is unknown. Therefore, we questioned whether the donor-specific CTL response is controlled by Tregs after renal transplantation.

Material and methods Blood was sampled from 12 patients after renal transplantation (mean 4.7 years ±1.2). We assessed the involvement of Tregs both by depleting them from patients’ PBMCs as well as by reconstituting them to the PBMCs depleted for Tregs. Patients’ cells were incubated with irradiated donor or 3^rd-party cells in the presence of IL-2, IL-7 and IL-15 to obtain optimal numbers of donor or third-party reactive effector memory CTLs. After 7 days of culture, a cell mediated lympholysis assay (CML) was performed by adding effector cells to a fixed number of europium labelled target cells (effector:target ratio starting from 40:1 to 0.6:1).

Results Donor-specific hyporesponsiveness compared to 3^rd-party response was found in all effector:target ratios (p=0.003). Depletion of Tregs from PBMC resulted in an increased donor-reactive CML response in 9/12 patients. Reconstitution of the Tregs to the CD25^- negative effector T cells, inhibited the donor-reactive CML response again in 9/12 patients. The potency to inhibit 3^rd-party reactivity was comparable: 11/12 were inhibited. When we characterised the Tregs by phenotypic analysis, we found that most of the CD4⁺CD127^- FoxP3⁺ Tregs expressed Helios, the marker of natural Tregs (nTregs Helios⁺: mean±SD; 68% ±4.1). Only 5% ±2.3 nTregs were capable to produce IFN-γ after stimulating the cells with PMA/ionomycin, while 28% ±13 of the iTregs (Helios^- induced Tregs) produced IFN-γ.

Conclusion Functional nTregs circulate long after renal transplantation, these cells inhibit the donor-specific CTL response.