IFNy plus and IFNy- Treg subsets with stable and unstable Foxp3 expression in kidney transplant recipients with good long-term graft function

Background: Treg are a heterogenous cell population. In the present study we attempted to identify Treg subsets that might contribute to stable and good long-term graft function. Method: Lymphocyte and Treg subsets were studied in 136 kidney transplant recipients with good long-term graft function and in 52 healthy control individuals using eight-color-fluorescence flow cytometry. Foxp3 TSDR methylation status was investigated in enriched IFNy + and IFNy Treg preparations using high resolution melt analysis. Results: Compared with healthy controls, patients showed strong associations of IFNy secreting Helios + and Helios Treg with Treg that co-expressed perforin and/or CTLA4 (CD152; p < 0.01). Moreover they showed associations of IFNy Helios + Treg with Treg that produced TGF13 and/or perforin and of IFNy Helios Treg with TGFp production (all p < 0.01). Only in patients, but not in healthy controls, were IFNy Helios + and Helios Treg associated with higher CD45 +, CD3 +, (CD4+), CD19 + lymphocyte counts (p < 0.001). In addition IFNy Helios + Treg were associated with CD16 + 56 + lymphocytes (p <0.001). Enriched IFNy Treg from female but not male patients showed an association of Foxp3 methylation with higher total Treg and higher Helios + IFNy CXCR3 +Lselectin + (CD183 + CD62L+), CXCR3 Lselectin + and CD28 + HLADR+ Treg subsets (p < 0.01). Enriched IFNy + Treg from male patients showed an association of demethylated Foxp3 with total Treg and IL10 TFG beta+ Treg counts, and in enriched IFNy Treg an association of methylated Foxp3 with APO1/FasR+FasL+ (CD95 +CD178+) Treg (p < 0.01). Conclusions: Kidney recipients with good long-term graft function possess IFNy+ and IFNy Treg with stable and unstable Foxp3 expression in the blood. They co-express CD28, HLADR, CTLA4, CXCR3, Lselectin, TGFI3, perforin and FasL and might contribute to the establishment and maintenance of good long-term graft function. (C) 2016 Elsevier B.V. All rights reserved.