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

•Treg with stable and unstable Foxp3 expression might regulate the immune system.•Both types of Treg can express IFNy and both IFNy- and IFNy+ Treg appear to have clinical relevance.•They modulate effector cells via TGFß, CTLA4, perforin, FasL and consumption of IFNy and IL2.•We believe that these Treg modulate alloreactivity of T- and B-lymphocytes as well as NK cells.•We believe that they have a potential of allowing reduction of immunosuppressive drugs to low maintenance levels.

BackgroundTreg 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.MethodLymphocyte 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.ResultsCompared 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 TGFβ and/or perforin and of IFNy − Helios − Treg with TGFβ 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 − TFGβ + Treg counts, and in enriched IFNy − Treg an association of methylated Foxp3 with APO1/FasR + FasL + (CD95 + CD178 +) Treg (p < 0.01).ConclusionsKidney 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, TGFβ, perforin and FasL and might contribute to the establishment and maintenance of good long-term graft function.