Fig. 4

Surface expression of the co-stimulatory molecules CD80 and CD86 marks a subset of activated Tregs in vivo. a, b Expression of the B7 molecules CD80 (a) and CD86 (b) was assessed ex vivo by flow cytometry in CD4⁺ T cells from four healthy donors. Scatter plots depict the frequency (median) of CD80⁺ and CD86⁺ T cells within the CD45RA⁻ Teff (mTeffs; black) and CD45RA⁻ CD127^lowCD25⁺ Treg (mTregs; blue) populations. c Scatter plot depicts the frequency (median) of FOXP3⁺ cells within total (black), CD80⁺ and CD86⁺ CD4⁺ T cells. d Gating strategy for the delineation of the Treg subsets according to the intracellular expression of the canonical Treg transcription factors FOXP3 and HELIOS. Scatter plot depicts the frequency of HELIOS⁺FOXP3⁺ (blue) and HELIOS⁻FOXP3⁺ (red) subsets in each of the four donors within total mTregs, CD80⁺ mTregs and CD86⁺ mTregs. e Co-expression of CD86 and the CD4⁺ T cell activation markers CTLA-4 and HLA-DR was assessed by flow cytometry in CD86⁺ (red) and CD86⁻ (blue) mTregs. f Scatter plots depict the frequency (median) of CTLA-4^hi HLA-DR⁺ cells within CD86⁺ and CD86⁻ mTregs. Expression of CTLA-4 was assessed by intracellular immunostaining