Fig. 5

Acquisition and maintenance of B7 molecule expression in CD4⁺ T cells in vitro is dependent on IL-2 signalling. a Gating strategy for the delineation of the mTeff and FOXP3⁺ mTreg populations. b, c Expression of CD80 (b) and CD86 (c) was assessed by flow cytometry in purified CD4⁺ T cells from two healthy donors incubated in vitro for up to 15 days in the presence of 0 U (square), 50 U (triangle) or 500 U (diamond) of IL-2. Single-parameter histograms represent the expression of CD80 and CD86 in mTreg incubated with 500 U IL-2 from a representative donor. Plots summarise the expression (median and 95% CI of the median) of the markers during the course of the experiment in FOXP3⁺ mTregs (red) and mTeffs (green). d Data shown depicts the frequency (median and 95% CI of the median) of CD80⁺ (blue) and CD86⁺ (red) cells within CD45RA⁻ CD4⁺ mTeffs. Data was obtained following in vitro stimulation with αCD3/CD28 beads (one bead to three cells ratio) every four days for two weeks in the presence (500 U; solid line) or absence (dashed line) of IL-2. e Frequency of CD80⁺ (blue) and CD86⁺ (red) cells within mTeffs was also assessed up to day 40 in resting cells, which were no longer re-stimulated with αCD3/CD28 after day 15. f Plots depict the frequency of membrane-bound CTLA-4⁺ (red) and HLA-DR⁺ (blue) within CD80⁺ and CD86⁺ mTeffs during the course of the experiment. g Immunophenotypic characterisation of the activation markers CD25, HLA-DR and CTLA-4 on B7⁺ mTeffs at day 40. Plots depict the frequency (median) of cells expressing these markers within CD80⁺ and CD80⁻ mTeffs (blue; left panel) and CD86⁺ and CD86⁻ (red; right panel) mTeffs, respectively. h Frequency (median) of CD80⁺ (blue) and CD86⁺ (red) cells in flow-sorted CD127^lowCD25^hi Tregs from three healthy donors, activated in vitro under specific Treg expansion conditions. B7 molecule expression was assessed by flow cytometry in the FOXP3⁺ T cells. For two of the donors, B7 molecule expression was also assessed for cells in the cycle, harvested in the middle of an expansion cycle (eight days after αCD3/CD28 restimulation). Tregs were expanded for between three (D3; square) and six (D1 and D2; circle and triangle, respectively) rounds of re-stimulation. i Expression of CD80 and CD86 was also determined at the mRNA level (NanoString) on the sorted Tregs ex vivo or following in vitro expansion