Figure 5.

Stimulation with Staphylococcus aureus in the absence of pre-existing regulatory T (Treg) cells induces activated FOXP3^low CD25⁺ CD127^low T cells. (a) CD4⁺ CD25^neg responder T cells were isolated and stored for 3 days at −70°. CD4⁺ CD25^neg/+ CD127⁺ (non-Treg cells), CD4⁺ CD25⁺ CD127^low T cells (Tregs) and remaining mononuclear cells from the same cord blood sample were sorted using flow cytometry. Next, non-Treg cells and the remaining mononuclear cells were co-cultured and stimulated with S. aureus for 3 days. On day 3 the responder cells were thawed and stained with CellTrace violet, stimulated with α-CD3 and α-CD28 and co-cultured in a ratio of 2 : 1, 4 : 1 or 5 : 1 (depending on the quantity of CD25⁺ CD127^low T cells that was collected during sorting), for 4–5 days with sorted autologous S. aureus-induced CD25⁺ CD127^low T cells (n = 3). (b) Representative histogram on the proliferation of CD4⁺ responder cells stimulated with α-CD3 and α-CD28 alone or co-cultured with induced CD25⁺ CD127^low T cells after stimulation with S. aureus in the absence of pre-existing regulatory T (Treg) cells (tTregs) during the stimulation phase in a ratio of 4 : 1. (c and d) The proportion of induced CD25⁺ CD127^low T cells that express FOXP3 in cultures in which non-Treg cells and remaining cord blood mononuclear cells were stimulated with S. aureus in the presence (n = 7) or absence (n = 4) of tTreg cells for 3 days, illustrated in (c) a graph and (d) zebra plots of one individual. The numbers represent the percentage of cells within the gate and for each sample, 8000–10 000 CD4⁺ T cells were collected. Each symbol in the graphs represents one individual and horizontal bars indicate the median value.