Figure 2.

Human p41⁺ Tr1 cell clones suppress expansion of CD4⁺ T cells. (a) T-cell clones were cocultured together with CFSE-labeled autologous CD4⁺CD25⁻ T cells (T_conv) (1:2 ratio) and irradiated p41-pulsed CD2-depleted APC (1:2:4 (clone:T_conv:APC)) in round bottom plates, coated with 0.5 μg ml⁻¹ anti-CD3 mAb. After three days, CFSE dilution was determined by flow cytometry. The histograms show CFSE dilution of T_conv in the absence and presence of one representative T_eff and Tr1 clone. Diagrams summarize %±s.d. suppression of T_conv proliferation in the presence Tr1 and T_eff clones (n=9 per group; three different donors) at a 1:2:4 ratio (clone:T_conv:APC). (b) CFSE-labeled PBMCs were cultured together with CellVue-Claret-labeled p41-specific T-cell clones at 2:1 ratio. After 24 h of culture and every second day, IL-2 were added to the culture. After seven days, the T cells were restimulated with p41-pulsed autologous DC at a ratio of 10:1 and cultured for additional six days in the presence of IL-7 and IL-15. Afterward, expansion of CFSE⁺CD4⁺p41⁺ T cells was analyzed by flow cytometry using a p41-specific PE-labeled tetramer. Dotplot analysis shows one experiment in the absence (left) and presence of a representative Tr1 clone (middle) and T_eff clone (right). Diagrams summarize %±s.d. suppression of CD4⁺CFSE⁺p41⁺ T-cell expansion in contact-dependent presence of Tr1 and T_eff clones (left) (n=6 per group; three different donors) as well as in contact-independent cocultures in transwell systems (right) (n=5 per group; three different donors).