IL-21 has a unique ability to expand DP thymocytes during positive selection. (A) Absolute numbers of DP (top panel) and SP (bottom panel) CD8 thymocytes after culture of 12.5 × 10³ DP3 OT-I thymocytes with EIINFEKL-pulsed OP9 cells supplemented with cytokines. Dashed lines represent the number of cells seeded per well at time 0. (B) Synergistic effect of IL-21 on CD8 T-cell differentiation. Five mice per group were tested. (C) CFSE content in DP1/DP2/DP3 thymocytes supplemented with 10 ng/mL of rIL-21 in the absence (black line) or presence (red line) of EIINFEKL-pulsed OP9 cells. (D) Representative flow cytometric analysis of intracellular BCL-6 and BLIMP-1 expression in unstimulated DP3 thymocytes. (E) Representative flow cytometric analysis of intracellular BCL-6 and BLIMP-1 expression in cocultures supplemented with various cytokines (2 ng/mL except for rIL-21, which was used at 10 ng/mL). Histograms are gated on SP CD8 T cells except for the 2 histograms gated on DP3 cells in the top panels. All data shown are representative of 3 separate experiments.