IL-21-induced GrB production in pDCs inhibits allogeneic CD4⁺ T cell proliferation. Freshly isolated pDCs from blood were preactivated for 2 days in medium with or without the TLR7 agonist R848 (10 μg/mL) and in the presence or absence of IL-21 (25 ng/mL) as indicated. After extensive washing, pDCs were cocultured with freshly isolated allogeneic CD4⁺ T cells (ratio pDC:T cell = 1:5) after labeling with the CellTrace violet dye. After 6 days, T cells were analyzed by flow cytometry for expression of CD3, the fluorescent CellTrace violet dye, and 7-AAD. Dot plots shown are gated on CD3+ T cells. Numbers represent percentages of cells in the indicated quadrants. CellTrace-violet^lo7-AAD⁻CD3⁺ cells (lower left panel) represent living T cells that have proliferated. CD4⁺ T cells activated with anti-CD3/CD28 beads are shown as a positive control for proliferation (black line histogram) in comparison with CD4⁺ T cells cultured with medium only (gray-filled histogram). The GrB inhibitor Z-AAD-CMK (5 μg/mL) was added during the pDC/T cell coculture. (B) CD4+ T cell expansion was measured as described in A. Shown are the mean percentages of CellTrace-violet^lo7-AAD⁻CD3⁺ cells from 4 donors (*P < .05). Error bars indicate standard deviation values.