RAG1⁺ pDC1 and RAG1^- pDC2 subsets express pDC-related genes but differ with respect to cytokine production and T-cell allostimulation. (A) RT-PCR analyses were performed with cDNA from freshly purified RAG1⁺ pDC1s, RAG1^- pDC2s, B220^-CD11c⁺ DCs, and Lin^-ckit⁺ cells. Primer pairs were used to detect transcripts for ICSBP, TLR9, TLR4, RelB, and SpiB along with β-actin as a control. (B) The 2 categories of pDCs (▦, pDC1; ▪, pDC2) were stimulated for 12 hours with CpG-ODNs. Supernatants were analyzed for IFNα, TNFα, IL-6, IL-12p70, and IFNγ levels by ELISA. Results are presented as the mean ± SD of 3 different experiments. (C) Expression of costimulatory molecules was assessed on pDC1s and pDC2s after 18-hour stimulation with CpG-ODNs. Dotted lines indicate unstimulated cells. (D) Preactivated RAG1⁺ pDC1s (□), RAG1^- pDC2s (○), and B220^-CD11c⁺ DCs (▵) were cultured for 6 days with 1 × 10⁵ allogeneic T cells. Proliferation of TCRβ⁺ cells was measured by BrdU incorporation as described in “Material and methods.” Of 3 experiments, 1 representative is shown. Results are presented as the mean ± SD of 3 different culture wells; one representative experiment of 3 is shown. (E) Intracellular cytokine accumulation was determined in T cells 6 days after priming with preactivated pDC1s or pDC2s, and 5-hour restimulation with PMA and ionomycin.