CD70 on CD8α⁺ thymic cDCs contributes to T_reg cell development. (A) Thymic sections were stained sequentially using anti-CD70 mAb, Alexa Fluor 568–conjugated anti-IgG, FITC-conjugated anti-CD11c mAb, and APC-conjugated anti-CD8α mAb. Arrowheads indicate cells with double staining for CD70 and CD11c. (B and C) DCs were enriched from WT or Cd70^Cre/Cre thymi and analyzed by flow cytometry. (B) Shown are representative plots of CD8α versus SIRPα expression on gated CD11c⁺B220^lo DCs. (C) CD8α⁺ and SIRPα⁺ DC subsets were purified from WT and Cd70^Cre/Cre thymi, RNA was isolated, and RT-PCR was performed on cDNA. Shown are PCR products from cDNA samples amplified with primers specific for CD70 and HPRT. (D) CD8α⁺ and SIRPα⁺ CD11c⁺B220^low/− cDCs and CD11c⁺B220⁺ pDCs were purified from the pooled thymi of two WT or Cd70^Cre/Cre mice. The purified DC subsets were co-cultured for 5 d with purified CD4⁺CD25⁻ WT thymocytes. After culture, gated CD4⁺ cells were analyzed for expression of Foxp3 and CD25. Shown is one representative plot of Foxp3 and CD25 expression on gated CD4⁺ cells in the presence of the indicated DC population. (E) Data from D, expressed as the percentage of Foxp3⁺CD25⁺ cells among gated CD4⁺ cells in the presence of the indicated DC populations. Results are from four to five separate wells over two independent experiments. The Mann–Whitney U rank sum test was used to analyze results (*, P < 0.05). Error bars are SEM.