Fas-independent T-cell apoptosis in DC-transferred MRL/<i>lpr</i> mice.

<p>(A) Experimental protocol of T cell apoptosis by repeated co-culture with DCs. Total T cells from MRL/<i>lpr</i> mice (5×10⁴) were repeatedly (three times) co-cultured with BMDCs (2.5×10⁵) from MRL+/+ or MRL/<i>lpr</i> mice for 8 hours without interval. BMDCs were stimulated with RANKL and CII for 24 hours before the co-culturing. (B) After the third co-culture, apoptosis of CD4⁺ and CD8⁺ T cells expressing annexin-V was detected by flow cytometry. Staining of T cell with FITC-labeled isotype control Ab is shown as a dotted line. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048798#s2" target="_blank">Results</a> are shown as representative of three independent experiments with similar results. (C) MFI of annexin-V on CD4⁺ T cells was calculated, and the data are shown as the means ± SD of triplicate samples. (D) Induction of T-cell apoptosis by repeated co-culturing with activated DCs. Purified CD4⁺, CD8⁺, DNT, and B220⁺ cells (5×10⁴) were repeatedly co-cultured with BMDCs (5 and 25×10⁴). Annexin-V⁺ cells are shown as the means ± SD of triplicate samples. The experiments were repeated three times with similar results. *p<0.05. (E) BMDCs were stimulated with RANKL and, OVA or BSA (10 µg/ml) for 24 hours before the co-culturing. Purified CD4⁺ cells (5×10⁴) were repeatedly co-cultured with BMDCs (5 and 25×10⁴). Annexin-V⁺ cells are shown as the means ± SD of triplicate samples. The experiments were repeated three times with similar results. (F) Purified CD4⁺ cells (5×10⁴) from ILNs in MRL+/+ mice were repeatedly co-cultured with <i>lpr</i> BMDCs (25×10⁴). Annexin-V⁺ cells are shown as the means ± SD of triplicate samples. The experiments were repeated three times with similar results.