Anti-4-1BB-activated PDCA-1⁺ B cells inhibit Ag-specific T cell responses.

<p><b><u>A</u></b> Purified PDCA⁺ B cells were treated for 3d with indicated markers and Annexin V assay was performed as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050272#s4" target="_blank">Materials and methods</a>. The events in the boxes represent cells undergoing apoptosis. None, no stimulation for 72 hrs. One of 2 independent experiments is shown (<i>n</i> = 2). <b>B</b> Purified PDCA⁺ B cells from the bone marrows of naïve Balb/C mice were stimulated for 3d with IL-7 (100 ng/ml) or soluble anti-4-1BB (5 µg/ml) and washed, and Ag uptake assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050272#s4" target="_blank"><i>Materials and Methods</i></a>. The cells were stained as indicated and flow cytometry was performed. One of two independent experiments is shown. <i>n</i> = 2. <b>C</b> Purified PDCA-1⁺ B cells were stimulated for 3d with IL-7 (100 ng/ml) or soluble anti-4-1BB (5 µg/ml), pulsed with 0.5 mg OVA^323–339 peptide, and incubated with purified CFSE-labeled DO11.10 CD4⁺ T cells for 3d. They were stained as indicated and flow cytometry was performed. One of two independent experiments is shown. <i>n</i> = 2. Gates were set around CFSE⁺ cells, and proportions of Vα2⁺ cells were enumerated. <b>D</b> Purified PDCA-1⁺ B cells from naïve Balb/C mice were pulsed with OVA^323–339 as in experiment <b>B</b>, and adoptively transferred (2×10⁶/mouse) into syngeneic DO11.10 mice. The recipient DO11.10 mice were further treated with BrdU as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050272#s4" target="_blank"><i>Materials and Methods</i></a>. Spleen cells from the treated mice were prepared 3d after cell transfer, stained as indicated, and flow cytometry was performed. One of two independent experiments is shown. Gates were set around Vα2⁺CD4⁺ cells, and Vα2⁺BrdU⁺ cells were enumerated.