Characterization of IDE ko mice.
2014-02-07T14:45:56Z (GMT) by
<p><b>A</b>, expression of MHC molecules on wt (black lines) and IDE ko (grey lines) NOD splenocytes. Live cells were gated on TcR-β+ cells, CD19+ cells, CD11c<sup>hi</sup> cells or CD11b+CD11c− cells, and analyzed for expression of H-2K<sup>d</sup> and I-A<sup>g7</sup>. Isotype controls are shown in open, and specific stainings in filled histograms. <b>B</b>, Splenocytes from IDE wt and ko C57BL/6 mice were subjected to a 90 s treatment with acid, incubated with 10% FCS complete medium for the indicated periods to allow for re-expression of MHC-I molecules, and then stained for H2-K<sup>b</sup> with mAb AF6-88.5, using gates on CD11c+ DCs (top panels, experiment represented as FACS plots) and CD19+ B cells (bottom panel, means ± SDEV of 2 experiments represented as histogram). Numbers on histograms in the top panels indicate MFI for AF6-88.5. <b>C</b>, 25,000 TCR-transgenic CFSE-labeled BDC2.5 CD4+ T cells were put in contact with 20,000 sorted splenic CD11c<sup>hi</sup> DCs incubated with graded amounts of the fusion proteins P3UOp31 or P3UO (Ctrl) in complexes with an anti-CD11c mAb. After 2 days (left panel) or 4 days (right panel), T cell activation was assessed by measuring the IL-2 concentration in the culture supernatant and proliferation was measured by flow cytometry as dilution of CFSE, respectively. The numbers next to the FACS plots indicate the division index of progenitor cells, calculated as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088365#pone.0088365-Kratzer1" target="_blank"></a>. One out of 2 experiments. <b>D</b>, priming of CD4<sup>+</sup> T cells in IDE-deficient mice. CFSE-labeled BDC2.5 T cells were injected into wt and IDE-deficient mice. Twenty-four hours later, 500 ng of the P3UOp31 or P3UO fusion proteins in complexes with a CD11c-specific mAb were injected s.c. Four days after antigen injection, CD4<sup>+</sup>/Vβ4<sup>+</sup> T cells recovered from draining and pancreatic lymph nodes were assessed for CFSE dilution by flow cytometry and the division index of injected precursors was calculated. Two independent experiments were performed, with a total of 5 or 6 mice per condition. Means and SDEV are shown.</p>