A proximal role for iNOS during T cell priming by Lymphoid Stroma.

<p>(<b>a</b>) Proliferation of OTI cells stimulated with peptide-loaded BMDCs in the presence or absence of FRC.5 cells and either a broad NOS inhibitor (L-NMMA) or the iNOS inhibitor,1400W. (<b>b</b>–<b>c</b>) Proliferation assays identical to that in (a) but measuring the effect of NOS inhibitors under various concentrations of FRC.5 cells. (<b>b</b>) % Blasts quantified, demonstrating FRC inhibition is reversible with iNOS blockade across a range of FRC concentration. (<b>d</b>) Proliferation of OTI cells stimulated with peptide-loaded iNOS-/- BMDCs with or without added FRC. All assays were repeated in a minimum of 2 trials, and typically 4–6 independent trials and all conditions were assayed in triplicate. Error bars represent SD. (<b>e</b>–<b>j</b>) C57/Bl6 or <i>iNOS^−/−</i> mice were immunized subcutaneously with either SL8 peptide-loaded BMDCs (<b>e</b>–<b>g</b>) or anti-DEC205-OVA conjugates (<b>h</b>–<b>j</b>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026138#s4" target="_blank">Methods</a>. Inguinal lymph nodes were isolated 3 days post-immunization, stained and analyzed by flow cytometry. (<b>e,h</b>) Relative recovery of total CD8⁺ cells in the inguinal lymph nodes of C57/Bl6 (n = 4) immunized mice relative to immunized <i>iNOS^−/−</i> mice (n = 4). (<b>f,i</b>) The percentage of tetramer positive cells in the CD8⁺ compartment post-immunization. (<b>g,j</b>) The number of tetramer positive cells in immunized C57/Bl6 and <i>iNOS^−/−</i> mice.