Regulation of T Cell Priming by Lymphoid Stroma Figure 6. Inhibition of T cell activation by LN stromal lines occurs largely at the level of cell-cycle progression. (a) Percentage of coupled OTI T cells and peptide-loaded BMDCs in the presence of various concentrations of FRC.5 cells. Cultures were analyzed at the times indicated post addition of OTIs. (b) Ratiometric measurement of cytosolic Ca2+ flux in FURA-2AM loaded OTI T cells, immediately after contact with peptide- bearing BMDCs in the presence or absence of FRC.5 cells. (c) Analysis of OTI and BMDC cluster area in the presence or absence of FRC.5 cells and iNOS inhibitor (1400W). (d) Images of T cell clusters under the conditions described in (c). Scale bar represents 100 um. (c,d) Cultures were incubated for 48 hours prior to imaging analysis. (e) Surface staining of OTI T cells for activation markers CD25 and CD69 after 24 hours when cultured with peptide-loaded BMDCs in the presence or absence of FRC.5 cells. Stains are compared to non-activated, na¨ıve OTI T cells. (f) Percent of OTI cells with upregulated CD25 and CD69 levels following culture with BMDCs and various concentrations of FRC.5 cells for 36 hours. (g) Propidium iodide (PI) incorporation and Ki67 levels in OTI T cells at 36 hours under identical culture conditions as those in (e). (h–i) PI incorporation and Ki67 levels in OTI cells with various concentrations of FRC.5 cells. All experiments are representative of a minimum of 3 similar trials. doi:10.1371/journal.pone.0026138.g006 PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e26138