Lymph Node Fibroblasts Limit T Cell Expansion Figure 5. Suppression by TRC is mediated via NO and COX-1/2 dependant factors. (A,B,C) Flow cytometric analysis of the T cell activation assay (as described in Fig. 1): (A) pLN2-TRC in the assay were present in the same well (‘normal’), separated by a permeable membrane (transwell) or 30% pLN2-TRC-conditioned-supernatant taken after 72 h from sub-confluent irradiated cultures was added to the cultures. The bar graph shows percentage of inhibition. (B) Pharmacological inhibitors against iNOS (1400W, 1 mM) and/or Cox-1/2 (indomethacin, 10 mM) were added to the co- cultures after optimization of their concentrations (tested range for 1400W: 1–10 mM; for indomethacin: 1–10 mM). The middle panel shows the percentage of divided OT-I cells (6 standard deviation) and the bar graph (right) shows the percentage of inhibition. (C) T cell activation assay was carried out in the presence of ex vivo isolated enriched TRC from WT or Inos2/2 mice along with the indicated inhibitors. The right panel shows the percentage of divided OT-I cells (6 standard deviation) and the bar graph (middle) shows the percentage of inhibition. (D) Immunohistochemical analysis of iNOS expression in pLN2 TRC lines. pLN2 cells were cultured alone (top) or in presence of OT-I and WT T cells together with LPS activated, SIINFEKL-pulsed BM-DC (ratios as in Fig. 1) (bottom). Insets of higher resolution images are shown to indicate the generally increased iNOS expression level in pLN2 co-cultured with BM-DC, antigen and T cells. (E) Bar graph showing NO22 levels in the co-culture supernatant from the T cell activation assay shown in (b,c) as determined using a Griess assay. (A,B,C,E) n = 3, (D) n = 3–4, (A,C) representative for 2, (B) representative for $3 experiments, (D) 1 experiment; * P,0.05, ** P,0.01, *** P,0.001, if not indicated otherwise P-values are relative ‘no stroma’. doi:10.1371/journal.pone.0027618.g005 PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27618