Lymph Node Fibroblasts Limit T Cell Expansion Figure 6. iNOS expression in TRC can be induced by IFNc. (A) Immunohistochemical analysis of iNOS expression in pLN2 TRC lines. pLN2 were cultured for 2 days either alone (1st row), in the presence of WT T cells without (2nd row), with anti-CD3/28 beads (3rd row), or in the in the presence of recombinant IFNc bottom row. The first column shows iNOS expression in red and nuclei stained with DAPI in blue, the second column shows iNOS in red and gp38+ TRC in green. Tiny nuclei are from T cells, larger nuclei from BM-DC and pLN2. Scale bar: 50 mm. (B) Flow cytometric analysis of intracellular iNOS expression in pLN2 TRC or BM-DC, cultured for 2 days either alone or together and with or without recombinant IFNc. The histograms show the % of iNOS-expressing TRC (gp38+CD452CD312; upper panel) or BM-DC (CD45+; lower panel) (6 standard deviation). (C) NO22 concentration in the supernatant from the co-cultures shown in (b). (D) Quantitative RT-PCR analysis for Inos mRNA of pLN2 cells stimulated with various cytokines or agonistic anti-LTbR (aLTbR) antibody for 7 h or 24 h. The expression levels relative to two housekeeping genes are shown. Different symbols indicate different experiments. (A,D) n = 3–5, representative for 2 experiments. (B,C) n = 2, 1 experiment; * P,0.05, ** P,0.01, *** P,0.001; if not indicated otherwise P-values are relative to ‘no stroma’. doi:10.1371/journal.pone.0027618.g006 PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27618