Lymph Node Fibroblasts Limit T Cell Expansion Figure 1. TRC dampen the expansion of antigen-specific CD8+ T cells. Flow cytometric analysis of T cell activation: CFSE-labeled OT-I T cells were mixed in a ratio of 1:50 with WT T cells (50% CFSE-labeled) and cultured with LPS-activated and OVA-peptide pulsed BM-DC. The assay was performed without stromal cells (‘no stroma’) or in the presence of TRC from distinct sources (1:1:200 ratio of TRC to BM-DC, and to T cells). (A) Total OT-I numbers on day 2, 3 and 4 in absence or presence of the pLN2 TRC line. (B) Proliferation of OT-I (black) or polyclonal T cells (gray) as assessed by CFSE dilution on day 3 or 4 with or without pLN2. Histograms (left) show the percentage (6 standard deviation) of divided OT-I cells and bar graphs the absolute OT-I cell numbers per CFSE dilution peak (right). (C) Dot plots (left) show CFSE dilution versus cell size (FSC) or CD44 expression of OT-I T cells after 3 days. Bar graphs (right) indicate the median FSC values or fluorescence intensity. (D) Histograms show CD62L expression and bar graphs the percentage of CD62Llow OT-I T cells. (E) As c, but showing CD25 expression on OT-I T cells after 4 days of co-culture with or without pLN2. (F,G) Bar graphs show the percentage inhibition of OT-I T cell proliferation as based on the proliferating OT-I cell numbers after 3 days of co-culture with different TRC lines (F) or ex vivo isolated and enriched TRC (G). The latter are derived from pLN of unmanipulated B6 mice or from mice s.c. injected with NP-CGG/Montanide 3 days prior to the TRC harvest (‘activated TRC’). Different symbols indicate different experiments. Data are representative of .10 (A–D), 3 (E,G), 2–3 (F) experiments; different symbols indicate different experiments. * P,0.05, ** P,0.01, *** P,0.001, P-values relate to ‘no stroma’ controls. doi:10.1371/journal.pone.0027618.g001 PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27618