Lymph Node Fibroblasts Limit T Cell Expansion Figure 4. TRC dampen T cell proliferation by modifying both T cells as well as BM-DC. (A) Flow cytometric analysis of T cell proliferation induced by anti-CD3/CD28 beads as based on CFSE dilution after 3 days of culture. WT T cells were cultured with beads and in presence of the pLN2 TRC line (T cell to TRC ratio of 100:1) or their absence (‘no stroma’). Representative histograms (top) show the percentage (6 standard deviation) of divided CD8+ T cells and bar graphs (bottom) the absolute CD8+ T cell numbers per division. The bar graph (right) shows percentage of inhibition (as in Fig. 1). Similar data were obtained for CD4+ T cells suggesting TRC may also suppress their expansion (data not shown). (B) Flow cytometric analysis of cell proliferation induced by TRC-conditioned BM-DC. LPS-activated and SIINFEKL-pulsed BM-DC were co-cultured with pLN2 TRC at a ratio of 1:1, or cultured alone. After overnight culture TRC-conditioned-BM-DC were re-isolated by MACS, counted and subsequently co-cultured for 3 days with CFSE labeled OT-I cells mixed 1:50 with unspecific WT T cells (of which 50% are CFSE labeled). Representative histograms (top) show the percentage (6 standard deviation) of divided OT-I cells and bar graphs (bottom) the absolute cell numbers per CFSE dilution peak. Percentage of inhibition (as in Fig. 1) is shown in the bar graph on the right. (A,B) n = 3, (A) representative for $3 experiments, (B) representative for 2 experiments. * 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.g004 iNOS (NOS2) or cyclooxygenase (Cox) 21 and 22 partially rescued T cell proliferation with the effects of the two pharmacological inhibitors used being partially additive (Fig. 5B). This result demonstrates that iNOS-dependent NO and Cox-1/2 dependent factors are responsible for most of the inhibitory effect observed. We focused our attention on iNOS as transcripts for Inos but not Cox2 were enhanced in stimulated TRC (as shown later). Strikingly, Inos2/2 TRC had lost all suppressive activity and showed even an enhancement of T cell proliferation, in contrast to the inhibitor 1400W that partially abolished the suppressive effect of ex vivo WT TRC (Fig. 5C), reminiscent of the effects seen for the pLN2 line (Fig. 5B). To show directly the expression of iNOS protein in TRC and assess the frequency of iNOS-expressing cells, antibodies to iNOS were used in immunofluorescence microscopy. Surprisingly, iNOS protein could only be detected in pLN2 co- cultures with BM-DC and activated T cells. However, iNOS was only detected in a small fraction of gp38+ TRC (Fig. 5D). Occasionally, iNOS protein staining was observed in BM-DC-like cells (gp382) but never in lymphocytes (not shown). These experiments establish that TRC are a source of iNOS that inhibits T cell proliferation. They also demonstrate that the inhibitory activity of TRC on T cell expansion is not due to metabolic effects of co-culture. As a readout of iNOS activity, we measured extracellular nitrite levels in the SN. In absence of TRC the SN of T cells activated by BM-DC contained very low levels of nitrite. Presence of pLN2 or ex vivo TRC in this assay increased the NO22 concentration 2- and 5-fold, respectively (Fig. 5E), further strengthening the notion that NO production by TRC leads to inhibition of T cell proliferation or survival. In the case of ex vivo TRC approximately half of the nitrite was due to iNOS activity in TRC as suggested by the use of 1400W or Inos2/2 TRC. They also suggest a contribution of NO production by one of the two other NOS isoforms which are insensitive to the concentrations of 1400W used and possibly expressed in TRC or in the few endothelial cells found within the adherent cell fraction. As the levels of iNOS protein correlate better with NO-mediated inhibition of T cell expansion than those of extracellular nitrite, the subsequent analysis was focused on the factors inducing iNOS expression in TRC. IFNc and other T cell derived cytokines induce iNOS protein expression in TRC To look at the role of T cells in inducing iNOS protein in TRC, pLN2 were co-cultured with T cells and fixed cells analyzed for intracellular iNOS protein by immunohistology. Non-activated T cells induced iNOS in only a few TRC whereas T cells activated with anti-CD3/CD28 beads up-regulated iNOS in a large proportion of TRC (Fig. 6A). This suggests that most TRC have the potential to express iNOS upon activation but typically only a small proportion shows detectable protein. IFNc is a T cell- derived cytokine known to induce iNOS expression in various cell types [33,34,35,36]. Indeed, adding recombinant IFNc was sufficient to induce iNOS expression in a fraction of pLN2 (Fig. 6A). Comparable results were obtained with ex vivo LN cells when the adherent cells enriched for gp38+CD312 TRC were used in presence of na¨ıve or activated T cells or IFNc (Fig. S4A). Consistent with these data a significant increase in nitrite was observed in the SN of TRC lines or ex vivo TRC upon IFNc stimulation or upon addition of T cells with or without anti-CD3/ 28 activation (Fig. S4B). To obtain a more quantitative assessment of iNOS-expressing TRC, iNOS-expressing cells were measured using intracellular staining and flow cytometry. This analysis showed that while IFNc induced few TRC to become iNOS- positive, there was a strong synergistic effect when both IFNc and PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27618