Lymph Node Fibroblasts Limit T Cell Expansion Figure 7. Lack of iNOS in vivo leads to increased an primary T cell response after viral infection. (A–D) WT or Inos2/2 mice were retro- orbitally grafted with 100’000 splenocytes from OT-I transgenic mice one day prior to subcutaneous infection with VSV-OVA. Draining pLN, blood or spleen from infected mice were harvested at the indicated time points after infection and cell suspensions generated by using collagenase digestion (A) or by mechanical homogenization (B, D). (A) Intracellular iNOS protein expression in TRC (gp38+CD452CD312CD352) or DC (CD11c+MHCII+CD45+) as analyzed by intracellular flow cytometry. Representative histograms show the percentage (6 standard deviation) of iNOS expressing TRC (top) and DC (bottom) in WT versus Inos2/2 mice. (B) The graph on the left side shows the total CD8+ T cell numbers, the graph in the middle the percentage of OT-I T cells among the CD8+ T cells and the graph on the right side the total OT-I number in pLN (pool of 6 draining LN). No significant difference was observed in total LN cell numbers between immunized WT and Inos2/2 mice (not shown). (C) Shows the percentage of OT-I T cells among the CD8+ T cells in the blood, (D) shows the total number of OT-I T cells in the spleen. (A–D) n$3, 1 out of 2 independent experiments is shown. * P,0.05, ** P,0.01, *** P,0.001. doi:10.1371/journal.pone.0027618.g007 self- and foreign-antigens. Whether they also modulate the deletion of self-reactive T cells still needs to be tested. Two previous reports have highlighted the capacity of spleen- derived stromal cells in inhibiting T cell activation. A mix of adherent stromal cells, possibly including TRC, was shown to promote development of BM-derived progenitor cells into IL-10+ regulatory DC [25] or the conversion of mature BM-DC into NO+ regulatory DC [24]. We did not observe consistent changes in the surface phenotype of TRC-conditioned DC, possibly because our co-cultures were less than 16 h allowing only short-term changes. In contrast, the previous reports were based on co-cultures of 1–2 weeks allowing developmental changes. While we have not yet identified the reason for the decreased stimulatory capacity of TRC-conditioned DC it may be due to the rapid induction of iNOS in BM-DC. Our results are reminiscent of observations with MSC known to modulate many aspects of innate and adaptive immunity, including T cell proliferation [30,31]. In vitro BM-derived MSC were shown to block mitogen- and DC-induced T cell prolifer- ation by many different pathways, including NO and PGE2 production [36,39,40]. In settings of allografts and autoimmune disease large numbers of injected MSC strongly suppress T cell responses due to their anti-inflammatory properties, and due to their efficient homing to inflammatory sites and lymphoid tissues [30,31,41]. Interestingly, mature mesenchymal cells such as fibroblasts from various non-lymphoid tissues also dampen T cell expansion [27,28,29,31] suggesting mesenchymal cells at several maturation stages share this property. Alternatively, MSC and fibroblasts may be hard to distinguish [28]. We propose that LN and spleen TRC have conserved this immunosuppressive property that they share with MSC and fibroblasts from non-lymphoid tissues. The extent of inhibition was highly dependent on the number of TRC being present in the culture (unpublished observation) which may explain the difference in the extent of NO-mediated suppression observed in vitro versus in vivo. Our preliminary results suggest that this inhibitory effect not only affects CD8+ but also CD4+ T cells. Besides affecting T cell expansion and possibly apoptosis, TRC presence partially reduced T cell effector differentiation in vitro but not in vivo. While the reason for this difference is not known, it may be explained by the presence of lower NO concentrations or additional factors found in vivo that positively influence CD8+ T cell differentiation. Given PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27618