that TRC and non-lymphoid fibroblasts can be grown easily from many accessible tissues in humans, such as tonsils or skin, they may represent an interesting source of immunomodulatory cells. A very recent study using skin fibroblasts has established their potential in efficiently suppressing the clinical signs of experimental arthritis in mice [29]. Different mechanisms of suppression have been described for MSC: they can either directly inhibit T cell activation or reduce the stimulatory capacity of DC by using several pathways [30,31]. MSC can produce high amounts of NO upon contact with T cells, which interferes with STAT-5 phosphorylation and therefore IL-2 signaling [42]. MSC-mediated T cell suppression could be partially neutralized using inhibitors of iNOS or Cox-2, or using Inos2/2 MSC [36,40] thus showing clear parallels in MSC- and TRC-mediated T cell mechanisms. In our in vitro experiments we have obtained evidence for an additive inhibitory effect of NO and COX-1/2-dependent factors (possibly PGE2), but additional factor(s) probably remain to be identified to explain the entire inhibitory effect. Blocking TGFb, PD-L1, IL-10, arginase-1 and IDO activity did not influence TRC mediated inhibition, however, additional studies will be necessary to fully rule out their contribution. Furthermore, we cannot exclude that the other two NOS isoforms, eNOS and nNOS, are expressed in TRC and contribute to the TRC mediated inhibition. The expression and activity of iNOS is tightly controlled at several levels with the best-characterized positive regulators being IFNc IFNab, TNFa IL-1 and LPS [35]. Indeed, iNOS expression in TRC was induced by all of these pro-inflammatory cytokines confirming and extending previous data using LN fibroblasts, MEF and MSC [33,34,35,36]. Interestingly, only a subset of IFNc-stimulated TRC expressed iNOS, even when a TRC clone was used (data not shown), in line with previous reports on other fibroblast types [33,34] as well as our observation on BM-DC reported here. Recent results suggest that IFNc is not restricted to immune synapses but more widely accessible throughout the T zone [43] which may explain why TRC obtain this signal in the absence of cognate interaction with primed T cells. Several reports showed that IFNc during T cell priming influences the degree of CD8+ T cell contraction (reviewed in [44]). While IFNc signaling within T cells may lead to this outcome [44], our results suggest that IFNc-signalling in TRC and DC leading to NO production early after infection might not only control the expansion, but also the contraction phase of CD8+ T cells, such as by nitrosylating and thereby altering key proteins of the IL-2 or TCR signaling pathway [42,45]. More experiments will be necessary to test this hypothesis and to understand the underlying mechanism. Strikingly, iNOS induction and NO22 production was highest when TRC, activated DC and IFNc were present together suggesting only adjacent TRC become licensed to suppress T cell expansion with this being a highly controlled and transient process. The DC-derived signals leading to iNOS expression in TRC remain to be identified. Good candidates are IL-1a/b and TNFa which may act synergistically with IFNc, similar to iNOS expression in MSC [36]. NO effects are known to be highly dose- dependent. Low NO doses can enhance T cell proliferation, as possibly suggested by the decreased T cell expansion in some of the BM chimera experiments (Inos2/2 into wt). Higher NO doses are known to induce cell-cycle arrest and apoptosis of T cells [35,46] indicating that the reduced expansion seen in presence of TRC may represent a combined effect of reduced proliferation and increased apoptosis of T cells. The dual role of NO may also explain the only two-fold higher expansion of T cells in the absence of iNOS. Given that NO is a highly reactive gas close Lymph Node Fibroblasts Limit T Cell Expansion proximity to TRC is likely to be critical in inhibiting T cell and DC activation. To further improve this process cytokine- stimulated TRC could actively recruit or retain primed T cells, as suggested for MSC [36]. Interestingly, TNFa-stimulated TRC were shown to express the IFNc-inducible chemokine CXCL10 that attracts activated but not na¨ıve T cells [47]. Both in vitro and in vivo, we found not only TRC but also DC to express iNOS and produce NO. It is possible that the iNOS+ DC subset identified in vivo represents the inflammatory TipDC (TNFa+iNOS+CCR2+) which are recruited into the splenic T zone early during infection with Listeria monocytogenes [48] or other pathogens [49] as they were also CD11cint in our analysis (unpublished observation). Interestingly, increased T cell prolifer- ation was also reported for Ccr22/2 mice lacking TipDC [48]. In vitro we found iNOS expression in BM-DC to be induced in a synergistic manner by IFNc and TRC presence, similar to synergistic effects of IFNc and BM-DC on iNOS expression by TRC. While the factors involved in the cellular crosstalk remain to be identified these findings point to the interdependence of these two cell types in inhibition of T cell expansion, an observation which needs further in vivo studies with cell-type specific Inos deletion. Based on our current results we propose the following working model (Fig. S7): Low levels of iNOS expression are induced in both immigrating DC and TRC when they meet in the T zone early during the immune response. This may increase the threshold required for T cell priming and thereby prevent the activation of low-affinity T cells that are often self-reactive. Once the first T cells are primed and produce IFNc they may boost iNOS expression in both cell types in a transient fashion thereby inducing a negative feedback loop dampening T cell expansion by either slowing down their proliferation or diminishing their survival. The immunosuppressive feature of mesenchymal cells is intriguing. It raises the question of why these cells have this function, especially when considering TRC localize to the T zone of SLO where inhibitory effects are less expected but well known from TipDC and regulatory T cells. We would like to propose two possible reasons: 1) Keeping the LN in a slightly suppressive state might be a mechanism to limit the activation of potentially auto- reactive cells while allowing strong immune responses to foreign antigens to occur. In line with this hypothesis we found Cox2 transcripts to be highly expressed in na¨ıve LN. Others showed that high iNOS and NO22 levels are frequently associated with autoimmune disease and Th1-mediated inflammation [35,46]. Consistent with a protective role of this pathway in certain autoimmune diseases, Inos2/2 and Ifng2/2 mice have a higher incidence, increased severity and less relapses of experimental autoimmune encephalitis that correlate with increased prolifera- tion of auto-reactive T cells relative to WT mice [50,51]. Similarly, in a mouse model for myasthenia gravis Inos2/2 mice develop more self-reactive T and B cells, including epitope spreading [52]. 2) In contrast to recirculating hematopoietic cells fibroblasts resident in lymphoid and non-lymphoid organs have a function for the organ itself: ensure its integrity and functionality. During injury, such as infection or inflammation, fibroblasts perform tissue repair to reestablish homeostasis. LN swelling during immune response can be viewed as a threat to the organ as it is characterized by the dramatic increase in organ size due to the influx of many na¨ıve lymphocytes as well as the rapid expansion of antigen-specific lymphocytes. The strong increase in lymphocyte numbers within the T zone may be harmful for the existing TRC network that functions as the structural platform for T cell priming and probably also T cell differentiation. Destruction of the TRC architecture, such as after LCMV infection, correlates well with PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27618