BM-DC were present in the co-culture with up to 25% TRC being positive (Fig. 6B). Given that BM-DC were occasionally observed to express iNOS in the co-cultures, we assessed the influence of IFNc and TRC on the ability of BM-DC to express iNOS. Interestingly, both IFNc and TRC induced iNOS protein expression in around 3% of BM-DC, but having both IFNc and TRC in the co-culture led to a strong synergistic iNOS expression in up to 30% of BM-DC (Fig. 6B). This synergy was reflected in the almost 10-fold increase in extracellular nitrite levels detectable in the SN (Fig. 6C). Interestingly, more than 50% of the nitrite seemed to be due to iNOS expression in TRC rather than BM-DC as assessed using Inos2/2 BM-DC. At present, a contribution by the two other NOS isoforms to nitrite production can not be excluded. Next we analyzed the speed of iNOS induction in pLN2 by looking at transcript levels. Already 7 h after IFNc stimulation Inos mRNA was induced 10-fold and further increased after 24 h indicating direct transcriptional regulation of the Inos promoter (Fig. 6D). Interestingly, several pro-inflammatory cytokines (IFNa, TNFa, LTa3) or LPS showed a similarly rapid and strong induction of Inos transcripts while their maintenance at 24 h differed. In summary, these results demonstrate that a subset of both TRC and BM-DC can be triggered to express Inos transcripts and proteins leading to the release of NO. Various pro- inflammatory signals can serve as triggers in TRC, including signals derived from newly primed T cells suggesting the possibility of a negative feedback loop leading to inhibition of T cell expansion in our in vitro co-culture assay. Inos deficiency leads to exaggerated CD8+ T cell responses in vivo To characterize the expression of iNOS during immune response in vivo, mice adoptively transferred with OT-I T cells were immunized with OVA-expressing vesicular stomatitis virus (VSV-OVA) and draining LN analyzed. By pressing LN across a 40 mm mesh a soluble fraction containing most hematopoietic cells was obtained as well as a non-soluble stromal cell fraction, including TRC and part of the DC [9,10]. Interestingly, one day after immunization Inos transcripts were induced in both fractions and dropped back to pre-immunization levels on day 2 and 4 (Fig. S5A). These levels were always 5–10-fold higher in the fraction enriched in stromal relative to hematopoietic cells. To identify more precisely the cell type and frequency of iNOS expressing cells in the draining LN intracellular iNOS protein expression was analyzed by flow cytometry along with lineage markers. A subset of at least 2.6% TRC were identified as major iNOS source appearing on day 1 and being again undetectable on day 2 and 4 post infection (Fig. 7A). DC showed a weak induction of iNOS expression on day 1 without reaching statistical significance relative to day 0. Few macrophages (CD11b+ CD11c2) were also found to express iNOS protein (data not shown). This result emphasizes the inducible and transient activation of iNOS both at the mRNA and protein level, consistent with pro-inflammatory molecules inducing it. It also strengthens the notion of more than one cell type expressing iNOS within the T zone of draining LN, most notably TRC and DC. Our attempts to identify and localize iNOS protein-expressing cells in situ were not successful (data not shown), consistent with the low iNOS expression detected by flow cytometry. As a next step the impact of Inos-deficiency on OVA- specific T cell expansion and survival was measured in the LN draining the site of VSV-OVA injection. While on day 4 after immunization the total CD8+ T cell number was similar in both mouse strains the percentage and number of OT-I T cells was 2- fold higher in Inos2/2 relative to WT mice (Fig. 7B). On day 6 and Lymph Node Fibroblasts Limit T Cell Expansion 8 after immunization, the difference in OT-I T cell numbers was minimal between the two strains presumably due to many effector cells having left the LN between day 5 and 8. This scenario is supported by the strong increase in OT-I cells in blood between day 4 and 6 as well as by the significantly higher frequency of OT- I cells in blood of Inos2/2 relative to WT mice (Fig. 7C). As a consequence 2-fold more effector OT-I T cells accumulated within day 8 spleen in Inos2/2 relative to WT mice (Fig. 7D). Interestingly, the differentiation into effector cells occurred efficiently in the absence of iNOS, as based on the analysis of IFNc expression and in vitro killing activity of effector CD8+ T cells from LN and spleen (Fig. S5 B,C). To address whether iNOS expression is critical in hematopoietic or non-hematopoietic cells bone marrow chimeras were generated. Unfortunately, a cell trapping defect was observed in inflamed LN if the stromal cell compartment was Inos-deficient, presumably due to a role of NO in vasodilation in irradiated mice but not straight Inos2/2 mice (Fig. S6). Therefore, no conclusions could be drawn from these experiments. In summary, the in vivo data indicate that acute inflammation associated with a viral infection leads to the transient expression of NO by TRC and DC found within the LN T zone, which slows down and lowers the antigen-specific T cell expansion but does not seem to impact on the differentiation and migration of effector T cells. Discussion Inflamed LN as well as the TRC network are generally considered as strongly immune-stimulatory for T cell immunity. Surprisingly, we obtained several lines of evidence demonstrating an inhibitory role of TRC in early T cell activation: 1) In vitro TRC limit the expansion of CD8+ T cells primed by antigen-pulsed BM- DC or anti-CD3/28 beads at TRC-T cell ratio’s as low as 1:100 with T cell effector function being reduced as well. 2) TRC reduce the T cell activation potential of antigen-loaded BM-DC. 3) TRC constitutively express transcripts for Cox-2, the enzyme required for the synthesis of PGE2 and related factors [37]. Inhibition of Cox-1/2 markedly reduces this suppressive effect. 4) When exposed to pro-inflammatory cytokines pLN2 and ex vivo TRC transiently express iNOS protein leading to NO22 synthesis. This correlates with rapid induction of Inos transcripts in activated pLN2. Inhibition of iNOS or use of Inos-deficient TRC markedly reduces the suppression with this effect being enhanced by a Cox- 2-inhibitor. 5) Upon immunization, iNOS protein gets transiently expressed in TRC and DC within the draining LN. 6) Absence of iNOS in vivo correlates with an exaggerated primary CD8+ T cell response, consistent with the previous observation of an exagger- ated memory T cell response in Inos2/2 mice [38]. Thus, while TRC may have several ways of enhancing T cell activation, our assays have only revealed attenuating effects. Attenuating effects of TRC on T cell immunity have been reported recently based on the expression of self-antigen in the context of MHC I as well as PD-L1 expression on TRC leading to modulation of CD8+ T cell responses [19,20,21]. Here we have used fibroblasts of the LN T zone to show that they are a source of soluble immunomodulators such as NO and COX-1/2 dependent factors strongly inhibiting T cell expansion in response to a foreign antigen, with no role observed for PD-L1. Another study had noted iNOS expression in reticular LN fibroblasts upon infection with Leishmania major parasites, but the precise nature and localization of these cells have remained unclear. In addition, NO was in that case shown to be important for the control of the parasite rather than for adaptive immunity [34]. Together these data suggest TRC may help control CD8+ T cell responses to both PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27618