cell lines each resulted in nearly complete inhibition of the induced proliferation. As T cell activation by DCs typically occurs in the T cell zone of lymph nodes, scaffolded by FRCs, we focused predominantly on these stromal cells and lines for subsequent studies. We first confirmed that freshly isolated gp38+CD312 CD452 FRCs were similarly inhibitory by assaying them in our T+DC stimulation cultures. In these assays, we compared FRC isolates to a similar number of total lymph node cells and found the FRCs (Fig 1d) again to be inhibitory for proliferation of CD8 T cells. To test for the specificity of inhibition, we also compared the FRC.5 line to mouse embryonic fibroblast lines (MEF) and fibroblasts (NIH-3T3) and found that whereas the latter lines also were modestly inhibitory for proliferation, the FRC.5 line consistently inhibited to a greater extent (Fig 1e). Given that the FRCs are present proximally in the LN during priming whereas BEC and LEC are not, we focused on the mechanism underlying their inhibition of the response. Inhibition of T cell activation by FRCs depends upon interferon gamma production during priming We first sought to determine the magnitude of T cell inhibition and if it involved interactions between FRCs and DCs or was a direct effect upon T cells, regardless of DC presence. In Fig 2a, we added FRC.5 cells at varying levels to CFSE-loaded T cells and primed with a selection of stimuli. A 10% fraction of FRCs provided nearly complete blockade of T cell proliferation in response to BMDCs loaded with SL8, plate-bound anti-CD3 +/2 soluble anti-CD28, or PMA plus ionomycin. However, when a smaller FRC concentration was used, there was little or no effect in some conditions but partial inhibition in the presence of aCD3 and aCD28. This implied that FRCs were not simply globally repressing responses but, rather, that they did so specifically in response to cues derived from the strength or type of activation of the T cells. We quantified this finding by a different criterion in Fig 2b–c. Here, we scored for the percentage of cells in the culture that were in or beyond the first division, and term this ‘‘% blasts’’. By this criterion, which measures the degree to which cells are allowed to begin cycling, a greater distinction in the threshold of FRC-based inhibition emerged. We interpret from this that FRCs can very effectively inhibit the first cell division in some conditions (i.e. at 2.5% or more) such that very few cells progress to becoming ‘blasts’. In contrast, at the lower fractions (i.e. 1%), they inhibit this to a lesser extent but, for some priming conditions (e.g. BMDC), can prevent the maximal extent of cell cycling. We used this same metric, while varying the number of BMDCs in the culture in order to test whether higher densities of DCs could overcome inhibition (Fig 2d). This revealed that the inhibition at the higher levels of FRCs could not be overcome with greater numbers of DCs, strongly suggesting that the inhibitory effect was not due to FRCs somehow confining DCs from T cells. The exclusion of such a mechanism was also supported by the ability of FRCs to inhibit T cell proliferation to the fully soluble PMA and Ionomycin stimulus (Fig 2a and Fig 2c). Since it has been shown that distinct priming conditions result in differences in both the quantity and quality of cytokines produced by blasting T cells[13,14], we sought to determine the effect of blocking select cytokines or adding them back to T+DC assays (Fig 2e). While we saw little effect of aIL4 or aIL-12 antibodies (data not shown), we found that addition of aIFNc almost fully restored proliferation of T cells cultured with DCs in the presence of FRCs. Interleukin 2 nor aIL-2 had a significant effect on FRC- mediated inhibition of CFSE dilution. This implied that, at a strength of stimulation resulting in strong IFNc secretion, FRCs Regulation of T Cell Priming by Lymphoid Stroma were licensed to inhibit the T cell response. That FRC might be responsive to IFNc was also supported by their robust expression of the IFNc receptor 1 (Fig 2f) When FRCs were separated from T cells and DCs by a semi- permeable membrane, the inhibitory effect was lost (Fig 2g). Addition of IFNc itself also could not restore the inhibition in the transwell context, suggesting that a lack of IFNc diffusion across the transwell didn’t explain this effect. Combined with Fig 2e, this implied that, while IFNc was involved, the inhibitory mechanism required cell-cell or very close proximity between T Cells, DC, and the modulating FRC population. Regulation of T cell priming by IFNc-promoted Stromal iNOS We therefore turned toward assessing mechanisms by which FRCs might inhibit T cell responses. Recent reports have shown that viral infection of FRCs results in their upregulation of PDL-1, an inhibitory molecule for T cells[12]. We assessed levels of PDL-1 as well as the costimulatory ligands CD86 (Fig 3a) and CD80 (data not shown). Whereas CD80 and CD86 levels were low and remained unchanged, PDL-1 levels were indeed upregulated in FRCs treated with IFNc. However, blocking these pathways did not appreciably alter the proliferation of T cells in our assays (Fig 3b), suggesting another mechanism. Since IFNc appeared to be necessary for inhibition, we next examined the transcription of nos2 gene and protein expression and the production of nitric oxide (NO) which had previously been shown to be IFNc- inducible in a selection of non-hematopoietic cells[15,16] as well as cells of hematopoietic origin[17,18]. The nos2 gene encodes the enzyme inducible nitric oxide synthase (iNOS), which is responsible for production of nitric oxide and can be specifically inhibited by the drug 1400W. As shown in Fig 3c, addition of IFNc directly to FRC lines in the absence of T cells or DCs results in an approximate 10-fold increase in transcription of the nos2 gene, an effect that was not directly altered by blocking the enzyme itself with 1400W. Using antibodies to iNOS protein, we were able to detect significant signal by immunofluorescence in FRC lines and these levels were both quantitatively higher and more localized to the cytosol in the presence of T plus DC (Fig 3d, arrowhead highlights T-DC couple). Flow cytometry con- firmed an upregulation of total iNOS levels in response to T+DC augmented, compared to control (DC alone), and that this augmentation could be blocked with antibodies to IFNc (Fig 3e). We next measured accumulation of nitrite, a relatively-stable end-product of nitric oxide, in our cultures using a standard Griess assay[19]. Here, we found that addition of exogenous IFNc to FRC cultures failed to increase production of nitrite to detectable levels (Fig 3f), an effect that may suggest that release or further activation of the enzyme requires additional signals delivered with T cell activation. However, nitrite was detected when T cells were stimulated in those cultures using SL8-loaded BMDCs, and this was considerably augmented by inclusion of FRC in a concentra- tion-dependent manner. Nitrite could not be detected when unstimulated FRCs were plated. Finally, the nitrite that was detected when FRCs were mixed with T cells plus peptide-loaded DCs was blocked by the iNOS-specific inhibitor 1400W. To test the emerging hypothesis that iNOS production was responsible for FRC inhibition of T cell responses, we mixed T cells plus SL8-loaded DCs together with FRCs in the presence or absence of iNOS inhibition. As shown in Fig 4a, we could completely restore proliferation of OTI CD8+ T cells by blocking iNOS function with 1400W. Similarly, we could restore prolifer- ation using the drug L-NMMA, a broader-spectrum NOS inhibitor. When quantified as % blasts, this drug completely released cells to PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e26138