Figure 5. FRC-mediated inhibition of IFNc producing CD4+ and CD8+ T cells but not Th2 cells. (a) Percentage of blasting IFNc2/ 2OTI T cells cultured with peptide-loaded BMDCs and various concentrations of FRC.5 cells with or without exogenous IFNc. (b) Dilution profiles of SE-eFluor670-labeled Th1 or Th2 skewed OTII cells when cultured with peptide-bearing BMDCs in the presence or absence of FRC.5 cells. (c) Quantification of SE-eFluor670 MFI (from (b)) as an index of proliferation (higher MFI represents reduced proliferation.) All experiments are representative of 2 similar trials. All conditions were assayed in triplicate. doi:10.1371/journal.pone.0026138.g005 inhibition of these measures. This effect was titratable: between 1 and 2.5% FRCs, both progression to G2/M and the expression of Ki67 were reduced (Fig 6g–i). From this we conclude that the interactions occurring after initial T-DC interaction, but prior to approximately 36 hours, lead to the predominant inhibition of T cell proliferation. From our other evidence, this is consistent with published reports that OTI T cells generate IFNc within the first 24 hours[27] and (our data) that T cell IFNc production leads to FRC expression of iNOS in a similar timeframe. Consistent with our previous data, inhibition of NO production reverses the block in cell-cycle and overall expansion of the T cells (Fig 6h–i) Regulation of T Cell Priming by Lymphoid Stroma Discussion A key finding of this study is that FRCs are inhibitory to T cell responses. This inhibition of cell-cycle progression clearly depends upon a number of features of the priming reactions. First, FRCs sense early IFNc. Thus, T cells that do not produce extensive cytokines of this class are less restricted. It is interesting, therefore, to note that Th2 cells are completely uninhibited by these effects. This may provide a basal bias towards non- inflammatory Th2 signaling, as T cells making these cytokines might be less likely to provoke inhibitory signaling from stromal sources. Speculatively, it may be that greater selective pressure exists to restrain local Th1-type responses given their ability to directly damage surrounding cells. In contrast, while grossly unrestrained Th2-responses are clearly detrimental, local con- trolled responses are more commonly associated with damage repair and tissue-beneficial roles. To this extent, the most important role of this mechanism might be tissue self-preservation in addition to T cell ‘tolerance’. How inhibitory are FRCs in situ? In our in vitro experiments, we observed inhibition with as few as 1% FRCs relative to T cells. A conservative estimate is that FRCs are on the order of 50–100 times more sparse than T cells on a per-cell basis, and thus our assays are likely in the physiological range of cell number. In vivo, however, effects are almost certainly local. NO itself will not persist in interstitial spaces, largely because it nitrosylates proteins locally and is thereby consumed. Thus, under typical priming situations, there is unlikely to be ‘inhibition at a distance’. Rather only those T cells that remain in contact with iNOS producing cells will be inhibited in this way. This prediction is borne out in our transwell experiments (Fig 2g), which show that inhibition is essentially undetectable when cells are across the barrier, although there are detectable levels of NO in supernatants (Fig 3f). One prediction of this finding is that T cell responses that lead to extensive local ‘stopping’ of motile T cells within regions of the lymph nodes would be more susceptible to this cross-talk as compared to more transient and dynamic priming reactions. As clear modulation of this remains difficult, we are presently unable to directly test this prediction but the concept may force a re-evaluation of whether it will always be beneficial for T cells to stably reside at a single site in the lymph node, particular when IFNc is elicited. A related and important feature of this mechanism is that it is likely to not only be spatial, due to the short-range action of NO, but also to be temporal due to a specific window of IFNc production and then a lag until FRCs respond by making iNOS. Bousso et. al recently showed that activated CD8 T cells produce IFNc within 24-hours[27], thus providing a mechanism for our early IFNc-iNOS axis. However, more typically, this cytokine may peak many hours or even days later. This delay may account for inhibition at 3+ days in vivo. In our hands, the method of priming even for immunization regiments altered the magnitude by which lymph node iNOS production affects priming. To this extent, FRCs may be induced to fully produce iNOS in some very late- stage responses and, given our finding that blasts can be inhibited and that inhibition of na¨ıve cells occurs via late blocks in cell-cycle progression, T cells may also be similarly inhibited by stroma in these responses. While FRCs are inhibitory, they are not alone in this capacity. As shown in Figure 1e, it is likely that many cell types, when added to T+DC priming reactions, may be inhibitory. This may be due to a selection of inhibitory factors or simply nutrient deprivation. However, the addition of IFNc blocking antibodies in the FRC culture shows that the mechanisms by which FRCs attenuate T cell responses are not ‘non-specific’ but rely on direct PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e26138