Regulation of T Cell Priming by Lymphoid Stroma were assayed in triplicate. Error bars represent SD. (e–j) C57/Bl6 or iNOS2/2 mice were immunized subcutaneously with either SL8 peptide-loaded BMDCs (e–g) or anti-DEC205-OVA conjugates (h–j) as described in Methods. Inguinal lymph nodes were isolated 3 days post-immunization, stained and analyzed by flow cytometry. (e,h) Relative recovery of total CD8+ cells in the inguinal lymph nodes of C57/Bl6 (n = 4) immunized mice relative to immunized iNOS2/2 mice (n = 4). (f,i) The percentage of tetramer positive cells in the CD8+ compartment post-immunization. (g,j) The number of tetramer positive cells in immunized C57/Bl6 and iNOS2/2 mice. doi:10.1371/journal.pone.0026138.g004 the stimulation of T cells using BMDC from mice genetically lacking the iNOS gene was nevertheless dramatically inhibited by stromal lines (Fig 4d). We noted that T cell inhibition by LEC or BEC stroma appears to operate via a similar iNOS/IFNc dependent mechanism (data not shown). Although it is currently not possible to generate a mouse in which deletion of iNOS is restricted to stroma and bone-marrow chimeras to approximate this fail to fully reconstitute the lymphoid compartment for unknown reasons (data not shown), we nevertheless sought to determine whether our findings were consistent with iNOS acting to attenuate T cell responses in lymph nodes. This was particularly important since previous reports had noted a require- ment for iNOS in augmenting immune responses, likely as a result of its potent anti-microbial activity[20–22]. We therefore turned to the use of a germline nos2 (iNOS) knockout and immunized in ways that should engage the pathway we had studied in vitro and, conversely, in ways that might not engage this mechanism. We additionally chose to use tetramer staining to examine the response derived from endogenous repertoire rather than using a TCR-transgenic adoptive transfer system that may bias the outcome due to abnormally high numbers of antigen-responsive cells. To recapitulate our in vitro studies in which BMDC prime IFNc production and the ensuing iNOS production, we first immunized control or iNOS2/2 mice in the flank using the same BMDCs pulsed with SL8 peptide. In this regimen, BMDC were always iNOS proficient and so any change due to the genetic background of the host would be due host cells beyond the APC. As shown in Fig 4e–g iNOS deficiency resulted in an overall trend towards an increased percentage of tetramer+ cells among total CD8 T cells and a statistically significant increase (1.8 fold) in the absolute number of tetramer+ CD8+ T cells in the lymph node at day 3. As a comparison, we repeated this type of experiment but sought to direct antigens to a resident DC population in the absence of additional overt inflammatory cues. For this, we targeted the relatively non-inflammatory DEC-205+/CD8+ DC subset as antigen presenting cells[23] using anti-DEC205-OVA antibody-protein conjugates[24]. In contrast to what was observed using inflammatory DCs as the priming antigen presenting cells, iNOS deficiency consistently resulted in a mild decrease in T cell expansion as assessed using tetramer staining (Fig 4h–j). Together this suggests that iNOS production during intra- lymph node priming in the first 3 days does represent a mechanism of regulating T cell responses, as predicted by our in vitro experiments. Interestingly, and subject to further study, it also indicates that iNOS is differentially engaged and utilized, depending on the type of challenge and we would propose that this may depend upon the cell types that are stimulated to produce it and the local responses to this. The engagement of this pathway during the first days of lymph node priming will represent fruitful future study to understand the likely multiplicity of its action. FRC inhibition depends on T cell production of IFNc and is Th1 specific A key prediction of these results is that T cells that do not make IFNc will not engage this regulatory mechanism. To formally test this, we assessed FRC-mediated inhibition of OTI cells lacking the interferon gamma gene (IFNc2/2). As shown in Fig 5a, OTI T cells lacking expression of IFNc were completely refractory to FRC-mediated inhibition, even when they were present at fractions as high as 10% of T cells. Confirming that this was truly due to the loss only of IFNc, addition of exogenous IFNc to these cultures re-established inhibition of T cell cycling. This observation raised the possibility that FRCs might specifically block certain types of T cells that make IFNc while sparing those that do not. A classic dichotomy exists in CD4 T cell subsets in which effector T helper 1 (Th1) cells produce IFNc but no IL-4, whereas T helper 2 (Th2) cells produce IL-4 but no IFNc. We therefore tested the prediction that FRCs would specifically block cycling of restimulated Th1 clones while having little or no effect on Th2 cells. As shown in Fig 5b–c, we found this to be the case: Th1 cells loaded with SE-efluor670 dye had a significantly higher MFI, indicative of less dilution and hence fewer rounds of cell division, when cultured with FRCs whereas Th2 cells showed no difference in MFI. Since blasts are heterogeneous in size and proliferate quickly from the start of this assay, we do not observe cells expressing undiluted levels of this dye. For this reason we did not quantify %Blasts for this assay. Figure 5c demonstrates that Th2 cells are insensitive to FRCs across a broad range of concentrations while Th1 cells proliferate less (higher SE- efluor670 MFI) as FRCs are added in increasing concentrations. FRCs block cell-cycle progression but do not block initial TCR recognition We next turned toward dissecting the dynamics of how iNOS functions during the priming response. We first assessed the ability of T cells to form stable conjugates with DCs over time and found no significant difference between T cells activated with BMDCs in the presence of upwards of 25% FRCs (Fig 6a). Similarly, analysis of calcium signaling profiles failed to detect significant differences in the kinetics of intracellular calcium rise over the first 12 minutes post BMDC contact, despite the readily available co-engagement of these cells (Fig 6b). When examining the dynamics of the priming reaction, the first sign of inhibition by FRCs occurred at approximately 24 hours when cultures were examined for homo- typic T-T clusters, correlating with successful activation[25,26]. This timeframe has previously been shown to correspond to early IFNc production in T cells[27]. OTI T cells cultured with SIINFEKL-pulsed DCs in the absence of FRCs displayed robust clusters averaging 1.26104 mm2 in size while average cluster size in the presence of FRC was 0.16104 mm2 (Fig 6c–d). This was, in part, mediated by the action of iNOS/NO production as homotypic clustering was partially restored by blockade with 1400W to an average size of 0.56104 mm2. The remaining inhibition may suggest a second mechanism that alters homotypic adhesiveness although this will be subject of future work. Interestingly, when we assessed CD25 and CD69 expression, markers of early T cell activation, we found only a small difference in CD25 upregulation at 24h when FRCs were added and no difference in CD69 upregulation at this time, regardless of how many FRC were in the culture (Fig 6e–f). In comparison, however, when cells were assessed at 36 hours for cell-cycle progression and the expression of Ki67, a marker of cell cycling, the profiles of T cells activated in the presence of FRC were identical to those of na¨ıve T cells, showing very significant PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e26138