Regulation of T Cell Priming by Lymphoid Stroma PMA and ionomycin and (d) various concentrations of total peptide-loaded BMDCs. (e) CFSE-based proliferation assay identical to that in (a) but also including soluble cytokines or cytokine-blocking antibodies that were added at the start of the assay as indicated in the legend. (f) Expression of the IFNc receptor in FRC.5 or freshly ex vivo isolated FRC was analyzed by FACS and compared to reagent control. (g) Proliferation assay of SE-efluor670 labeled OTI cells. 66105 OTI cells and 66104 peptide-loaded BMDCs cultured in contact with 66104 FRC.5 cells (proximal) or separated from FRC.5 cells by a 0.4um transwell membrane (distal). All assays were repeated in a minimum of 2 and typically 4–6 independent trials and all conditions were assayed in triplicate. Error bars represent SD. doi:10.1371/journal.pone.0026138.g002 proceed into cycle (Fig 4b–c) with only very modest inhibition of complete cycling of cells as assessed by CFSE peaks. As iNOS has clearly been described as being produced by a variety of macrophages[17,18], we wished to formally exclude that our BMDC were not in some way responsible for the production and inhibition. This was previously supported by our demonstra- tion that FRC inhibited responses to anti-CD3 plus anti-CD28 or PMA/Ionomycin (Fig 1) and also the demonstration that 1400W augmented responses in the presence of FRC, but had no effect on basal proliferation in their absence (Fig 4b–c). Consistent with this, Figure 3. IFNc dependent Induction of NOS2/iNOS/Nitrate production in Lymphoid stromal cells. (a) Costimulatory marker expression on the surface of FRC.5 cells following IFNc treatment. (b) CFSE dilution profiles of OTI T cells stimulated with peptide-loaded BMDCs in the presence or absence of FRC.5 cells and blocking antibodies to CD80, CD86, or PDL-1. (c) Real-time PCR expression of nos2 mRNA in FRC.5 cells cultured with either IFNc, an iNOS-specific enzyme inhibitor (1400W), or both. (d) Immunofluorescence staining of NOS2 protein in FRC.5 cells cultured with pre- labeled SL8-pulsed BMDC or in the presence of pre-labeled SL8-pulsed BMDC plus pre-labeled T cells. Teal lines around FRC in FRC/DC condition indicate the border of the FRC and white arrowhead indicates a T-DC couple. Scalebar 20 mm. (e) Intracellular flow cytometry for NOS2 protein expression by FRC.5 cells cultured with SL8-pulsed BMDC with or without T cells and with or without IFNc blockade for 48 hours. (f) Extracellular nitrite concentration measured by Griess assay in FRC.5 cultures either alone; with OTI T cells and peptide-loaded BMDCs; or OTI cells, peptide-loaded BMDCs, and 1400W. doi:10.1371/journal.pone.0026138.g003 PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e26138