sensing of the response. In that regard, too, iNOS responses like the ones discussed here have been previously reported in macrophages[17] as well as mesenchymal stem cells [15] and many other tissue stroma cells (e.g. in an accompanying paper by Luther et al.) A key distinction between our studies and those formerly mentioned is that we are now demonstrating this pathway to be actively engaged during specific types of T cell priming directly in the lymph node and via the lymph node structural cells, suggesting a more integral role, in the latter, in guiding the activation dynamics of Type 1-immune responses. Is iNOS production by stromal cells always inhibitory to T cell function? Our data using direct targeting to DEC-205 antigen presenting cells indicates it most certainly will not be for all types of priming in the T cell compartment. And, from the global immune response, there are many reasons an ongoing immune response benefits from iNOS production. Most prominently, iNOS is upregulated in neutrophils in response to bacteria[28], is critical for resistance to Leishmania[20], and Listeria infection[21]. In this context, iNOS may lead to increased antigen presentation and improved overall outcome. However and notably, these mice have previously been shown to exhibit enhanced Th1 responses, a result which now may be partly or fully explained by the mechanism we describe here. The role of iNOS in the immune response therefore continues to evolve and may play positive and negative regulatory roles when expressed in stromal cells. Methods Mice Ovalbumin-specific TCR transgenic OTI, OTII, OTI GFP and OTI IFNc 2/2 mice were bred in-house. iNOS2/2 mice[22] were gifts from L. Liu (University of California San Francisco) or purchased from the Jackson Laboratory (B6.129P2-Nos2tm1Lau/J). C57/Bl6 mice were purchased from the Simonsen Laboratory. All mice were maintained under specific pathogen-free conditions at the University of California San Francisco Animal Barrier Facility. Experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of California San Francisco under protocol authorization number AN081824- 03B. Antibodies, reagents, and flow cytometry Flow Cytometry and Cell Sorting. aCD31(clone 390), agp38(clone 8.1.1), aCD45(clone 30-F11), aCD25(clone PC61), aCD69(clone H1.2F3) and aCD8(clone 53-6.7) were purchased from BioLegend. aCD86(clone B7-2) and aCD279/PD1(clone J43) were purchased from eBioscience. aKi67(clone B56), aCD19(clone 1D3), aNOS2(anti-iNOS/NOS type II, polyclonal) and aIFNcR alpha chain(clone GR20) were purchased from BD. Tetramers specific for SIINFEKL peptide (SL8) bound to H-2Kb were purchased from ProImmune. For flow cytometry, cells were first blocked with aCD16/32 (clone 2.4G2) and then stained in PBS + 10% FCS with the antibodies listed above. For Intracellular staining of NOS2, cells were surface stained as described above (FACS only) and subsequently fixed 4% paraformaldehyde and then permeablized in PBS +10%FCS+0.5% Saponin: staining with aNOS2-FITC was then performed in this same permeabilization buffer at 4uC. Data was collected on either an LSRFortessa or a FACScalibur (BD) and analyzed using Flowjo (Treestar). In vitro and in vivo T cell activation. aCD3(clone 2C11, produced in-house), aCD28(clone PV-1, University of California San Francisco Hybridoma Core Facility, UCSF-HCF), SL8 peptide (Anaspec),Ovalbumin peptide (323-339) (Genscript) Regulation of T Cell Priming by Lymphoid Stroma PMA and ionomycin (Sigma-Aldrich). aCD40 (ebioscience), and IFNc (Peprotech.) aDEC205-OVA (DEC-OVA) conjugates were produced in-house, using established protocols3. In vitro blocking. Antibodies against IFNc(clone XMG1.2, BioXCell), IL2(clone JES6-1A12, ebioscience) and PD1(clone RMP1-14, Biolegend). Antibodies against CD80(16-10A1) and CD86(GL-1) were purchased from UCSF-HCF. Cytokine Stimulation and inhibitors. 1400W and L-NG- monomethyl Arginine citrate (L-NMMA) were purchased from Sigma-Aldrich. IFNc, IL4 and IL2 were purchased from Peprotech. IL2 was also produced in-house from transfected lines. The cellular dyes propidium iodide, CFSE, DDAO and FURA-2 AM were purchased from Invitrogen. Proliferation dye eFluor670 was purchased from eBioscience. Cell isolation OTI T cells were isolated from the lymph nodes and spleen of 6 to 8 week old OTI mice. Selection was carried out using a negative CD8 isolation kit (STEMCELL Technologies Inc.). Cells were cultured in RPMI supplemented with 1 mM L-glutamine, penicillin, streptomycin, 561025 M bME, and 10% FCS (R10). Th1 and Th2 cells were produced by isolating the spleen of OTII TCR transgenic mice and culturing 26106 cells with the following combination of reagents. Anti-CD28, anti-IL4, IL12, IL2 and Ovalbumin peptide (323–339) were added to produce Th1 cells. Anti-CD28, anti-IFNc, IL4, IL2 and Ovalbumin peptide (323– 339) were added to produce Th2 cells. Bone marrow-derived dendritic cells (BMDCs) were generated by culturing bone marrow cells for 7–10 days with GM-CSF in IMDM supplemented with 1 mM l-glutamine, penicillin, streptomycin, 561025 M bME, and 10% FCS. IL4 was added for the last 2 days of culture. Generation of stromal cell lines Peripheral lymph nodes from C57/Bl6 mice were isolated and ground between two microscope slides. Lymph node tissue was then digested for 20 minutes at 37uC in 2 ml of RPMI 1640 medium containing 2% FCS, collagenase IV (2 mg/ml; Sigma- Aldrich) and DNAseI (40 ug/ml; Roche). The tissue was then washed and resuspended in RPMI 1640 containing 2% FCS, collagenase D (1 mg/ml; Roche) and DNAseI (40 ug/ml) for further digestion. Throughout the digestion procedure fragments were gently sheared by passing through a 21.5 gauge needle every 10 minutes. When no visible fragments remained the sample was washed in RPMI 1640 medium containing 5 mM EDTA and finally resuspended in PBS containing 10% FCS at a concentra- tion of 26106/ml. The resulting cell suspension was incubated at room temperature with 0.1 mg of 2.4G2 for 15 minutes. Without washing aCD31, agp38, aCD45, and 1 mg DAPI were added to cells and incubated for an additional 30 minutes. Cells were then washed in sterile PBS + 10% FCS and sorted using a BD Biosciences FACSAria II Cell Sorter. Stromal populations were defined as FRC (FRC.5) CD31(2) gp38(+), LEC (LEC.6) CD31(+)gp38(+), and BEC (BEC.7) CD31(+) gp38(2) and sorted in sterile RPMI 1640 containing 50% FCS and subsequently washed and plated in sterile R10 media. RNA isolation and RT-PCR RNA was extracted using the RNeasy Mini Kit from Qiagen. First strand synthesis was completed using random hexamers and reagents from the SuperScript III kit purchased from Invitrogen. 100 ng of cDNA was amplified using a SYBR Green PCR Master kit (Applied Biosystems) on a CFX96 real-time thermal cycler (Bio- Rad). Expression of nos2 transcript is quantified as the amount relative to the expression of the housekeeping gene hypoxanthine PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e26138