immunodeficiency [6,32,53]. Limiting T cell expansion early in the immune response might be necessary to give TRC the time to adjust step-wise to the new space demands and to start proliferating thereby increasing the scaffold size accommodating T cells and DC. Such a process may ensure continuous functionality of the growing organ which is finally also to the benefit of efficient effector T cell generation. Materials and Methods Ethics Statement This study was carried out in strict accordance with the Swiss act for animal welfare. All mouse experiments, including the protocols, were authorized by the Swiss Federal Veterinary Office (Bern, Switzerland) with permits issued to S.A.L. (number 1612.2), B.J.M. (number 2216) and D.Z. (number 2308). All efforts were made to minimize suffering. Mice and immunizations Mice used were C57BL/6 (B6, CD45.2+); Janvier), B6 OT-I CD45.1+, Ubiquitin-gfp transgenic and Inos2/2 (Jackson laborato- ries). For immunization mice were subcutaneously injected at six sites in the back to target six pLN. 25 mg NP-CGG (Biosearch Technology) was diluted in Montanide ISA 25 (25% in PBS; Seppic). Alternatively, 0.336106 pfu VSV-OVA [54] were injected after retro-orbitally grafting 0.16106 OT-I splenocytes. Bone marrow (BM)-chimeras: BM from donor-mice was obtained from femur and tibia by crushing bones with a mortar. 156106 BM cells were injected retro-orbitally into recipient mice irradiated twice with 450 rad in a 4 h interval. The following 4 weeks mice received the antibiotic ‘Baytril 10%’ (1/1000) in the drinking water. WT mice used were either CD45.2+ or Cd45.1+ while Inos2/2 mice were CD45.1+. All mice were maintained in specific pathogen-free conditions. Flow cytometry 0.1–26106 cells were blocked (2% normal-mouse-serum (Sigma) or anti-CD16/32 antibody (2.4G2) and then stained with antibodies (see table S1) on ice. Dead cells were excluded using 7- AAD (7-Aminoactinomycin-D) or DAPI (4,6-diamidino-2-pheny- lindole), or Aqua (all from Invitrogen) in case of fixed cells. Intracellular staining: Cells were fixed with 4% PFA, pemeabilized using 0.1% saponin (Sigma) and incubated with anti-IFNc (XMG1.2) (45 min) or anti-iNOS antibody (rabbit antibody; Millipore) followed by Alexa488-coupled donkey-anti-rabbit IgG (Molecular Probes) (30 min each). For IFNc staining: cells were re- stimulated in vitro with 1 mM SIINFEKL peptide in the presence of Brefeldin-A (10 mg/ml, AppliChem) for 3–4 h at 37uC. Data were acquired on a FACSCanto or LSR II flow cytometer (both BectonDickinson) and were analyzed with FlowJo software (TreeStar). Ex vivo stromal cell isolation LN from CO2-killed mice were dissected, their capsule opened with a 26-gauge needle and the organs digested for 30 min at 37uC in DMEM (Invitrogen) containing collagenase IV (3 mg/ml; Worthington), DNAse-I (40 mg/ml; Roche), 2.5% FCS and 1.2 mM CaCl2, 10 mM HEPES and 50 IU/ml Penicillin, 50 mg/ml streptomycin. Subsequently, EDTA was added (5 mM final) and remaining clumps dissolved by pipetting, passed through a 40-mm mesh and re-suspended in complete RPMI containing 10% FCS. For cell isolation from femoral muscle, kidney and heart, the respective organs were dissected, cut into small pieces and digested as described above for 1 h at 37uC. Cell isolation Lymph Node Fibroblasts Limit T Cell Expansion from ears: Ears were split with forceps and digested in 0.5% trypsin (Sigma) and 5 mM EDTA for 20 min at 37uC to separate dermal and epidermal sheets. These were cut into small pieces and digested for 2 h as described. After digestion stromal cells were enriched by panning using antibodies against CD45 (M1/9.4.3), CD31 (GC-51), CD11c (N418) and CD11b (M1/70). Cells were counted using trypan blue dye and an automated cell counter (Countess, Invitrogen). Cell lines and bone-marrow-derived dendritic cells (BM- DC) Stromal cells were isolated as described above from various tissues of na¨ıve B6 or Ubiquitin-gfp B6 mice. After overnight culture in complete RPMI non-adherent cells were washed away. Adherent cells were cultured until confluent, then split on new dishes. Cells were used between passage 5 and 25. Other B6- derived cell lines used: B16-F10 and MC-38 tumor lines (kindly provided by A.Donda, Lausanne) [55], spleen and lung fibroblasts (C.Buckley [26]), MEF (M.Heikenwalder, Munich) and MSC (P.Nelson, Munich [56]). B6 BM-DC were generated as previously described [57]. T cell activation assay Stromal cells: Per 24-well 16104 stromal cells from lines were seeded in complete RPMI and after overnight culture irradiated with 1000rad. Initially, experiments with non-irradiated or irradiated pLN2 were performed with no difference in the outcome (data not shown). Ex vivo stromal cells were isolated as described above and non-adherent cells washed away after overnight culture (density comparable with lines); they were non- proliferative and therefore not irradiated. BMDC: They were activated with 0.5 mg/ml LPS (Sigma) for 6 h at 37uC. 2 h after LPS addition 1 mM SIINFEKL peptide was added. 16104 BM- DC were added per 24-well. T cells: They were obtained ex vivo from spleen and pLN dissected from CO2-killed WT B6 and OT-I transgenic mice and suspended by meshing. Erythrocytes were removed using red blood cell lysis buffer (Tris-Ammonium chloride based). CD8 cells were enriched by panning using antibodies to B220 (RA3-6B2), CD4 (H129.19.6), CD11b (M1/70) and CD11c (N418). OT-I cells were labeled with 2 mM CFSE (Invitrogen). Only 50% of WT B6 T cells were CFSE-labeled to identify the peak of undivided cells, with the 50% unlabeled T cells showing the background fluorescence of T cells. Per 24-well 0.046106 CFSE+ OT-I cells together with 0.986106 unlabeled B6 WT cells and 0.986106 CFSE+ B6 WT cells were added. The assay was performed in complete RPMI enriched with 16MEM (Invitrogen), 3 ng/ml murine IL-7 (Peprotech) and 10 U/ml human IL-2 (Merck Serono). After 2–4 days, cells were harvested, counted and analyzed by flow cytometry. Transwell assays used were 0.4 mm transwell chambers (HTS 24-well, Vitaris). Blocking experiments used 10 mM indomethacin (Sigma), 1 mM 1400W ( = dihydrochloride; Sigma), 10 mM 1-Methyl-L-tryptophan (1- MT; Sigma), 200 mM (S)-(2-Boronoethyl)-L-cysteine (BEC, Cal- biochem), 10 mg/ml anti-PD-L1 (MIH5, eBioscience), 20 mg/ml anti-IL-10 (kind gift from F.Tacchini, Lausanne, Switzerland) or 30 mg/ml anti-TGFb (clone 1D11.16.8; BioXCell). To better compare the decrease of T cell proliferation between experiments the attenuating effect by the stromal cells was defined as percent inhibition in divided OT-I T cell numbers relative to the ‘no stroma’ control: {1-(number of proliferated cells)stroma/(number of proliferated cells)no stroma}*100). The ‘no stroma’ control was considered as 0% inhibition; absence of proliferating OT-I cells was considered as 100% inhibition. PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27618