d with biotinylated anti-gp38-antibody followed by streptavidin-coupled magnetic beads. Cells were separated using MScolumns according to the manufactures instructions. Per 96-well 2500 TRC-conditioned-DC were co-cultured with 0.016106 CFSE labeled OT-I cells mixed with 0.486106 unspecific WT T cells for 3 days. Statistical analysis F-test was performed to determine usage of equal or unequal variance in the t-test, with P,0.05 considered as unequal variance. Statistical significance was determined with students t-test. Supporting Information T cell activation and cytokine stimulation of 92-61-5 stromal cells in chamber slides 3000 stromal cells were seeded per well in 8-well chamber slides and cultured overnight. T cell activation: 0.256106 WT T cells and 0.1256106 anti-CD3/28 beads were added or 5000 CFSE-labeled OT-I T cells mixed with 0.2456106 WT T cells together with 5000 LPS-activated and SIINFEKL-pulsed BM-DC. Cytokine stimulation: IFNc IL-1b, TNFa, LTa3, 500 U/ml IFNa or the agonistic antibody against LTbR were added to stromal cells for 7 h or 24 h. In vitro cytotoxicity assay Target cells: WT splenocytes were labeled with 0.16 mM or 0.5 mM eFluor670. The eFluro670high population was pulsed with 1 mM SIINFEKL for 1 h at 37uC. Per 96-well 5000 eFluor670high and 5000 eFluor670low cells were added. Effector cells were harvested from the T cell activation assay on day 4 or from spleen or pLN from VSV-infected mice. Cells were mixed in different E/T ratios and after overnight culture analyzed by flow cytometry. Alternatively, they were isolated from homogenized spleen or pLN from VSV-infected mice. Effector cells were added to target cells in different ratios and incubated overnight. The ratio of target cells was analyzed by flow cytometry, as were the input numbers of OT-1 effector T cells allowing the calculation of the effective E/T ratio. Percentage specific lysis = 100 with specific survival = eFluor670high/eFluorlow,. Nitrite detection NO22 in cell culture supernatants was measured using the Griess assay. 0.1% N-1-naphtylethylenediamine dihydrochloride was mixed with p-aminobenzensulfonamide in 5%phosphoric acid; 100 ml of this mixture were incubated with 100 ml cell culture supernatant. After 10 min incubation at RT absorbance was measured at 550 nm and background absorbance at 690 nm 
was subtracted as well as the absorbance of complete RPMI. In each measurement a NO22 -standard series was included. and ex vivo isolated stromal cells. Flow cytometric analysis of the surface phenotype of non-lymphoid cell lines or ex vivo stromal cells isolated from dermis, epidermis and kidney, and used in the T cell activation assay shown in 12 November 2011 | Volume 6 | Issue 11 | e27618 Immunofluorescence microscopy Staining and microscopy of cryosections were performed as previously described. Cells cultured in chamber slides: chambers were removed according to the manufactures description, staining Lymph Node Fibroblasts Limit T Cell Expansion production in ex vivo TRC, while stimulation of pLN2 with different cytokines does not induce Cox2, Ccl21 or Ccl19 transcription. Immunohistological analysis of iNOS protein expression in ex vivo TRC. TRC-enriched cells from pLN of WT mice were cultured for 2 days either alone, in the presence of WT T cells without 21123673” or with anti-CD3/28 beds or in presence of 10 ng/ml recombinant 11714831” IFNc. For the first four rows the first column shows iNOS expression and DAPI+ nuclei, while for the last row, the first column sh
