T cell activation by anti-CD3 and anti-28 beads 2500 stromal cells were seeded per 96-well and after overnight culture irradiated with 1000 rad. 2.56105 T cells mixed with 1.256105 anti-CD3/28 beads (Dynabeads, Invitrogen) were added. After 2–3 days of co-culture cells were harvested by gentle pipetting. Live cells were counted and analyzed by flow cytometry. T cell activation by TRC-conditioned BM-DC 10’000 stromal cells were seeded per 24-well and after overnight culture 10’000 LPS-activated and SIINFEKL-pulsed BM-DC were added. After overnight culture BM-DC were separated by magnetic cell sorting (MACS): Cells were stained with biotinylated anti-gp38-antibody (clone 8.1.1) followed by streptavidin-coupled magnetic beads (Miltenyi). Cells were separated using MS- columns (Miltenyi) 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. T cell activation and cytokine stimulation of stromal cells in chamber slides 3000 stromal cells were seeded per well in 8-well chamber slides (Falcon) 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 (all 10 ng/ml, from Peprotech), 500 U/ml IFNa (PBL, Interferon source) or the agonistic antibody against LTbR (4H8 WH2; provided by C.Ware) were added to stromal cells for 7 h or 24 h. In vitro cytotoxicity assay Target cells (T): WT splenocytes were labeled with 0.16 mM or 0.5 mM eFluor670 (eBioscience). 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 (E) 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 = {12(specific survi- val)sample/(specific survival)control}*100 with specific survival = e- Fluor670high/eFluorlow, (control: target cells only). Nitrite detection NO22 in cell culture supernatants was measured using the Griess assay. 0.1% N-1-naphtylethylenediamine dihydrochloride (Sigma) was mixed with p-aminobenzensulfonamide (Sigma) 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 (100- 0.1 mM NaNO2; Sigma) was included. Immunofluorescence microscopy Staining and microscopy of cryosections were performed as previously described [9]. Cells cultured in chamber slides: chambers were removed according to the manufactures description, staining Lymph Node Fibroblasts Limit T Cell Expansion and microscopy was performed as done for cryosections (for antibodies used see table S2). Images were acquired with a Zeiss Axioplan microscope at RT and treated with ImageJ (http:// rsbweb.nih.gov/ij/) and Photoshop software (Adobe). Transcript analysis RNA isolation reverse transcription and quantitative real-time PCR was performed as described [9]. For additional primers used see table S3. Efficiency-corrected expression of Inos, Cox2, Ccl19 and Ccl21 was normalized by dividing with the geometric mean of expression of two housekeeping genes. 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 Figure S1 Surface phenotype of TRC lines and ex vivo isolated TRC. Flow cytometric analysis of the surface phenotype of TRC lines (A) or TRC isolated ex vivo from pLN (B) used in the T cell activation assay shown in Figure 1. (A) The first row shows dot plots of CD31 versus CD45 expression and the percentage of CD452 CD312 cells. (6 standard deviation). Following rows show histograms with the indicated surface markers on cells pregated on CD452 CD312 cells. pLN2 and pLN1 are two different TRC lines derived from pLN-pools derived from distinct mice. pLN2 cells also express CD54 (ICAM-1), CD140b (PDGF-Rb) and CD105 (Endoglin) (not shown). (B) CD452 CD312 cells were analyzed in dot plots for CD31 versus gp38 expression (first row). The following rows show the indicted surface markers on CD452 CD312 CD352 gp38+ cells (lower quadrant on the right in the gp38 versus CD31 dot plot; displayed as black line). Grey shadowed curves show the ‘no primary antibody’ control. (A,B) representative for $3 independent experiments. (TIF) Figure S2 Surface phenotype of non-lymphoid cell lines and ex vivo isolated stromal cells. Flow cytometric analysis of the surface phenotype of (A) non-lymphoid cell lines or (B) ex vivo stromal cells isolated from dermis, epidermis and kidney, and used in the T cell activation assay shown in Figure 3. (A) The first row shows dot plots of CD31 versus CD45 expression on the cell lines, the following rows show histograms with the indicted surface markers on CD452 CD312 cells. (B) CD452 CD352 cells were analyzed in dot plots for CD31 versus gp38 expression (first row). The following rows show histograms with the indicated surface markers on CD452 CD352 CD312 gp38+ cells as black line, and gp382 CD312 cells as dotted line (includes epithelial cells. Grey shadowed curves show the ‘no primary antibody‘ control. (A,B) representative for 2–3 independent experiments. (TIF) Figure S3 Suppression of T cell proliferation by TRC is not mediated by IDO, PD-L1, TGFb, IL-10 or arginase-1. Flow cytometric analysis of the T cell activation assay (as described in Fig. 1) in the presence of pharmacological inhibitors of IDO (10 mM 1-MT), arginase-1 (200 mM BEC) or blocking antibodies against PD-L1 (10 mg/ml), TGFb (30 mg/ml) or IL-10 (20 mg/ml). One out of several tested inhibitor concentrations is shown along with the percentage of inhibition (as in Fig. 1). The dotted line shows the average inhibition by pLN2 TRC without inhibitor/ blocking antibodies. n$3, representative for 2–3 experiments. (TIF) PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e27618