Lymph Node Fibroblasts Limit T Cell Expansion Figure 3. Stromal cells from various non-lymphoid organs decrease antigen-specific T cell proliferation. The T cell activation assay (see legend of Fig. 1) was performed in presence of either stromal cell lines (A,B,D) or ex vivo isolated stromal cells (C). OT-I T cells co- cultured for 3 days were analyzed for CFSE dilution using flow cytometry. Representative histograms of CFSE dilution (left panel) and the number of proliferating OT-I cells as percentage inhibition are presented (right panel, as in Fig. 1). Shown are assays with a (A) mouse embryonic fibroblast line (MEF) and lung fibroblast line, (B) lines derived from different non-lymphoid organs (dermis, heart, femoral muscle, kidney), (C) ex vivo isolated adherent cells from the dermis, epidermis or kidney, (D) Mesenchymal stem/stromal cell (MSC) line and the tumor cell lines B16-F10 (melanoma) and MC-38 (colon carcinoma). (A–D) n$3, representative for $2 experiments. Different symbols indicate different experiments. * P,0.05, ** P,0.01, *** P,0.001, if not indicated otherwise p-values are relative to ‘no stroma’. The dotted line indicates the inhibition observed by pLN2 cells within the same experiment. doi:10.1371/journal.pone.0027618.g003 Figure 2. CD8+ T cells primed in presence of TRC still produce IFNc and kill target cells. The T cell activation assay (see legend of Fig. 1) was performed without stroma (‘no stroma’) or in presence of the pLN2 TRC line for 4 days followed by flow cytometric analysis of OT-I T cell effector function. (A) Dot plots (left) show intracellular IFNc deposition versus CFSE dilution in OT-I T cells after in vitro re- stimulation with 1 mM SIINFEKL peptide in presence of brefeldin A. The right panel shows the median fluorescence intensity of IFNc expression in OT-I T cells. (B) The cytotoxic capacity of OT-I T cells was assessed by co-incubating OT-I T cells ( = effector cells, E) from the T cell activation assay in the indicated E:T ratios with SIINFEKL-pulsed-eFluor670high- labeled splenocytes ( = target cells, T) mixed 1:1 with unpulsed- eFluor670low-labeled splenocytes ( = internal control). After overnight culture the percentage of eFluor670high versus eFluor670low spleno- cytes was analyzed and plotted as histograms. Indicated as percentage is the survival index for the target cells, as based on the ratio of peptide- pulsed eFluor670high relative to unpulsed eFluor670low cells (6 standard deviation). (A, B): n = 3, representative of 3 independent experiments. * P,0.05, ** P,0.01, *** P,0.001, P-values are relative to the corresponding ‘no stroma’ control. doi:10.1371/journal.pone.0027618.g002 TRC limit T cell expansion by producing NO To gain insight into the nature of the TRC-derived inhibitory factor T cells and BM-DC were either separated from the pLN2 by a permeable-membrane (transwell) or simply incubated with supernatant (SN) from pLN2. Both settings led to a decrease in T cell expansion, but this was smaller than in co-cultures where all the three cell types are intermingled (Fig. 5A). These results suggest a role for either a soluble factor which acts only efficiently at short distance, or for at least two inhibitory factors, one being soluble and the other being cell-cell contact dependent. It also argues against a role for TRC in presenting antigen to T cells and thereby inhibiting T cell proliferation. To identify the molecules responsible for suppressing T cell expansion in our assay several candidates or their synthesis pathway were blocked, including PD-L1, TGFb, IL-10, IDO and arginase-1 but none of them neutralized the TRC-mediated inhibition (Fig. S3). However, blocking the enzymatic activity of PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27618