Figure S4 IFNc induces iNOS expression and NO production in ex vivo TRC, while stimulation of pLN2 with different cytokines does not induce Cox2, Ccl21 or Ccl19 transcription. (A) 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 (top row), in the presence of WT T cells without (second row) or with anti-CD3/28 beds (third row) or in presence of 10 ng/ml recombinant IFNc (last two rows). For the first four rows the first column shows iNOS expression (red) and DAPI+ nuclei (blue), while for the last row, the first column show the ‘no primary antibody control’ (red), the second column shows gp38+ CD312 TRC and gp38+ CD31+ lymphatic cells. Arrows show examples of gp38+ CD312 TRC expressing iNOS. Scale bar: 50 mm. (B) Analysis of NO22 concentration in the supernatants from the co-cultures shown in (A). (C) Quantitative RT-PCR analysis of Cox2, CCl21 and CCl19 mRNA levels in pLN2 stimulated with various cytokines or agonistic antibody to LTbR (aLTbR) for 7 h or 24 h. The relative expression levels are shown. Different symbols indicate different experiments. (A–C) n = 3–4, (A–C) respresentative for 1–2 experiments. * p,0.05,**p,0.0,***p,0.001, p values are relative to unstimulated pLN2. (TIF) Figure S5 Inos expression and CD8+ T cell effector functions in VSV-OVA infected WT or Inos2/2 mice. WT or Inos2/2 mice were retro-orbitally grafted with 100’000 splenocytes from OT-I transgenic mice one day prior to subcutaneous infection with VSV-OVA. (A) Regional pLN from infected mice were harvested 1, 2 or 4 days after infection and analyzed for Inos mRNA by by quantitative real time-PCR on crude fractions of LN. Normalized Inos mRNA levels are shown. (B, C) Draining pLN and spleen were harvested on day 4 and 6 after infection, homogenized and analyzed for intracellular IFNc protein expression in OT-I T cells using flow cytometry (B) or for killing activity (C). (B) Histograms show the percentage of OT-I T cells (6 standard deviation) expressing IFNc. Bar graphs show the median fluorescence intensity of the IFNc staining within OT-I T cells. (C) Cells from day 6 were used in an in vitro killing assay. The specific lysis of target cells is shown for the respective effector to target ratios. (A–C) n = 3 (A,B) representative for 2 experiments, (C) 1 experiment (similiar data were obtained for day 8 LN and spleen, not shown). (TIF) Figure S6 Bone marrow chimeras lacking Inos in the non-hematopoietic system show a trapping defect in immunized lymph nodes. To assess the relative contribution of hematopoietic versus non-hematopoietic cells as iNOS source, BM chimeras were generated having Inos-deficiency in either the hematopoietic system (Inos2/2 BM into WT hosts) or non- hematopoietic system (WT in Inos2/2 and were compared with control chimeras (WT into WT). 2 months after reconstitution, BM-chimeras were infected with VSV-OVA (as described in Figure 7) and 4 days after infection pLN were collected, single cell suspensions counted and stained before analysis using flow cytometry. As comparison non-immunized BM-chimeras are shown. Inos2/2 into Inos2/2 chimeras have not been made. Surprisingly, none of the chimeras showed an increased OT-I expansion. Rather, Inos -deficiency in either the hematopoietic or the non-hematopoietic system led to a strong decreased OT-I expansion. Surprisingly, lymphocyte trapping did not occur in the case of Inos -deficiency in the non-hematopoietic compartment, in contrast to the other two groups and the non-chimeric Inos2/2 mice (Figure 7). Therefore, the expansion of OT-I T cells cannot Table S1 (DOCX) Table S3 ysis. (DOCX) Lymph Node Fibroblasts Limit T Cell Expansion PLoS ONE | www.plosone.org 13 November 2011 | Volume 6 | Issue 11 | e27618 be interpreted for that group of mice. The lack of OT-I expansion in the group of mice having Inos deleted in the hematopoietic system indicates also a positive role of Inos in T cell proliferation, presumably in a low but not high concentration, as previously suggested [46]. In all BM chimeras the chimerism was .85% as assessed by measuring ratio’s of CD45.2 (WT) versus CD45.1 (Inos2/2 or WT) expression on total LN cells using flow cytometry. 2–4 mice were in each group. (TIF) Figure S7 Model showing how inflammatory cytokines may induce iNOS expression in TRC and create a negative feedback loop limiting antigen-specific T cell expansion. During the early phase of immune response antigen- specific CD8+ T cells interact with antigen-bearing DC within the context of the TRC network within the T zone of the draining LN. Upon prolonged cognate interaction T cells start to produce IFNc and possibly other cytokines that induce strong but transient iNOS expression in neighboring TRC as well as DC. The local production of NO creates a negative feedback loop in which NO and possibly other inhibitory factors limit the expansion of neighboring antigen- specific T cells. This effect is due in part to direct inhibition of T cell proliferation or survival, in part to a decrease in the stimulatory capacity of DC. NO is known to lead to nitrosylation of cysteine- and tyrosine-containing proteins thereby altering their function, including in T cells where reduced T cell proliferation was reported in presence of NO [35]. High NO concentrations may also reduce T cell survival. An alternative model is that in vivo innate immune cells, such as NK cells, become activated early during the response and release IFNc that induces iNOS in TRC and DC. Together, these processes may prevent overshooting antigen-specific T cell expansion while affecting much less T cell differentiation. This selective negative regulation of T cell numbers by TRC and DC may allow gradual organ and stromal cell growth thereby achieving a compromise between preservation of functional organ structure and fast effector T cell differentiation. It is reminiscent of the role of TRC in controlling na¨ıve T cell numbers [9,13]. The early and transient induction of iNOS may have also an impact on later aspects of the immune response, such as the contraction phase and memory T cell generation, as they are thought to be controlled by the conditions encountered during the T cell priming phase [1,44]. (TIF) Staining reagents used for flow cytometry. Table S2 Staining reagents used for immunofluores- cence microscopy. (DOCX) Primer sequences for quantitative PCR anal- Acknowledgments We would like to thank: Fabienne Tacchini-Cottier for discussions and critical reading of the manuscript; Alena Donda, Greta Guarda, Catherine Ronet, Carl Ware (NIH AI033068), Anne Wilson (Flow cytometry facility of the University Lausanne) and Michael Sixt for reagents and advice; Sarah Enouz for technical assistance. Author Contributions Conceived and designed the experiments: SAL SS DZ HA-O. Performed the experiments: SS H-YH C-YY LS LC. Analyzed the data: SS H-YH C-YY HA-O DZ SAL. Contributed reagents/materials/analysis tools: SS LS SE MH PJN CDB BJM. Wrote the paper: SAL SS H-YH DZ.