Fibroblastic Reticular Cells From Lymph Nodes Attenuate T Cell Expansion by Producing Nitric Oxide Stefanie Siegert1, Hsin-Ying Huang 1 , Chen-Ying Yang1, Leonardo Scarpellino1, Lucie Carrie2, Sarah Essex3, Peter J. Nelson 4 , Matthias Heikenwalder5, Hans Acha-Orbea1, Christopher D. Buckley3, Benjamin J. Marsland 6 , Dietmar Zehn2, Sanjiv A. Luther1* 1 Department of Biochemistry, University of Lausanne, Epalinges, Switzerland, 2 Swiss Vaccine Research Institute, and Centre Hospitalier Universitaire Vaudois (CHUV), Service of Immunology and Allergy, Lausanne, Switzerland, 3 School of Immunity and Infection, Institute for Biomedical Research, Medical Research Council Center for Immune Regulation, University of Birmingham, Birmingham, United Kingdom, 4 Medical Policlinic, Ludwig-Maximilians University/Helmholtz-Zentrum Mu¨ nchen, Munich, Germany, 5 Institute for Virology, Technical University Munich (TUM), Munich, Germany, 6 Service of Pneumology, Centre Hospitalier Universitaire Vaudois (CHUV), University of Lausanne, Lausanne, Switzerland Abstract Adaptive immune responses are initiated when T cells encounter antigen on dendritic cells (DC) in T zones of secondary lymphoid organs. T zones contain a 3-dimensional scaffold of fibroblastic reticular cells (FRC) but currently it is unclear how FRC influence T cell activation. Here we report that FRC lines and ex vivo FRC inhibit T cell proliferation but not differentiation. FRC share this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. We identified FRC as strong source of nitric oxide (NO) thereby directly dampening T cell expansion as well as reducing the T cell priming capacity of DC. The expression of inducible nitric oxide synthase (iNOS) was up-regulated in a subset of FRC by both DC-signals as well as interferon-c produced by primed CD8+ T cells. Importantly, iNOS expression was induced during viral infection in vivo in both LN FRC and DC. As a consequence, the primary T cell response was found to be exaggerated in Inos2/2 mice. Our findings highlight that in addition to their established positive roles in T cell responses FRC and DC cooperate in a negative feedback loop to attenuate T cell expansion during acute inflammation. Citation: Siegert S, Huang H-Y, Yang C-Y, Scarpellino L, Carrie L, et al. (2011) Fibroblastic Reticular Cells From Lymph Nodes Attenuate T Cell Expansion by Producing Nitric Oxide. PLoS ONE 6(11): e27618. doi:10.1371/journal.pone.0027618 Editor: Jo¨ rg Hermann Fritz, McGill University, Canada Received September 15, 2011; Accepted October 20, 2011; Published November 14, 2011 Copyright: 2011 Siegert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Swiss National Science Foundation grants #PPOOA-116896/1 and #31003A-130488/1 to S.A.L; by the Boehringer Ingelheim Fonds to S.S.; and by the Taiwan National Science Council to H-Y.H.. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Sanjiv.Luther@unil.ch Introduction Adaptive immune responses are initiated most efficiently within secondary lymphoid organs (SLO), such as the spleen or lymph nodes (LN), where pathogens (or foreign antigens) are filtered from body fluids and presented to recirculating T cells. Typically, dendritic cells (DC) residing in peripheral tissues capture foreign antigens and danger signals inducing their maturation, including up-regulation of the chemokine receptor CCR7 allowing DC to migrate via lymphatic vessels into the paracortex (T zone) of the draining SLO. There they present antigen-derived peptides in the context of surface MHC molecules to thousands of na¨ıve recirculating T cells. Only the rare antigen-specific T cells become activated, start secreting cytokines and undergo multiple rounds of cell division. Finally, they differentiate into effector cells and leave the LN as a large cohort that migrates specifically to the site of inflammation [1,2,3,4]. Both in mice and humans, the microenvironment where T cells encounter DC is spanned by a 3-dimensional (3D) network of T zone fibroblastic reticular cells (T-FRC or TRC) known to produce the extracellular matrix scaffold, including microvessels called conduits [2,4,5,6,7,8,9]. More recently it has become clear that TRC are not only cells providing a 3D microenvironment but play an active role in adaptive immunity. They physically guide lymphocytes during their several hours-long migration across the T zone by forming a ‘road system’ [2]. TRC also actively recruit CCR7 expressing T cells and DC into the T zone by constitutively secreting CCL19 and CCL21 [9,10,11]. These chemokines not only retain T cells in the T zone but also promote their motility [12]. Furthermore, incoming and resident DC adhere to TRC as well as their associated matrix structures [2,4,8]. Finally, TRC are the major constitutive source of IL-7 in LN and access to LN TRC is critical for na¨ıve T cell survival [9,13]. As the processes of selection, amplification and differentiation of antigen-specific T cells all take place within the TRC environment, it raises the possibility that TRC positively influence these steps. Several lines of evidence support this hypothesis: First, the TRC network appears to increase the frequency of DC-T cell encounters leading to a faster selection of antigen-specific T cells whose frequency is estimated to be around 1 out of 100’000 T cells for a given protein antigen. Both physical and chemical guidance cues provided by TRC are thought to contribute to this effect [2,4]. Second, the homeostatic chemokines CCL19 and CCL21 act as co- stimulatory signals for T cell activation and proliferation in vitro [14,15]. These chemokines also increase DC maturation and function (reviewed in [16]). Third, IL-7 enhances T cell responses to PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27618