Regulation of T Cell Priming by Lymphoid Stroma Omar Khan1, Mark Headley1, Audrey Gerard1, Wei Wei2, Limin Liu2, Matthew F. Krummel1* 1 Department of Pathology, University of California San Francisco, San Francisco, California, United States of America, 2 Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California, United States of America Abstract The priming of immune T cells by their interaction with dendritic cells (DCs) in lymph nodes (LN), one of the early events in productive adaptive immune responses, occurs on a scaffold of lymphoid stromal cells, which have largely been seen as support cells or sources of chemokines and homeostatic growth factors. Here we show that murine fibroblastic reticular cells (FRCs), isolated from LN of B6 mice, play a more direct role in the immune response by sensing and modulating T cell activation through their upregulation of inducible nitric oxide synthase (iNOS) in response to early T cell IFNc production. Stromal iNOS, which only functions in very close proximity, attenuates responses to inflammatory DC immunization but not to other priming regimens and preferentially affects Th1 cells rather than Th2. The resultant nitric oxide production does not affect T cell-DC coupling or initial calcium signaling, but restricts homotypic T cell clustering, cell cycle progression, and proliferation. Stromal feedback inhibition thus provides basal attenuation of T cell responses, particularly those characterized by strong local inflammatory cues. Citation: Khan O, Headley M, Gerard A, Wei W, Liu L, et al. (2011) Regulation of T Cell Priming by Lymphoid Stroma. PLoS ONE 6(11): e26138. doi:10.1371/ journal.pone.0026138 Editor: Matthew L. Albert, Institut Pasteur, France Received September 14, 2011; Accepted September 20, 2011; Published November 14, 2011 Copyright: 2011 Khan 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 work was supported by NIH R01 CA134622-01A1 (MFK), The Foundation Rothshild (MFK), and CA122359(LL). 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: matthew.krummel@ucsf.edu Introduction Na¨ıve T cells are activated through surface presentation of peptide-MHC fragments by tissue-draining dendritic cells (DC). While the cell biology and dynamics of this isolated interaction have been extensively scrutinized[1–5], the interaction typically takes place against the background of lymphoid stroma, a collection of structural cells whose influence on the process has not been fully investigated. Four cell types constitute the majority of stroma in secondary lymph nodes. Blood endothelial cells (BECs) are the structural cells that are assembled to form capillaries and high-endothelial venules (HEVs), structures T cells must traverse in order to enter the lymph node. Lymphatic endothelial cells (LECs), assemble the afferent and efferent lymphatic vessels, are largely contiguous with lymphatics themselves, and provide entry sites for dendritic cells arriving via lymph[6]. Within the lymph node, B cell zones are isolated and supported by follicular dendritic cells (FDCs) whereas fibroblastic reticular cells (FRCs) largely scaffold the T cell zone. FRCs are thus the dominant stromal cell present at the site of T cell priming. These cells engulf reticular collagen fibers that weave throughout the T cell zone. The hollow core of the ensuing FRC network provides a conduit for soluble material to penetrate into the lymph node[7,8]. FRCs also provide structure to the T cell zone and are hypothesized to act as ‘tracks’ for T cells to survey the contents of a node and on which chemokines may be immobilized for the purpose of guiding T cells[9]. They are also major sources of CCL19, CCL21 and IL-7, key factors guiding T cell motility and survival in the lymph node[10]. Finally, FRC appear to be direct targets of some viruses, resulting in viral modulation of CCL21 expression and possibly modulation of lymphoid homeostasis[11,12]. In these various settings, FRC are seen largely to promote T cell survival and activation. In this study, we investigated whether FRCs directly communi- cate with T cells activating in their midst and whether they are capable of providing specific feedback in the T cell priming process. We find that inclusion of FRC lines or freshly purified cells to T-DC priming cultures results in significant inhibition of activation. This inhibition does not rely on the FRC directly presenting antigens. FRC s are highly responsive to T cell-produced interferon gamma (IFNc) and respond by upregulating their transcription of the nos2 gene, encoding the enzyme inducible nitric oxide synthase (iNOS). The resulting nearby production of nitrite results in a block in T cell cell-cycle progression. We find that that iNOS inhibition during specific types of priming reactions in the lymph nodes plays a significant role in restricting T cell activation to immunization with inflammatory DC populations, consistent with a role in regulating the response. FRCs, prior and apart from this crosstalk, have no discernable effect on T cell priming at the level of signaling or expression of early activation markers. In sum, this places FRCs as regulators of T cell activation through direct communication with IFNc producing T cells. Results Lymph node stromal cell populations inhibit naive T cell activation In order to study the function of lymphoid stromal cells during T cell priming, we first established cell lines from the CD45 negative fraction of the LN, representing FRCs (‘‘FRC.5’’: gp38+, CD312), LECs (‘‘LEC.6’’: gp38+CD31+) and BECs (‘‘BEC.7’’: gp382 CD31+) (Fig 1a). These lines, once established, were PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e26138