Fibroblastic Reticular Cells Guide T Lymphocyte Entry into and Migration within the Splenic T Cell Zone This information is current as Marc Bajénoff, Nicolas Glaichenhaus and Ronald N. of September 24, 2021. Germain J Immunol 2008; 181:3947-3954; ; doi: 10.4049/jimmunol.181.6.3947 http://www.jimmunol.org/content/181/6/3947 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2008/08/29/181.6.3947.DC1 Material References This article cites 41 articles, 16 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/181/6/3947.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Fibroblastic Reticular Cells Guide T Lymphocyte Entry into and Migration within the Splenic T Cell Zone1 Marc Baje´noff,*†‡ Nicolas Glaichenhaus,† and Ronald N. Germain2* Although a great deal is known about T cell entry into lymph nodes, much less is understood about how T lymphocytes access the splenic white pulp (WP). We show in this study that, as recently described for lymph nodes, fibroblastic reticular cells (FRCs) form a network in the T cell zone (periarteriolar lymphoid sheath, PALS) of the WP on which T lymphocytes migrate. This network connects the PALS to the marginal zone (MZ), which is the initial site of lymphocyte entry from the blood. T cells do not enter the WP at random locations but instead traffic to that site using the FRC-rich MZ bridging channels (MZBCs). These data reveal that FRCs form a substrate for T cells in the spleen, guiding these lymphocytes from their site of entry in the MZ into the PALS, within which they continue to move on the same network. The Journal of Immunology, 2008, 181: 3947–3954. Downloaded from he “spleen is quantitatively the most important organ in blood content (8). This critical function is ensured by specialized the lymphoid system, with more lymphocytes passing populations of macrophages termed marginal zone macrophages through this organ each day than all the other lymphoid and marginal zone metallophilic macrophages (MMMs), as well as T 3 tissues combined” (1). In lymph nodes (LNs), blood-borne lym- by dendritic cells designated marginal zone dendritic cells (9–11). phocytes enter the paracortex via high endothelial venules, spe- Located between the MZ and the WP, the MZ sinus and its sinus- http://www.jimmunol.org/ cialized blood vessels that support the rolling, arrest, and diape- lining cells are believed to be the place where recently incoming desis of T and B lymphocytes across the endothelium into the lymphocytes can access the neighboring WP (4, 8, 12, 13), al- surrounding parenchyma (for reviews, see Refs. 2, 3). The molec- though there is little direct experimental support for this model. ular events involved in this migration from blood to LN are well Based on histological observation of human spleen sections, an characterized and sequentially involve selectins, chemokines, and alternative scheme has been proposed in which specialized fibro- integrins. In the spleen, high endothelial venules are absent (4) and blasts guide CD4ϩ T cell entry into the periarteriolar lymphoid although a key role for chemokines such as CCL21 and CXCL13 sheath (PALS) (14). are appreciated in the intrasplenic localization of T (5, 6) and B Recently, we demonstrated that a fibroblastic reticular cell cells (7), respectively, there is scant evidence for any special role (FRC) network supports and guides T and B cell motility in the T by guest on September 24, 2021 of nonhematopoietic structural elements of this organ in guiding cell area of LNs (15), dictating the apparent random migratory lymphocytes to their sites of accumulation once they have entered behavior of these cells. Lymphocytes adapt their shape to the cell from the vasculature. bodies and processes of these large stellate fibroblastic cells and The spleen has a complex and well-described microanatomy follow the supporting fibers of the FRCs during migration within (see Fig. 1A). The white pulp (WP), where T and B cell popula- the paracortical region (T cell zone) that is itself defined by the tions segregate, is surrounded by the red pulp (RP), a loose mesh- extent of this FRC network (15). In the spleen, the only known work of reticular fibers and fibroblasts where blood is filtered and function of the FRCs is their ability to create a conduit system that old erythrocytes removed (4). Localized between the WP and the transports blood-derived material inside the PALS in the same way RP, the marginal zone (MZ) creates a transit area for recently that the comparable FRC-based conduit system can transport immigrating blood lymphocytes as well as a filtering zone for lymph content in the paracortex of the LN (16, 17). In this study, we characterize the exact location and describe ad- ditional functions of the FRC network in the spleen. As anticipated *Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy from our previous work, we show that these stromal cells are located and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and †Institut National de la Sante´et de la Recherche Me´dicale and ‡Centre National de la Recherche in the splenic PALS and support T cell motility in this region. Sur- Scientifique, Universite´de Nice-Sophia Antipolis, Valbonne, France prisingly, we also found that FRCs connect the PALS to the MZ only Received for publication October 30, 2007. Accepted for publication July 14, 2008. where the MZ sinus and MZ macrophages rims are interrupted (i.e., The costs of publication of this article were defrayed in part by the payment of page the so-called MZ bridging channels (MZBCs)) (18). Using T cell charges. This article must therefore be hereby marked advertisement in accordance homing experiments, we show that T cells entering the PALS do not with 18 U.S.C. Section 1734 solely to indicate this fact. cross the MZ randomly but only use these FRC-rich bridging chan- 1 This research was supported in part by the Intramural Research Program of National nels. Thus, by their unique location, FRCs not only support T cell Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, by the Institut de la Sante´et de la Recherche Me´dicale motility in the PALS but also provide access roads to this area for T (INSERM), and by the Centre National de la Recherche Scientifique (CNRS). cells that have recently immigrated into the spleen. 2 Address correspondence and reprint requests to Dr. Ronald N. Germain, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11N-311, 10 Center Drive, MSC 1892, Bethesda, MD 20892. E-mail ad- Materials and Methods dress: [email protected] Mice 3 Abbreviations used in this paper: LN, lymph node; WP, white pulp; RP, red pulp; MZ, marginal zone; MMM, marginal metallophilic macrophage; PALS, periarteriolar C57BL/6 and C57BL/6 ubiquitin-GFP mice (UBI-GFP/BL6, strain 4353) lymphoid sheath; FRC, fibroblastic reticular cell; MZBC, marginal zone bridging were purchased from The Jackson Laboratory and maintained in the channel; 2P, two photon. National Institutes of Health animal facilities. Hu-CD2 GFP mice were www.jimmunol.org 3948 REGULATION OF T CELL MIGRATION IN THE SPLENIC WP originally a gift from D. Kioussis (Mill Hill, London, U.K.). For the gen- eration of chimeras, C57BL/6 ubiquitin-GFP mice were gamma-irradiated with a single dose of 950 rads (or twice with 500 rads) from a cesium source and were reconstituted with 2 ϫ 106 C57BL/6 bone marrow cells. At 8 wk after reconstitution, mice were tested for chimerism. Chimeras were used for subsequent experiments only if analysis of blood leukocytes showed the presence of less than 2% of CD3ϩ T cells of host origin. All procedures performed on animals in this study have been approved by the Animal Care and Use Committee, National Institute of Allergy and Infec- tious Diseases, National Institutes of Health. Adoptive transfers T cells were purified from the LNs of wild-type mice with a pan T cell isolation kit while B cells were purified from their spleens with a pan B cell isolation kit (Miltenyi Biotec). Cells were stained with either 5-chlorom- ethyl fluorescein diacetate-2 ␮M, carboxy-fluorescein diacetate, succinimi- dyl ester-2 ␮M, CellTracker red CMTPX (2.5 ␮M), or SNARF-1 (2.5 ␮M) (Invitrogen) at 37°C for 15 min. The indicated numbers of cells were trans- ferred into host mice by i.v. injection. Downloaded from Antibodies ERTR-7 Ab specific for an unknown FRC-secreted molecule and anti- desmin serum were purchased from Acris Abs. RA3–6B2 Ab specific for B220, 17A2 specific for the CD3 complex, and MECA-89 specific for MadCAM-1 were from BD Pharmingen. MOMA-1 Ab specific for MMM was purchased from Cedarlane. A goat polyclonal anti-murine CCL21/ 6cKine was purchased from R&D Systems.