The Journal of Immunology Ly49H؉ NK Cells Migrate to and Protect Splenic White Pulp Stroma from Murine Cytomegalovirus Infection1 Vasileios Bekiaris,* Olga Timoshenko,† Tie Zheng Hou,* Kai Toellner,* Saba Shakib,* Fabrina Gaspal,* Fiona M. McConnell,* Sonia M. Parnell,* David Withers,* Chris D. Buckley,* Clive Sweet,† Wayne M. Yokoyama,‡ Graham Anderson,* and Peter J. L. Lane2* In this study, we show that in the absence of a protective NK cell response, murine CMV causes destruction of splenic white and red pulp pulp areas in the first few days of infection. Destruction of T zone stroma is associated with almost complete loss of dendritic cells and T cells. We provide evidence that the virus replicates in red and white pulp stroma in vivo and in vitro. Control of white pulp viral replication is associated with migration of murine CMV-specific activated NK cells to white pulp areas, where they associate directly with podoplanin-expressing T zone stromal cells. Our data explain how NK cells protect the lymphoid-rich white pulp areas from CMV, allowing protective adaptive T cell-dependent immune responses to develop, and how this mechanism might break down in immunocompromised patients. The Journal of Immunology, 2008, 180: 6768–6776. n immunocompetent individuals, acute infection with the planin-expressing stromal cell lines to levels similar to that found ␤-herpes virus, CMV, is generally asymptomatic, but it in the fibroblasts that are used to propagate the virus in vitro. To causes morbidity and mortality in NK cell-deficient mice and investigate why MCMV replication was suppressed in C57BL/6 I 3 ϩ humans (1–3). Mice infected with murine CMV (MCMV) are mice, we analyzed the location and activation of Ly49H NK either susceptible or resistant to disease, depending on their genetic cells. There was selective up-regulation of perforin and IFN-␥ in background (4). Resistance maps to a single genetic locus encod- Ly49Hϩ NK cells following infection. Furthermore, a significant ing the Ly49 family of NK lectin receptors (5, 6). Specific resis- fraction of activated Ly49Hϩ NK cells moved from the red pulp tance is through Ly49H, expressed on a subset of NK cells, present (their location in noninfected mice) to the T cell areas, where they in resistant C57BL/6 mice, but not in susceptible BALB/c (7, 8). were found specifically associated with podoplanin-expressing fi- This receptor directly recognizes the MCMV-encoded protein broreticular cells. These studies suggest a mechanism for the ac- m157 on the surface of infected cells (9, 10), resulting in a quired T cell immunodeficiency found in humans that fail to control MCMV-specific NK response (11). Although both perforin and CMV infection, and demonstrate how NK cells protect T zone stroma. IFN-␥ expression by NK cells contributes to early protection from by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. MCMV (12), NK perforin expression is crucial to the prevention Materials and Methods of the immunopathology observed in the spleen (13). Mice Recent studies have indicated that the murine pathogen, lym- All experiments were performed in accordance with United Kingdom laws phocytic choriomeningitis virus, infects the chemokine-expressing and with the approval of the University of Birmingham ethics committee. fibroreticular cells in the splenic white pulp, accounting for disor- All strains of mice were bred in our animal facility. ganization of lymphoid structures and consequent immunodefi- Infections and virus ciency observed in that disease (14). In this study, we demonstrate that infection of susceptible BALB/c mice with MCMV is also For infections, we used wild-type MCMV K181 that was grown in BALB/c mice and isolated from salivary glands. Each mouse was injected i.p. with associated with white and red pulp MCMV infection, and that viral 5 http://classic.jimmunol.org ϩ 50 ␮lof4ϫ 10 PFU MCMV. For in vitro infections, the same stock of replication in both sites is suppressed in Ly49H C57BL/6 mice. virus was applied for 4 ϫ 105 cells at multiplicities of infection (MOIs) 1.6, Second, we show that MCMV can infect and replicate in podo- 3.2, 1.6 ϫ 10Ϫ2, and 3.2 ϫ 10Ϫ4, and cells were harvested 6, 5, 10, or 9 days later, respectively. Otherwise, 4 ϫ 105 cells were infected at MOI 3.3 and harvested 2, 4, or 6 days later. *Medical Research Council Centre for Immune Regulation, Birmingham Medical Immunostaining and confocal microscopy School and †School of Biosciences, Birmingham, United Kingdom; and ‡Washington Downloaded from University School of Medicine, St. Louis, MO 63110 The procedure for preparing and staining mouse spleen tissue for light and confocal microscopy has been described before (15, 16). Ly49Hϩ cells Received for publication January 8, 2008. Accepted for publication March 6, 2008. were detected with a FITC (fluorescein)-conjugated mouse anti-Ly49H The costs of publication of this article were defrayed in part by the payment of page mAb (7). Pixel analysis and confocal image acquisition were performed, as charges. This article must therefore be hereby marked advertisement in accordance previously described (16). with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a Wellcome Programme grant to P.J.L.L. and G.A. NK cell depletion 2 Address correspondence and reprint requests to Dr. Peter J. L. Lane, Medical Re- NK cells were depleted using a single i.p. injection of 50 ␮l of anti-asialo search Council Centre for Immune Regulation, Institute for Biomedical Research, GM1 (Wako Chemicals) from a 1-ml stock. The efficiency of the depletion Birmingham Medical School, Birmingham B15 2TT, U.K. E-mail address: was checked by NK cell staining in the blood before infecting the mice [email protected] (always 1 day after anti-asialo GM1 injection). 3 Abbreviations used in this paper: MCMV, murine CMV; DAPI, 4Ј,6Ј-diamidino- 2-phenylindole; DC, dendritic cell; LT␤R, lymphotoxin-␤ receptor; MEF, mouse em- Preparation of DNA and cDNA bryonic fibroblast; MOI, multiplicity of infection. Total genomic DNA from frozen 15-␮m-thick spleen sections, or laser- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 microdissected tissue or cells was prepared using the DNeasy blood and www.jimmunol.org The Journal of Immunology 6769 by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. http://classic.jimmunol.org FIGURE 1. White pulp disintegration in the absence of NK cell immunity following MCMV infection. A, Spleen sections from uninfected (day 0, d0) and day 4 (d4)-infected C57BL/6 or BALB/c mice were stained for IgM (brown) and either CD11c (blue) or CD3 (blue), and analyzed by light microscopy; photographs were taken with a ϫ4 objective. B, Spleen cells from day 0- or day 4-infected BALB/c and C57BL/6 mice were stained for T and NK cells using CD3 and either DX5 or NK1.1 and analyzed by flow cytometry; dot plots are from live-gated cells based on forward/side scatter; numbers in quadrants indicate percentages of Downloaded from total live cells; results are representative of two experiments with three to five mice per experiment. C and D, Summarizes the results in B for T and NK cells, respectively; Ⅺ ϭ BALB/c, f ϭ C57BL/6; columns ϭ mean of three to five mice, error bar ϭ SD. E, Activated splenic Ly49Hϩ NK cells from day 0- or day 4-infected C57BL/6 mice were identified by staining for intracellular IFN-␥ or perforin; cells were costained for Ly49H and NK1.1; NK1.1ϩ-gated cells are shown. tissue kit (Qiagen), according to the manufacturer’s instructions. cDNA housekeeping gene in a duplex reaction (5Ј33Ј): forward, GCC-GCT- was prepared from cells using the ␮MACS One-step cDNA kit (Miltenyi AGA-GGT-GAA-ATT-CTT-G; reverse, CAT-TCT-TGG-CAA-ATG- Biotec), according to the manufacturer’s instructions, or from 8-␮m-thick CTT-TCG; probe, VIC-CCG-GCG-CAA-GAC-GGA-CCA-GA-TAMRA. spleen sections using the RNeasy micro kit (Qiagen), according to the The following genes were also detected with the same method (5Ј33Ј): manufacturer’s instructions. CCL21, forward, TCC-CGG-CAA-TCC-TGT-TCT-C; reverse, TTC- TGC-ACC-CAG-CCT-TCC-T; probe, 6-FAM-CCC-CGG-AAG-CAC- Quantitative real-time PCR TCT-AAG-CCT-GAG-CTA-T-BHQ-1; CCL19, forward, CCT-TCC- Quantitative real-time PCR (TaqMan) reactions were performed, as de- GCT-ACC-TTC-TTA-ATG-AAG; reverse, ACA-GAG-CTG-ATA- scribed previously (16). The target MCMV gene was glycoprotein L GCC-CCT-TAG-TGT; probe, 6-FAM-TGC-AGG-GTG-CCT-GC- (5Ј33Ј): forward, CCC-CGC-CGT-GTT-TTC-A; reverse, GCC-ATC- TAMRA; and CXCL13, forward, ACA-TCA-TAG-ATC-GGA-TTC- ACG-GTC-TCT-TTC-GT; probe, 6FAM-ACC-CGA-CAA-CAC-TAT- AAG-TTA-CG; reverse, TCT-TGG-TCC-AGA-TCA-CAA-CTT-CAG; CGC-CCT-CAA-TAT-CA-BHQ1. The 18S rRNA was used as the control probe, 6-FAM-CCT-GGG-AAT-GGC-TGC-CCC-AAA-TAMRA. To 6770 WHITE PULP DISINTEGRATION DURING CMV INFECTION detect CCL19, lymphotoxin-␤ receptor (LT␤R), and IL-7 in cultured white pulp stromal cells, we used the following primers (5Ј33Ј): CCL19, forward, GGG-GTG-CTA-ATG-ATG-CGG-AA; reverse, CCT-TAG- TGT-GGT-GAA-CAC-AAC-A; LT␤R, forward, GAG-CAG-AAC- CGG-ACA-CTA-GC; reverse, GAA-GGT-AGG-GAT-GAG-CAC-C; and IL-7, forward, TTC-CTC-CAC-TGA-TCC-TTG-TTC-T; reverse, AGC- AGC-TTC-CTT-TGT-ATC-ATC-AC. The control gene was ␤-actin: for- ward, ATC-TAC-GAG-GGC-TAT-GCT-CTC-C; reverse, CTT-TGA- TGT-CAC-GCA-CGA-TTT-CC.