Cytomegalovirus Infection Splenic White Pulp Stroma from Murine NK

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

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: forward, ATC-TAC-GAG-GGC-TAT-GCT-CTC-C; reverse, CTT-TGA- TGT-CAC-GCA-CGA-TTT-CC. These reactions were performed using SYBR Green, as previously described (17). CXCL10 and CXCL11 were used as ready-made TaqMan gene expression assays (CXCL10, Mm00445235_m1; CXCL11, Mm00444662_m1). Laser-capture microdissection Spleen tissue was cut at 5-␮m-thick sections in PALM membrane slides (Zeiss) and then stained with a 1% w/v cresyl violet solution made from cresyl violet acetate (Sigma-Aldrich) dissolved in molecular grade ethanol (Sigma-Aldrich). Slides were dipped sequentially into 100, 70, and 50% solutions of ethanol and stained immediately with cresyl violet for 6 min, and then dipped in the ethanol solutions in the reverse order. Laser microdissection was performed using a Microbeam HT microscope (PALM Mi- crolaser Technologies). Dissected areas were used to obtain genomic DNA. Flow cytometry Splenocytes were prepared, as previously described (16), and stained with anti-CD3␧ PE (eBioscience), and either anti-DX5 FITC (eBioscience) or anti-NK1.1 allophycocyanin (eBioscience). CCR7 was detected with anti- CCR7 PE (BD Biosciences). CXCR3 was detected with anti-CXCR3 (rabbit; Invitrogen), followed by anti-rabbit FITC (Southern Biotechnology Associates). White pulp stromal cells were stained for podoplanin with hamster anti-podoplanin (clone 8.1.1), followed by anti-hamster FITC (Southern Biotechnology Associates) or isotype FITC (Southern Biotech- nology Associates), for VCAM-1 with anti-VCAM-1 PE (Southern Bio- technology Associates) or isotype PE (Southern Biotechnology Associ- ates), and for CD248 with rabbit anti-CD248 (gift from C. Isacke, Breakthrough Breast Cancer Research Centre, London, U.K.), followed by anti-rabbit PE (Southern Biotechnology Associates) or isotype PE (South- ern Biotechnology Associates). For intracellular staining, spleen cells were cultured for4hinthepresence of GolgiStop (BD Biosciences), and they were stained for intracellular IFN-␥ PE (BD Biosciences) or perforin PE (eBioscience) using the Cytoﬁx/Cytoperm kit (BD Biosciences), according by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. to the manufacturer’s instructions. Samples were acquired using a BD FACScan ﬂow cytometer, and the results were analyzed using FlowJo software. Generation of white pulp stromal cell line To generate white pulp stromal cell lines, spleen cells from 7- to 10-day- old C57BL/6 mice were cultured for 2–3 days and then nonadherent cells FIGURE 2. Disintegration of the white pulp is not NK or T cell medi- were washed out. Adherent cells were cultured for an additional 5–6 mo, Ϫ ϩ ated, and NK cells are required for its protection during MCMV infection. at which time they were positively MoFlo sorted for CD45 podoplanin A, Spleen sections from day 0- and day 4-infected C57BL/6 or BALB/c cells. Podoplaninϩ stromal cells were kept in culture for an additional 5 mo

http://classic.jimmunol.org mice and C57BL/6 or BALB/c mice that were previously depleted of NK before used for in vitro MCMV infection experiments. cells were stained for B220 (green) and CD3 (red) and analyzed by con- Statistical analysis focal microscopy. B, Dot plots indicate the efficiency of anti-asialo GM1 to deplete NK cells in C57BL/6 and BALB/c mice. C, Spleen sections from All statistical analyses were performed with the nonparametrical Mann- day 0- and day 4-infected nude (BALB/c background) mice were stained Ͻ Whitney U test using StatView 5.0 ( p 0.05 is considered significant). and analyzed as in A. A and C, Confocal micrographs were taken with a ϫ10 objective (scale bar ϭ 100 ␮m). Results Downloaded from Disintegration of splenic white pulp in the absence of protective NK cell immunity following MCMV infection CMV infection is associated with secondary T cell immunodefi- 1, A–C) and NK (Fig. 1, B and D) cells. At this time point, there ciency (18). To investigate the role of NK cells in protection from was preferential activation of Ly49Hϩ NK cells in C57BL/6 mice, MCMV, we compared day 0 and day 4 spleens from resistant shown by their expression of IFN-␥ and perforin (Fig. 1E). These Ly49Hϩ C57BL/6 or susceptible BALB/c mice (Fig. 1A). Before data showed that absence of a specific NK response was linked to infection, B and T cell white pulp areas were comparable in both near complete loss of T cells and DCs, and disintegration of the strains (Fig. 1A). However, 4 days postinfection, there were structure of the splenic white pulp areas. marked differences between the two strains of mice with regard to The splenic immunopathology observed was not dependent on preservation of white pulp areas. In C57BL/6 mice, there was re- nonspecific NK cell activation, because infection of NK-depleted tention of CD11cϩ dendritic cells (DCs) and CD3ϩ T cells (Fig. BALB/c and C57BL/6 mice resulted in the same immunopathol- 1A). In contrast, there was marked destruction of splenic white ogy (Fig. 2, A and B). Moreover, MCMV-infected BALB/c nude pulp areas in BALB/c mice (Fig. 1A), with loss of both CD11cϩ mice (few T cells) exhibited similar immunopathology (Fig. 2C), DCs (Fig. 1A), and a ϳ10-fold reduction in the number of T (Fig. suggesting that destruction of the white pulp is not T cell mediated. The Journal of Immunology 6771

FIGURE 3. Localization of MCMV in the white and red pulp. A, Quantita- tive real-time PCR for MCMV in total p ϭ 0.01 comparing B6 at ,ء ;spleen p ϭ 0.07 comparing ,ءء ;days 0 and 4 BALB/c at days 0 and 4. B, A spleen section from a day 4-infected C57BL/6 mouse stained with cresyl violet and indicating the laser-microdissected red and white pulp areas. C, Quantitative real-time PCR for MCMV in laser-microdissected white p ϭ 0.045 comparing B6 at ,ء ;pulp -p ϭ 0.0045 compar ,ءء ;days 0 and 4 ing BALB/c at days 0 and 4. D, Quan- titative real-time PCR for MCMV in p ϭ ,ء ;laser-microdissected red pulp 0.1 comparing B6 at days 0 and 4; p ϭ 0.3 comparing BALB/c at ,ءء days 0 and 4. A, C, and D,B6ϭ C57BL/6, ϪNK.B6 ϭ NK-depleted C57BL/6; a value of 1 represents equal expression of the MCMV gene and the control housekeeping gene; solid line ϭ median; each symbol represents a mouse.

Taken together, the above data support the importance of specific control in both locations. This series of experiments indicated that NK activation for protection of white pulp areas. the immunopathology associated with white pulp areas could be attributable to MCMV replication at this site. Evidence that MCMV replicates in both red and white pulp areas MCMV destroys white and red pulp stroma in vivo in BALB/c mice by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. Using quantitative PCR primers for glycoprotein L, a late expressed MCMV gene (19), we evaluated viral titers in whole spleen sections Both red and white pulp stroma express VCAM-1, although ex- (Fig. 3A). Viral titers were ϳ10-fold greater on day 2 and ϳ100-fold pression levels are much higher in the red pulp, where there is a greater on day 4 in BALB/c compared with C57BL/6 mice (Fig. 3A). high density of stromal cells (Fig. 4). In the white pulp, VCAM-1 In C57BL/6 mice, viral titers were reduced at day 4 compared with expression is restricted to fibroreticular stromal cells that ensheath day 2, indicating NK-mediated protection (Fig. 3A). In contrast, in the splenic conduit (21, 22), which guides lymphocyte movement BALB/c mice, viral titers were increased at day 4 compared with day (23). Splenic T zone stroma also expresses the transmembrane 2 (Fig. 3A). Depletion of NK cells in C57BL/6 mice resulted in com- mucin-like glycoprotein, podoplanin, which is strongly up-regu- parable viral titers to BALB/c mice (Fig. 3A). lated following MCMV infection in both C57BL/6 and BALB/c http://classic.jimmunol.org The main site of MCMV viral replication has been identified as mice (Fig. 4, A–D, and Benedict et al. (20)). Although the podo- the splenic red pulp (20). To investigate whether the destruction of planin network is increased in BALB/c mice, very few live cells splenic white pulp areas in susceptible BALB/c mice was second- could be identified at day 4 postinfection using a 4Ј,6Ј-diamidino- ary to MCMV infection, we performed laser capture microdissec- 2-phenylindole (DAPI) nuclear counterstain, compared with unin- tion of splenic white and red pulp areas, and evaluated MCMV fected and day 4 C57BL/6 mice (Fig. 4E). Furthermore, whereas viral load. Cresyl violet-stained spleen sections identified white VCAM-1 expression was preserved in C57BL/6 red and white

Downloaded from pulp areas as darker violet compared with red pulp (Fig. 3B). At pulp stroma, it was greatly reduced in BALB/c mice in both red day 2 postinfection, MCMV could be detected in both the splenic and white pulp areas (Fig. 4, B and D), consistent with infection red and white pulp areas of both C57BL/6 and BALB/c mice (Fig. and destruction of these cells by MCMV. Accordingly, the same 3, C and D). Red pulp titers at day 2 were significantly higher in stromal cell phenotype was exhibited by C57BL/6 and BALB/c BALB/c mice (Fig. 3D). At this time point, MCMV was also mice that were depleted of NK cells and by T cell-deficient nude readily detected in the white pulp areas: there was no significant BALB/c mice (data not shown) in agreement with white pulp dis- difference in MCMV titers between C57BL/6 and BALB/c mice integration in these mice (see Fig. 2) and viral-, but not immune- (Fig. 3C). By day 4, the situation had changed substantially: red mediated destruction. pulp titers were falling in C57BL/6 mice, whereas viral titers re- There is evidence that viral infection can cause down-regulation mained unchanged in BALB/c mice (Fig. 3D). In the white pulp, of homeostatic chemokines, particularly CCL21 (20), and this can there was a significant reduction in viral titers associated with pres- be secondary to immune activation through IFN-␥ (24). We found ervation of white pulp areas in C57BL/6 mice, whereas titers had that the destruction of the white pulp in BALB/c mice was asso- continued to rise in BALB/c mice (Fig. 3C). NK-depleted ciated with much lower levels of CCL21 and CCL19 compared C57BL/6 mice showed similar red and white pulp viral titers to with C57BL/6 (Fig. 5, A and B), whereas the difference in BALB/c mice (Fig. 3, C and D), consistent with NK-mediated viral CXCL13 expression was significant, but not as marked (Fig. 5C). 6772 WHITE PULP DISINTEGRATION DURING CMV INFECTION

FIGURE 4. Destruction of white and red pulp stroma by MCMV. Spleen sections from day 0 BALB/c (A) and C57BL/6 (C)or day 4-infected BALB/c (B) and C57BL/6 (D) mice were stained for B220 (blue), podoplanin (red), and VCAM-1 (green), and analyzed by confocal microscopy; confocal micrographs were taken with a ϫ10 objective (scale bar ϭ 100 ␮m). E, High magni- ﬁcation (ϫ63 objective, scale bar ϭ 20 ␮m) confocal micrographs showing podoplaninϩ (red) white pulps counterstained with DAPI (gray) in day 0- or day 4-infected C57BL/6 and BALB/c mice. by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

This grossly reduced chemokine expression in BALB/c mice is in vitro, and podoplanin-expressing stromal cells derived from day consistent with destruction of the white pulp chemokine-express- 7–10 murine spleen (see Materials and Methods). The podoplanin- ing stroma. expressing stromal line was very similar to freshly isolated podoplanin-expressing stroma (Fig. 5, D and E) (25). Besides podoplanin, this In vitro evidence that MCMV infects white pulp stroma cell line expressed VCAM-1, but lacked CD248 (Fig. 5D), which To test directly whether white pulp podoplanin-expressing stromal speciﬁcally marks red pulp stroma (26). In addition, at the mRNA cells could be infected with MCMV, we compared viral replication level, those cells expressed CCL19, IL-7, and Lt␤R (Fig. 5E), all of in mouse embryonic ﬁbroblasts (MEFs), used to propagate MCMV which are associated with white pulp stroma (25). Following infection The Journal of Immunology 6773 by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd.

FIGURE 5. MCMV down-regulates stromal cell-expressed chemokines and infects white pulp stroma in vitro. A–C, Quantitative real-time PCR in total spleen for CCL21 (A), CCL19 (B), and CXCL13 (C) at days 0 and 4 after infection for BALB/c (Ⅺ) and C57BL/6 (f) mice; columns ϭ mean of three to ﬁve mice, error bar ϭ SD. D, White pulp podoplanin-expressing stromal cells were stained for the indicated markers and analyzed by ﬂow cytometry;

http://classic.jimmunol.org plots are from live-gated cells based on forward/side scatter; shaded histograms are isotype control Abs. E, Quantitative real-time PCR for the indicated genes in white pulp stromal cells. F, Quantitative real-time PCR for MCMV in white pulp stromal cells (f) or MEFs (Ⅺ) at the indicated MOIs.

in vitro with MCMV, the virus replicated to comparable levels in both found in the white pulp areas by day 2, and they were clustered in MEFs and the podoplanin-expressing cell line (Fig. 5F), consistent bridging channels of the marginal zone (Fig. 6), where CD11cϩ DCs

Downloaded from with the idea that MCMV infected podoplanin-expressing stroma are located normally, and where they enter the white pulp areas after in vivo. immunization. By day 4, significant numbers of Ly49Hϩ cells were found in the T zone areas, where they were engaged with podoplanin- ϩ Migration of Ly49H NK cells in the white pulps of expressing stroma cells (Fig. 6, bottom high magnification micro- MCMV-infected mice graph), illustrating how Ly49Hϩ NK cells might inhibit viral repli- The implication of our data is that the specific resistance to cation in T zone stroma. Consistent with the requirement of both MCMV provided by Ly49Hϩ NK cells acts not only by control- IFN-␥ and perforin expression in NK cells for protection from de- ϩ ling viral replication (7), but by preserving the structure of the struction (13), Ly49H NK cells selectively up-regulated expression splenic white pulp as well. To try to understand how might of these molecules postactivation (Fig. 1E). Ly49Hϩ NK cells protect the white pulp, we investigated their response and localization properties following MCMV infection. Expression of CXCR3 by NK cells and induction of its ligands, We found that this subpopulation was preferentially localized in the CXCL10 and CXCL11, by spleen, white pulp, red pulp, and splenic red pulp in MCMV naive mice (Fig. 6, day 0). However, white pulp stroma following infection in C57BL/6 mice, Ly49Hϩ NK cells relocated to Unlike T cells, murine NK cells do not express CCR7 (Fig. 7A), the white pulp areas of the spleen (Fig. 6). A few Ly49Hϩ cells were and therefore cannot respond to CCL19 and CCL21, which are 6774 WHITE PULP DISINTEGRATION DURING CMV INFECTION

FIGURE 6. Migration of Ly49Hϩ NK cells in the white pulp. Spleen sections from day 0-, day 2-, or day 4-infected C57BL/6 mice were stained for IgM (blue), podoplanin (red), and Ly49H (green), and analyzed by confocal microscopy; top two confocal micrographs were taken with a ϫ10 objective (scale bar ϭ 100 ␮m); bottom left confocal micro- graph was taken with a ϫ63 objective (scale bar ϭ 20 ␮m) and represents a white pulp area at day 4 after infection. To quantify Ly49Hϩ cell migration, positive pixels for Ly49H were calculated, as described before (16), per ␮m2 of white or red pulp; total (white plus red pulp); columns are the means from 6 to 15 confocal micrographs repre- senting 2–5 mice (three micrographs per mouse); error bar ϭ SD.

produced by T zone stroma. It has been shown before that CXCR3 were found in both BALB/c and C57BL/6 mice, but by day 4 white can mediate NK cell migration to lymph nodes (27) and that pulp titers had fallen in C57BL/6 mice, but had risen substantially Ϫ Ϫ CXCR3 / mice have elevated MCMV titers in spleen and liver (ϳ10-fold) in BALB/c-infected mice.

by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. (28). We found that NK cells express CXCR3 before and after The above data taken together were consistent with red and MCMV infection (Fig. 7A). Importantly, the CXCR3 ligands, white pulp stromal cells being targets for MCMV replication. Al- CXCL10 and CXCL11, were up-regulated following infection in though viral titers were higher in red pulp than white pulp areas, total spleen tissue (Fig. 7B), white pulp (Fig. 7C), and red pulp this is probably due to the difference in stromal cell density. The (Fig. 7D), as well as podoplanin-expressing stromal cells (Fig. 7, expression of the stromal marker, VCAM-1, is far more intense in E and F). This suggests a mechanism whereby, upon infection, NK the red pulp, because white pulp VCAM-1 expression is restricted cells can be recruited first to the spleen and second to distinct areas to the B and T cell conduit. A striking observation was the pres- within the spleen, including the white pulp and T zone. ervation of podoplanin expression in the T zone, but staining of live nuclei with DAPI demonstrated profound loss of viable cells http://classic.jimmunol.org Discussion in susceptible mice. In this study, we report that MCMV infection in Ly49H-deficient Direct evidence that MCMV could infect white pulp stroma was BALB/c mice is associated with splenic white pulp destruction and provided by our observation that a podoplaninϩ stromal cell line subsequent loss of CD3ϩ T and CD11cϩ DCs. Recent studies have derived from day 7 neonatal spleen, which expresses CCL19, IL-7, indicated that viral infection nonspecifically leads to down-regu- and the LT␤R, supported the replication of MCMV to levels com- lation of homeostatic chemokines (mainly CCL21, but also parable to those attained in mouse embryonic fibroblasts, normally Downloaded from CXCL13) via a mechanism dependent on IFN-␥ production from used to propagate the virus in vitro. These data taken together CD4 T cells (24). We found that down-regulation of CCL21 was imply that MCMV infects and destroys podoplanin-expressing exacerbated in the absence of a specific NK response, which could be explained if viral replication occurred in the CCL21-expressing white pulp stroma with consequent abrogation of expression of T zone fibroreticular cells, as reported for lymphocytic choriomen- VCAM-1 and CCL21. ingitis virus (14). It has already been shown that MCMV replicates NK cells are normally found in the red pulp of spleen. To un- ϩ derstand how white pulp areas were protected in C57BL/6 mice, in red pulp areas of the spleen in Ly49H C57BL/6 mice (20), and ϩ consistent with a cytopathic effect, we found extensive destruction we analyzed Ly49H NK cells before and after infection. As ex- ϩ of VCAM-1-expressing red pulp stromal cells in the absence of pected, preinfection Ly49H cells were located in the red pulp. By ϩ Ly49Hϩ NK cells. We also noted, however, that there was loss of day 4 postinfection, Ly49H NK cells were preferentially ex- VCAM-1 expression from the podoplanin-expressing white pulp panded and expressed both perforin and IFN-␥, both linked with ϩ stroma, consistent with viral infection of these cells in BALB/c, protection from MCMV (12, 13). Some Ly49H cells were clus- but not C57BL/6 mice. Using laser capture microscopy of red and tered in marginal zone bridging channels by day 2, and on days 2 white pulp areas, we demonstrate viral replication in white pulp and 4, Ly49Hϩ cells were also found in white pulp areas, associ- areas. At day 2 postinfection, comparable levels of MCMV virus ated with podoplanin-expressing stromal cells. The Journal of Immunology 6775

FIGURE 7. Expression of CXCR3 by NK cells and induction of its ligands, CXCL10 and CXCL11, after MCMV infection. A, Spleen cells from day 0 or 4 MCMV-infected C57BL/6 mice were stained for NK1.1, CD3␧, and either CXCR3 or CCR7; NK1.1ϩCD3␧Ϫ (NK cells)- or NK1.1ϩCD3␧ϩ (T cells)-gated cells are shown. B–D, Quantitative real- time PCR in total spleen (B), laser- microdissected white pulp (C), and laser-microdissected red pulp (D) for CXCL10 and CXCL11 at day 0 (f) and day 4 (Ⅺ) after infection; error bar ϭ SD; B, columns ϭ mean of four to five mice (one of two experiments shown); C and D, columns ϭ mean of four to six mice, n.d. ϭ not detected. E and F, Quantitative real- time PCR for CXCL10 (E) and CXCL11 (F) in white pulp stromal cells that were uninfected or infected at MOI 3.3 and cultured for 2, 4, or 6 days; columns ϭ mean of two cell lines, error bar ϭ SD. by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. http://classic.jimmunol.org The chemokine receptor CXCR3 has been implicated in the mi- Disclosures gration of NK cells in secondary lymphoid tissue (27). Accord- The authors have no financial conflict of interest. ingly, we found that NK cells express CXCR3 and that MCMV infection induces expression of its ligands, CXCL10 and CXCL11. References The expression of CXCR3 by NK cells and the up-regulation of 1. Biron, C. A., K. S. Byron, and J. L. Sullivan. 1989. Severe herpesvirus infections

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