• No results found

Perforin-Deficient CD8+ T Cells Mediate Fatal Lymphocytic Choriomeningitis despite Impaired Cytokine Production

N/A
N/A
Protected

Academic year: 2019

Share "Perforin-Deficient CD8+ T Cells Mediate Fatal Lymphocytic Choriomeningitis despite Impaired Cytokine Production"

Copied!
9
0
0

Loading.... (view fulltext now)

Full text

(1)

0022-538X/06/$08.00⫹0 doi:10.1128/JVI.80.3.1222–1230.2006

Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Perforin-Deficient CD8

T Cells Mediate Fatal Lymphocytic

Choriomeningitis despite Impaired Cytokine Production

Pernille Storm, Christina Bartholdy, Maria Rathman Sørensen,

Jan Pravsgaard Christensen, and Allan Randrup Thomsen*

Institute of Medical Microbiology and Immunology, University of Copenhagen,

Copenhagen, Denmark

Received 12 September 2005/Accepted 9 November 2005

Intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) is one of the most studied models for virus-induced immunopathology, and based on results from perforin-deficient mice, it is currently assumed that fatal disease directly reflects perforin-mediated cell lysis. However, recent studies have revealed additional functional defects within the effector T cells of LCMV-infected perforin-deficient mice, raising the possibility that perforin may not be directly involved in mediating lethal disease. For this reason, we decided to reevaluate the role of perforin in determining the outcome of i.c. infection with LCMV. We confirmed that the expansion of virus-specific CD8T cells is unimpaired in perforin-deficient mice. However, despite the fact that the virus-specific CD8effector T cells in perforin-deficient mice are broadly impaired in their effector function, these mice invariably succumb to i.c. infection with LCMV strain Armstrong, although a few days later than matched wild-type mice. Upon further investigation, we found that this delay correlates with the delayed recruitment of inflammatory cells to the central nervous system (CNS). However, CD8effector T cells were not kept from the CNS by sequestering in infected extraneural organ sites such as liver or lungs. Thus, the observed dysfunctionality regarding the production of proinflammatory mediators probably results in the delayed recruitment of effector cells to the CNS, and this appears to be the main explanation for the delayed onset of fatal disease in perforin-deficient mice. However, once accumulated in the CNS, virus-specific CD8T cells can induce fatal CNS pathology despite the absence of perforin-mediated lysis and reduced capacity to produce several key cytokines.

CD8⫹T cells are key mediators in the immune response to many viral infections. Following activation in the regional sec-ondary lymphoid organs, cytotoxic T lymphocytes migrate to the site(s) of infection, kill virus-infected cells by the granule exocytosis- and/or Fas/FasL-mediated pathways (29, 59, 62), and secrete proinflammatory cytokines such as gamma inter-feron (IFN-␥) and tumor necrosis factor alpha (TNF-␣) (15, 33, 43, 49, 55). The relative importance of these effector path-ways varies with the virus studied (28, 46, 47, 53, 54). However, the same molecular effector systems may also form the basis for immunopathology and contribute to the tissue damage usually associated with viral infection. Whether the net effect of the immune response is protection or immunpathology de-pends on a number of factors, such as viral tropism, the intrin-sic cytopathogenecity of the virus, and the relative kinetics of immune response versus virus spreading (64). The present study focuses on the immunological effector mechanisms, which determines the outcome of the intracranial infection of mice with lymphocytic choriomeningitis virus (LCMV).

LCMV infection is a classical model for studying the dichot-omous role of the antiviral immune response. It represents an ideal tool in this respect, since the virus itself is noncytopathic and the pathology incurred during infection is exclusively caused by the immune response, mainly the effector CD8⫹T cells (19). In vivo studies using depleting antibodies, transgenic

mouse strains, and adoptive transfer have revealed an essential role of cytotoxic T lymphocytes in the control of acute LCMV infection, and high viral loads are found in infected perforin-deficient mice, many of which do not thrive and eventually die (8, 27, 39, 51, 61, 62, 66). Production of IFN-␥is also important for the control of LCMV infection, and the requirement for this cytokine in the clearance of acute infection is strongly influenced by the tropism and invasiveness of the infective virus strain (4, 44, 51, 60, 63).

The central nervous system (CNS) is a very sensitive and vital organ, and presumably to spare it from wanton immuno-pathology, lymphocyte trafficking through the CNS is minimal under normal circumstances (25, 35). However, during a wide range of infectious and autoimmune neurological diseases such as virus-induced meningoencephalitis and multiple sclerosis, large numbers of circulating lymphocytes gain access to the CNS (7, 26, 58).

During acute infection of the CNS, LCMV replicates pre-dominantly in the choroid plexus, ependyma, and meninges; however, some virus-infected cells are also found in the outer layers of the brain parenchyma (10, 16, 24, 42). It is estimated that upon intracerebral (i.c.) injection of LCMV, about 90% of the inoculum escapes to the periphery, resulting in priming of virus-specific CD8⫹T cells in the secondary lymphoid organs, particularly the spleen, wherefrom activated cells are recruited to the CNS (2, 12, 19, 20, 36). The i.c. infection itself seems to induce a low expression of chemokines and adhesion mole-cules, which allows the initial recruitment of relevant effector cells to the CNS. The recruited cells then reinforce the inflam-matory response through the production of proinflaminflam-matory

* Corresponding author. Mailing address: Institute of Medical Mi-crobiology and Immunology, The Panum Institute, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark. Phone: 45 35327871. Fax: 45 35327891. E-mail: a.r.thomsen@immi.ku.dk.

1222

on November 8, 2019 by guest

http://jvi.asm.org/

(2)

cytokines and chemokines, attracting more mononuclear cells, mainly monocytes/macrophages and activated CD8⫹T cells (9, 38). Along with the increase in the number of effector cells in the CNS, the blood brain barrier is disrupted, brain edema evolves, and in immunocompetent mouse death occurs 7 to 9 days postinfection (p.i.) (1, 40).

Regarding the precise mechanism underlying lethality, var-ious possibilities have been suggested (1, 37, 48, 57): cell con-tact-dependent killing of virus-infected cells, production of proinflammatory cytokines that could induce progressive cere-bral edema, and incarceration, as well as a combination of the two. However, in recent years, it has been widely accepted that LCMV-induced immunopathology correlates directly with the perforin-mediated cytotoxic action of virus-specific CD8⫹ T cells on virus-infected cells in the meninges and that perforin is pivotal for the development of lethal choriomeningitis (29). This paradigm is primarily based upon a study which showed that mice deficient in perforin (Pfp⫺/⫺mice) survive i.c.

infec-tion with the viscerotropic virus strain LCMV-WE (29). While this could point to the direct involvement of perforin in the processes terminating in fatal disease, it might just as well reflect a scenario where the associated extraneural infection is out of control due to the lack of perforin. Under these condi-tions, effector T cells might initially be sequestered in extra-neural sites (56). Furthermore, the high viral load and ongoing antigen stimulation would drive the T cells toward functional inactivation and deletion (21, 22, 23, 32, 45). Thus, the role of perforin in the pathogenesis of LCMV-induced CNS disease is not clearly established. Recently, our group found by using chemokine receptor knockout mice that the presence of virus-specific CD8⫹T cells in the outer layers of the brain paren-chyma (10) correlates better with mortalility than overall cell influx into the CNS, including meningeal infiltration. This find-ing led us to question whether it is simply perforin-mediated damage to the meninges that causes fatal outcome of this disease.

Hence, the present study was undertaken to better define the role of perforin in determing the outcome of i.c. infection with LCMV. For this purpose, Pfp⫺/⫺ and wild-type (WT)

mice were challenged i.c. with a moderate dose of the slowly replicating and neurotropic Armstrong strain of LCMV. Sur-prisingly, we found that Pfp⫺/⫺ mice die from CD8

T-cell-mediated inflammation of the LCMV infected CNS, despite a severely reduced capacity to secrete proinflammatory cyto-kines in addition to the defect in contact-dependent cell killing.

MATERIALS AND METHODS

Mice.Pfp⫺/⫺mice (C57BL/6-Pfptm1Sdz) were the progeny of breeder pairs obtained from The Jackson Laboratory (Bar Habor, ME). Mice deficient in both perforin and IFN-␥were generated as previously described (51) and bred at the Panum Institute, University of Copenhagen. WT (C57BL/6) mice were pur-chased from Taconic M&B (Ry, Denmark). All mice were housed under specific pathogen-free conditions, and sentinels were tested regularly for unwanted in-fections according to Federation of European Laboratory Animal Science As-sociations standards; no unwanted infections were detected. Mice from outside sources were always allowed to acclimatize for at least a week before entering into an experiment. Mice entered experiments when⬃7 to 10 weeks old.

Virus.LCMV of the neurotropic Armstrong strain (clone 53b) was kindly provided by M.B.A. Oldstone, Scripps Clinic and Research Foundation, La Jolla, CA (52). Virus was grown in BHK cells and titered in an immune focus assay as previously described (10). In most experiments, mice were infected i.c. with 200 PFU of virus in a volume of 0.03 ml. In some experiments, the same dose was

given intravenously (i.v.) in a volume of 0.3 ml. To determine virus titers in organs, these were first homogenized in phosphate-buffered saline (PBS) to yield 10% (vol/wt) organ suspensions and serial 10-fold dilutions were prepared, and then these were titered in duplicate using the immune focus assay.

Survival study.Mortality was used to evaluate the clinical severity of acute LCMV-induced meningitis. Mice were checked twice a day for a period of up to 28 days after infection

In vivo depletion of CD8T cells.The␣CD8 monoclonal antibody (clone 53.6.72) was used. Mice to be depleted of CD8⫹T cells received a dose of 0.1 ml of clarified ascitic fluid in 0.5 ml PBS intraperitoneally on days⫺1, 0, 2, 5, and 9 days relative to infection (13).

CSF cell count.Mice were deeply anesthetized and exsanguinated. Cerebro-spinal fluid (CSF) was obtained from the fourth ventricle as previously described (14, 18). Total number of inflammatory cells was determined by counting in a hemocytometer (background level in uninfected mice,⬍100 cells/␮l CSF), and phenotypic analysis was performed using flow cytometry (see below)

Preparation of total RNA.Brains, livers, and lungs from mice that were deeply anesthetized and exsanguinated were immediately removed, snap frozen in liquid nitrogen, and stored in a liquid nitrogen freezer. Total RNA was extracted from homogenized organs by use of the RNeasy midi kit (QIAGEN, Hilden, Ger-many).

Detection of mRNA in the brain by real-time quantitative PCR.One microliter of purified RNA was converted to cDNA using the RevertAid First Strand cDNA synthesis kit (MBT Fermentas). All setups were analyzed using Brilliant SYBRGreen quantitative PCR Master Mix (Stratagene, AH Diagnostics).

Detection of mRNA in the organs by RNase protection assay.Using a custom-made template set, cell subset markers (CD3ε, CD4, CD11b, CD8␤, F4/80) were detected by the RiboQuant multiprobe RNase protection assay system (Pharm-ingen). The template set also included templates for the murine housekeeping genes L-32 (a ribosomal protein) and glyceraldehyde-3-phosphate dehydroge-nase (GADPH) to serve as loading controls. The RNase protection assay was performed according to the manufacturer’s instructions. Briefly, [␣-32 P]UTP-labeled antisense RNA transcript was generated from the template sets using T7 RNA polymerase. RNA from each sample was allowed to hybridize to the labeled probe for 16 to 20 h at 56°C. Single-stranded RNA was digested with an RNase/T1 mixture, and the hybrids were analyzed on a denaturing urea-polyac-rylamide gel. Protected fragments were visualized by autoradiography by placing dried gels on film (Biomax MS-1; Kodak, New Haven, CT) in cassettes with intensifying screens (Biomax MS; Kodak), which were then exposed at⫺80°C. For quantitative results, gels were subjected to PhosphorImager analysis (Am-ersham Pharmacia Biotech), and the data were subsequently analyzed using ImageMaster TotalLab software (Amersham Pharmacia Biotech).

Quantification of IFN-.IFN-␥levels in serum and CSF were determined by using a sandwich enzyme-linked immunosorbent assay kit from R&D Systems Europe Ltd. (Abingdon, United Kingdom) per the manufacturer’s instruction.

Cell preparation.Single cell suspensions from spleens, livers, and lungs were prepared as previously described (2, 5, 34).

Monoclonal antibodies for flow cytometry.The following monoclonal antibod-ies were purchased from BD Pharmingen (San Diego, CA) as rat anti-mouse antibody: Cy-chrome-conjugated anti-CD8a (53-6.7, immunoglobulin G subclass 2a [IgG2a]), fluorescein isothiocyanate (FITC)-conjugated anti-CD44 (IM7), FITC-conjugated anti-Mac-1 (CD11b), FITC- and phycoerythrin (PE)-conju-gated anti-IFN-␥(XMG1.2, IgG1), PE-conjugated anti-TNF-␣ (MP6-XT22), and PE-conjugated IgG1 isotype control (R3-34)

Detection of antigen-specific CD8T cells by major histocompatibility com-plex (MHC) class I dextramer.LCMV-specific CD8⫹T cells were enumerated by binding of PE-conjugated H-2Db

/GP33-41 and H-2Db

/NP396-404 dextramers obtained from DakoCytomation (Glostrup, Denmark).

Flow cytometric analysis.Staining of cells for flow cytometry was performed according to standard laboratory procedure (2, 3). For the enumeration of LCMV-specific, cytokine-producing CD8⫹T cells, splenocytes were incubated in vitro for 5 h at 37°C in 5% CO2with a combination of GP33-41 and NP396-404 peptides (both at 0.1␮g/ml) in the presence of monensin (3␮M, Sigma Chemical Co., St. Louis, MO) and murine recombinant interleukin-2 (IL-2, 10 U/well; R&D Systems Europe Ltd., Abingdon, United Kingdom). After incubation, cells were stained with antibodies for surface markers (CD8 and CD44) for 20 min at 4°C, washed, and permeabilized using 0.5% saponin. Cells were stained intra-cellularly with anti-IFN-␥, anti-TNF-␣, or isotype control for 20 min at 4°C (15). Samples were analyzed using a Becton Dickinson FACSCalibur cytometer, and at least 104

mononuclear cells were gated using a combination of low-angle and side scattering to exclude dead cells and debris. Data analysis was conducted using Cell Quest Pro (B&D Biosciences).

on November 8, 2019 by guest

http://jvi.asm.org/

(3)

Statistical analysis. Quantitative results were compared using the Mann-Whitney U test.

RESULTS

Lethal CD8T-cell-mediated CNS disease in i.c. infected Pfp/

mice. The current mechanistic paradigm for fatal LCMV-induced meningitis is a perforin-mediated killing of virus-infected meningeal cells (29). The aim of the present study was to reevaluate the importance of perforin in deter-mining the outcome of i.c. infection with LCMV. Accordingly, Pfp⫺/⫺and WT mice were challenged with a moderate dose of

neurotropic LCMV Armstrong, and the disease pattern of the mice was registered. Surprisingly, we found that Pfp⫺/⫺mice

invariably succumbed to infection, and most of them exhibited classical tonic-clonic convulsions around the time of expira-tion, which was delayed compared to WT mice: Pfp⫺/⫺mice

died 9 to 12 days postinfection, i.e., 2 to 5 days later than the WT mice (Fig. 1A). To rule out that death was the result of generalized immunopathology due to disseminated viral repli-cation in extraneural organs, we also challenged Pfp⫺/⫺mice

with the same dose of virus intravenously. Since i.v. infected Pfp⫺/⫺mice did not die during a 2-week observation period,

we concluded that i.c. infected Pfp⫺/⫺ mice succumb from

localized inflammation in the CNS.

In normal immunocompetent mice, virus-specific CD8⫹T cells are responsible for the immunopatholgy induced during i.c. infection with LCMV (19). However, in i.c. infected ␤2

-microglobulin-deficient mice lacking CD8⫹ T cells, CD4⫹T

cells have also been found to be capable of inducing fatal disease (65). To ascertain that fatal disease in Pfp⫺/⫺mice

requires CD8⫹T cells, Pfp⫺/⫺mice were challenged i.c. and

half the mice received CD8-depleting antibodies intraperito-neally on days⫺1, 0, 2, 5, and 9 relative to infection; the other half of the mice was injected with PBS for control. Although antibody-depleted mice suffered a transient weight loss (data not shown), depletion of CD8⫹T cells completely prevented fatal disease, whereas all sham-treated mice succumbed to the infection (Fig. 1B). Thus, CD8⫹T cells were pivotal for lethal CNS disease in Pfp⫺/⫺mice.

Lethal CNS disease despite multidysfunctional CD8T cells.The above results were quite surprising, particularly in light of recent studies revealing that LCMV-specific CD8⫹T cells in Pfp⫺/⫺ mice are deficient not only in their cytotoxic

ability but also regarding the secretion of cytokines, probably as a result of functional exhaustion subsequent to extensive and persistent infection (22, 23, 27). Therefore, to ascertain that these findings were pertinent under our experimental con-ditions as well (a low dose of slowly disseminating virus), the total number of splenic CD8⫹T cells specific for GP33-41 plus NP396-404 (two of the major immunodominant LCMV epitopes in H-2bmice) was determined 6 and 8 days

postin-fection. In addition, we analyzed the ability of virus-specific (GP33-41 plus NP396-404) CD8⫹T cells to produce cytokines. Notably, in order to be able to examine WT mice on day 8 postinfection, some of these mice were infected intravenously with the same dose of virus as that given i.c. to the other groups of mice.

Expanding on earlier studies (29, 62, 66), we found that the initial activation and expansion of virus-specific CD8⫹effector T cells tended to be augmented in the absence of perforin (Fig. 2A and B). However, the quality of the generated effector cells was clearly impaired in Pfp⫺/⫺ mice (Fig. 2C and D). The

mean fluorescence intensity (MFI) of IFN-␥staining was sig-nificantly lower in cells from mice lacking perforin expression (Fig. 2C), indicating a reduced capacity of the individual ef-fector cell to synthesize this cytokine. Furthermore, the ability of virus-specific cells from Pfp⫺/⫺mice to coproduce TNF-

was markedly impaired compared to WT mice on both days examined (Fig. 2D).

Since a reduced capacity to produce critical proinflamma-tory cytokines in itself could explain the delay in mortality, thereby potentially excluding any role for perforin in CNS pathology, we found it pertinent to further define the func-tional capacity of the virus-specific CD8⫹T cells generated in Pfp⫺/⫺mice. For this reason, we harvested splenocytes from

Pfp⫺/⫺ and WT mice and cultured the cells in vitro with or

without added viral peptides. Cytokine production was evalu-ated during the first 1.5 h as well as during the standard 5 h of incubation. If the virus-specific cells from Pfp⫺/⫺mice were

stimulated as a result of the persistent infection, we would expect immediate cytokine production in the absence of pep-tide stimulation. Moreover, if in vivo activation limited the capacity of the effector cells to go on producing cytokine, prolonged in vitro incubation would not result in a further increase in the intracellular cytokine level.

As can be seen in Fig. 3, both predictions were fulfilled. In the case of cells from Pfp⫺/⫺mice, the frequency of

[image:3.585.68.260.67.328.2]

cytokine-producing CD8⫹T cells did not differ much between peptide

FIG. 1. Mice deficient in perforin succumb to CD8⫹ T-cell-medi-ated brain disease. (A) Pfp⫺/⫺and WT mice were infected i.c. with 200

PFU of LCMV Armstrong, and mortality was registered (n ⫽ 10 mice/group). (B) Mice received CD8-depleting antibodies or PBS in-traperitoneally on days⫺1, 0, 2, 5, and 9 relative to i.c. infection with 200 PFU LCMV Armstrong (n5 mice/group).

on November 8, 2019 by guest

http://jvi.asm.org/

(4)

and unstimulated cultures, whereas few WT cells produced cytokine without peptide stimulation. Furthermore, whereas the amount of cytokine per cell (as evaluated in terms of MFI) did not increase substantially with longer incubation for cells

“spontaneously” producing cytokine, peptide stimulation of normal effector cells (i.e., from WT mice) resulted in markedly increased MFI with time. Peptide-stimulated effector cells from Pfp⫺/⫺mice behaved as if representing a mixture of in

vivo- and in vitro-activated cells. Taken together, these results are consistent with the assumption that many effector T cells from Pfp⫺/⫺mice are stimulated already under in vivo

condi-tions and have a limited capacity for continued cytokine syn-thesis. Nevertheless, these cells still suffice for the induction of lethal CNS disease in mice infected i.c. with LCMV.

Delayed leukocyte recruitment to the CSF in i.c. infected Pfp/

mice. Based on the above results, there were at least two nonmutually exclusive explanations for the delayed death pattern in i.c. infected Pfp⫺/⫺ mice: (i) the recruitment of

[image:4.585.310.535.69.425.2]

effector cells was delayed due to a reduced capacity to induce local inflammation and/or (ii) the individual effector cell had a

FIG. 2. Virus-induced CD8⫹T-cell expansion is unimpaired, but cytokine production is reduced in mice deficient in perforin. For eval-uation on day 6 p.i., all mice were infected i.c. with 200 PFU of LCMV Armstrong. For evaluation on day 8 p.i., Pfp⫺/⫺mice were infected i.c.

and WT mice were infected i.v. On the indicated days, splenocytes were isolated and the frequency of LCMV-specific (MHC class I dex-tramer⫹) (A) and IFN-␥-producing (by intracellular cytokine staining following stimulation for 5 h with GP33-41 and NP396-404) (B) CD8⫹ T cells was determined. Medians⫾ranges of 10 mice/group are de-picted. There were no significant differences between Pfp⫺/⫺and WT

mice in any of the measured parameters. (C) Mean fluorescence in-tensity of IFN-staining of gated IFN-⫹CD8⫹T cells is shown. Note that only analyses carried out on the same day can be directly com-pared. (D) Fraction of IFN--producing CD8⫹ T cells coproducing TNF-␣ is shown. Medians⫾ ranges of 10 mice/group are shown; statistical comparison of Pfp⫺/⫺and WT mice was performed using the

Mann-Whitney U test.ⴱ,P⬍0.05.

FIG. 3. Spontaneous cytokine synthesis in Pfp⫺/⫺mice correlates

inversely with the capacity for continued production. Pfp⫺/⫺and WT

mice were infected i.v. with 200 PFU of LCMV Armstrong, and 8 days later, splenocytes were incubated in vitro with or without viral peptides (GP33-41 and NP396-404). After 1.5 and 5 h of incubation, cells were harvested and cytokine synthesis was evaluated by intracellular cyto-kine staining. Representative plots of gated CD8⫹T cells are depicted; values represent the percentage of CD44highCD8T cells that produce

IFN-␥(upper right quadrant) and mean fluorescence intensity of the staining. Averages⫾standard deviations of four mice/group are pre-sented.

on November 8, 2019 by guest

http://jvi.asm.org/

[image:4.585.74.257.72.497.2]
(5)

lesser capacity to mediate disease in situ due to a reduced production of one or more essential effector molecules.

In order to evaluate the first possibility, we compared the formation of the inflammatory exudate in the CSF of Pfp⫺/⫺

and WT mice infected i.c. with LCMV Armstrong. The mice were sacrificed on days 6 and 8 postinfection, CSF was tapped, and the inflammatory cells were counted. Since WT mice al-ready succumbed to infection by day 7 p.i., only Pfp⫺/⫺mice

could be analyzed on day 8 after virus challenge. Six days after i.c. infection, high numbers of leukocytes, in particular, mono-cytes/macrophages (⬃60%), were recovered from the CSF of WT mice, whereas only a few cells had invaded the CSF of similarly infected Pfp⫺/⫺ mice (Fig. 4A and B). Eight days

after infection, the number of infiltrating cells in the CSF of Pfp⫺/⫺ mice matched that observed for WT mice on day 6

postinfection, but unlike results for the latter mice, the major-ity of the inflammatory cells were CD8⫹T cells (⬃75%) (Fig. 4B). Thus, independent of the genotype, roughly the same number of mononuclear cells was found in the CSF at the time of expiration; however, CD8⫹T cells dominated the cellular infiltrate to a much higher degree in Pfp⫺/⫺mice.

Delayed inflammation of the CNS is not a result of compe-tition between the CNS and extraneural organ sites for inflam-matory cells but correlates with reduced expression of VLA-4.

Impaired virus control in the absence of perforin results in high viral loads in several major organs (29, 51, 62), and it can be seen from Fig. 5A that a significant difference in virus clear-ance can be observed already 6 days after infection. This could lead to competition between the CNS and peripheral organ sites for inflammatory cells (56), which might explain why the recruitment of cells to the CSF is delayed. To determine if sequestering of effector T cells actually explains the delayed onset of inflammation in Pfp⫺/⫺mice, we first quantified the

expression of mRNA for CD8␤in brains, livers, and lungs of mice infected 6 days earlier, i.e., at a time point when a marked difference in meningeal infiltration could be observed (cf. Fig. 4); expression levels were determined using both a quantitative PCR and an RNase protection assay. Except for a reduced expression in the brains of Pfp⫺/⫺ mice which matched the

reduced inflammation in these mice, we found no significant differences between the genotypes (data not shown).

To ascertain that this result was valid with regard to virus-specific cells, we also isolated lymphocytes from the livers and lungs of Pfp⫺/⫺and WT mice infected i.c. with LCMV 6 days

[image:5.585.332.505.84.602.2]

earlier, and by use of MHC/peptide dextramers for GP33-41 and NP396-404, we compared the number of LCMV-specific CD8⫹T cells retained in these organ sites. As can be seen in Fig. 5B, we could not detect any difference between the geno-types in this respect, indicating that the reduced inflammatory reaction in the CNS of Pfp⫺/⫺mice at this time was not simply

caused by sequestering of the relevant cells in the visceral organs.

Finally, since previous studies have indicated that the adhe-sion molecule VLA-4 play an important role in targeting ef-fector T cells to areas of viral infection including the brain (6, 12), we evaluated the expression of this adhesion molecule on LCMV-specific CD8⫹T cells harvested from the spleen on day 6 p.i. As can be seen in Fig. 5C, a substantial fraction of virus-specific CD8⫹T cells in Pfp⫺/⫺mice had a lower

expres-sion of VLA-4 than effector T cells from WT mice. However,

FIG. 4. Delayed recruitment of effector cells to the CNS in the absence of perforin is IFN-␥independent. Pfp⫺/⫺, IFN-/Pfp⫺/⫺, and

WT mice were infected i.c. with 200 PFU LCMV Armstrong. (A) On the indicated days, CSF was harvested and the cells were counted; medians⫾ranges are depicted (n⫽7 to 9 mice/group). All WT mice had died by day 7 p.i. (B) On the indicated days, CSF was harvested and the cells were stained with anti-CD8 and anti-Mac-1 (n ⫽ 5 mice/group). (C and D) Concentration of IFN-␥in CSF (C) (n⫽6 to 8 mice/group) and serum (D) (n⫽ 3 or 4 mice/group). Statistical comparison of knockout and WT mice was performed using the Mann-Whitney U test.ⴱ,P⬍0.05.

on November 8, 2019 by guest

http://jvi.asm.org/

(6)

despite this, the frequency of virus-specific cells with an ex-pression level matching that of effector cells from WT mice (Fig. 5C, upper right quadrant) did not fall below the fre-quency found in the latter mice, suggesting that this difference cannot by itself explain the delayed recruitment to the CNS.

CNS inflammation in Pfp/

mice is IFN-independent.To determine the role of IFN-␥in mediating the delayed local inflammatory response in Pfp⫺/⫺mice, we infected Pfp⫺/⫺and

WT mice and followed the production of IFN-␥in CSF and serum. Consistent with published results (23, 27) as well as the results of our ex vivo analysis, we found that Pfp⫺/⫺mice had

a higher level of IFN-␥in serum (Fig. 4D). However, unlike the situation in the general circulation, the concentration of this cytokine in CSF tended to be lower than in similarly infected WT mice on day 6 p.i. (Fig. 4C). Notably, matching the delayed massive influx of CD8⫹T cells in Pfp⫺/⫺mice, very

high levels of IFN-␥were measured in the CSF on day 8 p.i., coinciding with the onset of fatal disease. To establish whether this cytokine response was the cause of inflammation in Pfp⫺/⫺

mice or merely an effect of the delayed inflammatory reaction, IFN-␥/Pfp⫺/⫺mice were infected i.c., and CSF infiltration in

these mice was analyzed. As can be seen in Fig. 4A and B, the influx of cells into the CSF of i.c. infected mice followed the same time course in Pfp⫺/⫺mice and Pfp⫺/⫺mice lacking the

ability to produce IFN-␥, and qualitative analysis of the inflam-matory exudate revealed a similar cellular composition. Thus, factors other than IFN-␥sufficed for the development of local inflammation and CD8⫹T-cell recruitment in Pfp⫺/⫺ mice.

Unfortunately, we cannot formally prove that double-deficient mice could develop lethal meningitis because both i.c. and i.v. infected IFN-␥/Pfp⫺/⫺ mice died following LCMV infection

(data not shown).

DISCUSSION

Perforin is pivotal for clearance of LCMV, which seems to be eliminated primarily through the contact-dependent killing of virus-infected cells (29, 62). For this reason, perforin is also expected to contribute to LCMV-induced immunopathology (29, 30, 62), and for more than a decade, it has been widely accepted that perforin is the mediator in the development of lethal CNS pathology following i.c. infection with LCMV (29). However, in the present study, we demonstrate that following i.c. challenge with LCMV, mice can develop lethal CD8⫹ T-cell-mediated meningitis in the absence of perforin, although in a delayed fashion compared to WT mice. In light of the facts that virus injected i.c. readily accesses the bloodstream and that some Pfp⫺/⫺mice eventually succumb to extraneural

in-fection with LCMV (8, 27, 39, 51, 62), one could speculate if in fact the mice die from more generalized immunopathology. However, this does not appear to be the case, since Pfp⫺/⫺

mice challenged with the same dose of virus i.v. survived for at least 2 weeks, whereas all i.c. infected Pfp⫺/⫺mice died within

12 days after infection. Since Pfp⫺/⫺mice are completely

im-paired in virus clearance (29, 51, 62), we find it likely that the absence of CNS disease in the previous study reflects a case of rapid collapse of the immune response due to massive viral replication in internal organs (45). To avoid such a scenario, we exclusively employed a low dose of a slowly invasive and neurotropic strain of LCMV. Under these conditions,

alterna-FIG. 5. (A) Pfp⫺/⫺and WT mice were infected i.c. with 200 PFU of

LCMV Armstrong, and on days 4 and 6 postinfection, spleen virus titers were determined. Points represent individual mice. Statistical comparison of Pfp⫺/⫺and WT mice was performed using the

Mann-Whitney U test.ⴱ,P⬍0.05. (B) Pfp⫺/⫺and WT mice were infected i.c.

with 200 PFU LCMV Armstrong, and 6 days later, lymphocytes were isolated from the liver and lungs. Cells were stained with MHC class I dextramers (H-2Db/GP33-41 and H-2Db/NP396-404), and the frequen-cies of CD8⫹T cells binding these dextramers were determined using a flow cytometer. From this frequency and the total numbers of mono-nuclear cells recovered, the total numbers of LCMV-specific cells present in these organ sites were calculated. Medians⫾ranges of five mice/group are depicted; there were no significant differences between Pfp⫺/⫺ and WT mice in either organ site. (C) Pfp⫺/⫺and WT mice

were infected i.c. with 200 PFU LCMV Armstrong, and 6 days later, splenocytes were analyzed for the expression of VLA-4 on virus-spe-cific CD8⫹T cells (producing IFN-␥in response to stimulation for 5 h with GP33-41 and NP396-404). Representative plots of gated CD8⫹T cells are presented (n⫽5 mice/group); percentages refer to antigen-specific cells with high and low expression of VLA-4, respectively.

on November 8, 2019 by guest

http://jvi.asm.org/

[image:6.585.72.251.87.522.2]
(7)

tive effector systems clearly suffice for the induction of fatal disease, questioning the pivotal role of cell lysis in the patho-genesis of this disease.

Why then do Pfp⫺/⫺ mice die later than WT mice? We

observed a distinct correlation between time of death and influx of inflammatory cells into the CSF, indicating that a delayed onset of local inflammation is an important part of the explanation. It is apparent from our results that the delayed onset of inflammation is not due to an impaired generation of virus-specific CD8⫹effector T cells in the secondary lymphoid organs. This result confirms previous studies revealing similar or even significantly elevated numbers of activated CD8⫹ T cells in the absence of perforin (23, 27, 29, 31, 62). Thus, there are at least as many virus-specific T cells generated in infected Pfp⫺/⫺mice as in WT mice, but in Pfp⫺/⫺mice, these cells are

somehow kept from entering or are not effectively recruited to the CNS within the first 6 to 7 days of infection.

To explain this, one could consider that the effector cells became sequestered in other organs, e.g., liver or lungs. How-ever, we did not find evidence supporting this possibility. Al-ternatively, one might entertain the thought that perforin could be directly involved in the extravasation of effector cells at sites of inflammation, but that seems a rather remote pos-sibility. Interestingly, confirming and expanding on recent find-ings by several other groups (22, 27), we noted a marked functional difference between effector CD8⫹T cells generated in Pfp⫺/⫺and WT mice. The reason for this aberrant

func-tional state is likely to be found in the unrestrained virus replication taking place in the absence of perforin. This is suggested by the fact that a similar cellular phenotype may be found in WT mice infected with much higher doses of rapidly replicating virus strains (21, 23, 32), but not in Pfp⫺/⫺mice

infected with a virus (vesicular stomatitis virus) that is not controlled through a perforin-dependent mechanism (11). Our present results provide additional supporting evidence that CD8⫹T cells from Pfp⫺/⫺mice synthesize cytokine already

under in vivo conditions and, notably, that this activity corre-lates with a decreased capacity for prolonged cytokine synthe-sis in vitro, indicating that the cells become exhausted through chronic stimulation in vivo. The latter interpretation is consis-tent with results from the group of John Harty (17) showing that CD8⫹T cells may undergo on/off cycling, but if the initial Ag stimulus is maximal, they cannot produce IFN-␥after an-tigen reexposure.

The generation of dysfunctional CD8⫹ T cells in Pfp⫺/⫺

mice is important for two reasons. First, impaired production of several key proinflammatory mediators could explain why the onset of inflammation is delayed in Pfp⫺/⫺mice compared

to WTs. Additionally, if the overall effector potential on a per-cell basis is exhausted before the cells accumulate locally, one would predict that it would take more cells to induce to the same degree of local immunopathology, and this fits well with the findings that Pfp⫺/⫺mice tended to live longer after the

onset of inflammation and tolerate a higher number of CD8⫹ T cells to accumulate in the CSF, before death is induced.

The above-mentioned results raised the possibility that im-paired production of IFN-␥by the CD8⫹T cells from Pfp⫺/⫺

mice could be the key factor in the delayed onset of disease in these mice. Although it has been found that IFN-␥ is not essential for the induction of lethal disease in otherwise

im-munologically intact (i.e., Pfp⫹/⫹) mice infected with LCMV

Armstrong (50), the possibility that IFN-␥is redundant only in the presence of an intact Pfp pathway and in high concentra-tions would also induce fatal disease cannot be ruled out. However, following i.c. challenge of IFN-␥/Pfp⫺/⫺mice, these

double knockout mice develop meningitis with the same kinet-ics as Pfp⫺/⫺mice, demonstrating that severe CNS

inflamma-tion can be induced in the absence of both perforin and IFN-␥. Unfortunately i.v. infection of IFN-␥/Pfp⫺/⫺mice also results

in lethal disease, which prevents us from being able to formally exclude IFN-␥as an alternative mediator of lethal CNS disease in i.c. infected Pfp⫺/⫺mice. It is quite interesting that

LCMV-infected IFN-␥/Pfp⫺/⫺mice die even following i.v. infection.

Several studies have indicated that neutralization of IFN-␥ protects Pfp⫺/⫺mice from the development of severe systemic

immunopathology following i.v. LCMV infection (23, 27, 51), which is quite the opposite of what we found in this study. Probably the reason for this discrepancy lies in differences in peak viral load in the infected organs. Thus, unlike the previ-ous studies, we here used a low dose of a slowly invasive LCMV strain under which conditions the elimination of IFN-␥ could primarily augment immunopathology by leading to a more disseminated infection.

Most importantly, our work demonstrates that even in the absence of their key cytotoxic molecule and with a reduced capacity for the secretion of several proinflammatory cyto-kines, once accumulated in sufficient numbers, virus-specific CD8⫹T cells are capable of and responsible for the develop-ment of lethal CNS disease. The precise molecular mechanism is still unknown, but an earlier study has shown that LCMV-infected neurons are susceptible to killing through Fas/FasL interaction (41). We have recently presented data indicating that a fatal outcome of i.c. LCMV infection requires CD8⫹ T-cell infiltration into the neural parenchyma (10). One pos-sibility is therefore that the Fas/FasL pathway may play a critical role in LCMV-induced CNS disease when perforin-induced lysis is prevented. Most importantly, contrary to cur-rent thinking, the present results underscore that perforin is not pivotal; thus, once again, experimental analysis has re-vealed that extensive redundancy exists when it comes to key processes in antimicrobial host responses.

ACKNOWLEDGMENTS

We thank Christina Jespersgaard and Jørgen Schøller (DakoCyto-mation, Glostrup, Denmark) for generously providing the MHC/pep-tide dextramers used in this study.

This work was supported in part by the Danish Medical Research Council, the Lundbeck Foundation, and the Novo Nordisk Founda-tion. P.S. is the recipient of a scholarship from Novo Nordisk, M.R.S. is the recipient of a Ph.D. scholarship from the Faculty of Health Science, University of Copenhagen, and C.B. is the recipient of a postdoctoral fellowship from the Danish Medical Research Council.

REFERENCES

1.Andersen, I. H., O. Marker, and A. R. Thomsen.1991. Breakdown of blood-brain barrier function in the murine lymphocytic choriomeningitis virus infection mediated by virus-specific CD8⫹T cells. J. Neuroimmunol.31: 155–163.

2.Andersson, E. C., J. P. Christensen, O. Marker, and A. R. Thomsen.1994. Changes in cell adhesion molecule expression on T cells associated with systemic virus infection. J. Immunol.152:1237–1245.

3.Andreasen, S. O., J. P. Christensen, O. Marker, and A. R. Thomsen.1999. Virus-induced non-specific signals cause cell cycle progression of primed CD8(⫹) T cells but do not induce cell differentiation. Int. Immunol.11: 1463–1473.

on November 8, 2019 by guest

http://jvi.asm.org/

(8)

4.Bartholdy, C., J. P. Christensen, D. Wodarz, and A. R. Thomsen.2000. Persistent virus infection despite chronic cytotoxic T-lymphocyte activation in gamma interferon-deficient mice infected with lymphocytic choriomenin-gitis virus. J. Virol.74:10304–10311.

5.Bartholdy, C., W. Olszewska, A. Stryhn, A. R. Thomsen, and P. J. Openshaw. 2004. Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis. J. Gen. Virol.85:3017–3026.

6.Bartholdy, C., A. Stryhn, N. J. Hansen, S. Buus, and A. R. Thomsen.2003. Incomplete effector/memory differentiation of antigen-primed CD8⫹T cells in gene gun DNA-vaccinated mice. Eur. J. Immunol.33:1941–1948. 7.Bilzer, T., and L. Stitz.1994. Immune-mediated brain atrophy. CD8⫹T cells

contribute to tissue destruction during borna disease. J. Immunol.153:818– 823.

8.Binder, D., M. F. van den Broek, D. Kagi, H. Bluethmann, J. Fehr, H. Hengartner, and R. M. Zinkernagel.1998. Aplastic anemia rescued by ex-haustion of cytokine-secreting CD8⫹T cells in persistent infection with lymphocytic choriomeningitis virus. J. Exp. Med.187:1903–1920. 9.Ceredig, R., J. E. Allan, Z. Tabi, F. Lynch, and P. C. Doherty.1987.

Pheno-typic analysis of the inflammatory exudate in murine lymphocytic chorio-meningitis. J. Exp. Med.165:1539–1551.

10.Christensen, J. E., A. Nansen, T. Moos, B. Lu, C. Gerard, J. P. Christensen, and A. R. Thomsen.2004. Efficient T-cell surveillance of the CNS requires expression of the CXC chemokine receptor 3. J. Neurosci.24:4849–4858. 11.Christensen, J. E., D. Wodarz, J. P. Christensen, and A. R. Thomsen.2004.

Perforin and IFN-gamma do not significantly regulate the virus-specific CD8⫹T cell response in the absence of antiviral effector activity. Eur. J. Immunol.34:1389–1394.

12.Christensen, J. P., E. C. Andersson, A. Scheynius, O. Marker, and A. R. Thomsen.1995. Alpha 4 integrin directs virus-activated CD8⫹T cells to sites of infection. J. Immunol.154:5293–5301.

13.Christensen, J. P., C. Bartholdy, D. Wodarz, and A. R. Thomsen.2001. Depletion of CD4⫹T cells precipitates immunopathology in immunodefi-cient mice infected with a noncytocidal virus. J. Immunol.166:3384–3391. 14.Christensen, J. P., O. Marker, and A. R. Thomsen.1993. T-cell

responsive-ness to LCMV segregates as a single locus in crosses between BALB/cA and C.B-17 mice. Evidence for regulation by a gene outside the Igh region. Scand. J. Immunol.38:215–224.

15.Christensen, J. P., J. P. Stenvang, O. Marker, and A. R. Thomsen.1996. Characterization of virus-primed CD8⫹T cells with a type 1 cytokine profile. Int. Immunol.8:1453–1461.

16.Cole, G. A., D. H. Gilden, A. A. Monjan, and N. Nathanson.1971. Lympho-cytic choriomeningitis virus: pathogenesis of acute central nervous system disease. Fed. Proc.30:1831–1841.

17.Corbin, G. A., and J. T. Harty.2005. T cells undergo rapid ON/OFF but not ON/OFF/ON cycling of cytokine production in response to antigen. J. Im-munol.174:718–726.

18.Doherty, P. C.1973. Quantitative studies of the inflammatory process in fatal viral meningoencephalitis. Am. J. Pathol.73:607–622.

19.Doherty, P. C., J. E. Allan, F. Lynch, and R. Ceredig.1990. Dissection of an inflammatory process induced by CD8⫹T cells. Immunol. Today11:55–59. 20.Doherty, P. C., and R. M. Zinkernagel.1974. T-cell-mediated

immunopa-thology in viral infections. Transplant. Rev.19:89–120.

21.Fuller, M. J., A. Khanolkar, A. E. Tebo, and A. J. Zajac.2004. Maintenance, loss, and resurgence of T cell responses during acute, protracted, and chronic viral infections. J. Immunol.172:4204–4214.

22.Fuller, M. J., and A. J. Zajac.2003. Ablation of CD8 and CD4 T cell responses by high viral loads. J. Immunol.170:477–486.

23.Gallimore, A., A. Glithero, A. Godkin, A. C. Tissot, A. Pluckthun, T. Elliott, H. Hengartner, and R. Zinkernagel.1998. Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visual-ized using soluble tetrameric major histocompatibility complex class I-pep-tide complexes. J. Exp. Med.187:1383–1393.

24.Gilden, D. H., G. A. Cole, A. A. Monjan, and N. Nathanson.1972. Immu-nopathogenesis of acute central nervous system disease produced by lym-phocytic choriomeningitis virus. I. Cyclophosphamide-mediated induction by the virus-carrier state in adult mice. J. Exp. Med.135:860–873.

25.Hickey, W. F.2001. Basic principles of immunological surveillance of the normal central nervous system. Glia36:118–124.

26.Huseby, E. S., D. Liggitt, T. Brabb, B. Schnabel, C. Ohlen, and J. Goverman. 2001. A pathogenic role for myelin-specific CD8(⫹) T cells in a model for multiple sclerosis. J. Exp. Med.194:669–676.

27.Jordan, M. B., D. Hildeman, J. Kappler, and P. Marrack.2004. An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8⫹T cells and interferon gamma are essential for the disorder. Blood104:735–743. 28.Kagi, D., and H. Hengartner.1996. Different roles for cytotoxic T cells in the

control of infections with cytopathic versus noncytopathic viruses. Curr. Opin. Immunol.8:472–477.

29.Kagi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K. J. Olsen, E. R. Podack, R. M. Zinkernagel, and H. Hengartner.1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature369:31–37.

30.Kagi, D., B. Ledermann, K. Burki, R. M. Zinkernagel, and H. Hengartner.

1996. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol.14:207–232.

31.Kagi, D., B. Odermatt, and T. W. Mak.1999. Homeostatic regulation of CD8⫹T cells by perforin. Eur. J. Immunol.29:3262–3272.

32.Kristensen, N. N., J. P. Christensen, and A. R. Thomsen.2002. High num-bers of IL-2-producing CD8⫹T cells during viral infection: correlation with stable memory development. J. Gen. Virol.83:2123–2133.

33.Kristensen, N. N., A. N. Madsen, A. R. Thomsen, and J. P. Christensen. 2004. Cytokine production by virus-specific CD8(⫹) T cells varies with ac-tivation state and localization, but not with TCR avidity. J. Gen. Virol. 85:1703–1712.

34.Liu, Z. X., S. Govindarajan, S. Okamoto, and G. Dennert.2000. NK cells cause liver injury and facilitate the induction of T cell-mediated immunity to a viral liver infection. J. Immunol.164:6480–6486.

35.Lowenstein, P. R.2002. Immunology of viral-vector-mediated gene transfer into the brain: an evolutionary and developmental perspective. Trends Im-munol.23:23–30.

36.Lynch, F., P. C. Doherty, and R. Ceredig.1989. Phenotypic and functional analysis of the cellular response in regional lymphoid tissue during an acute virus infection. J. Immunol.142:3592–3598.

37.Marker, O., M. H. Nielsen, and N. H. Diemer.1984. The permeability of the blood-brain barrier in mice suffering from fatal lymphocytic choriomeningitis virus infection. Acta Neuropathol. (Berlin)63:229–239.

38.Marker, O., A. Scheynius, J. P. Christensen, and A. R. Thomsen.1995. Virus-activated T cells regulate expression of adhesion molecules on endo-thelial cells in sites of infection. J. Neuroimmunol.62:35–42.

39.Matloubian, M., M. Suresh, A. Glass, M. Galvan, K. Chow, J. K. Whitmire, C. M. Walsh, W. R. Clark, and R. Ahmed.1999. A role for perforin in downregulating T-cell responses during chronic viral infection. J. Virol. 73:2527–2536.

40.McGavern, D. B., D. Homann, and M. B. Oldstone.2002. T cells in the central nervous system: the delicate balance between viral clearance and disease. J. Infect. Dis.186(Suppl. 2):S145–S151.

41.Medana, I. M., A. Gallimore, A. Oxenius, M. M. Martinic, H. Wekerle, and H. Neumann. 2000. MHC class I-restricted killing of neurons by virus-specific CD8⫹T lymphocytes is effected through the Fas/FasL, but not the perforin pathway. Eur. J. Immunol.30:3623–3633.

42.Mims, C. A.1960. Intracerebral injections and the growth of viruses in the mouse brain. Br. J. Exp. Pathol.41:52–59.

43.Morris, A. G., Y. L. Lin, and B. A. Askonas.1982. Immune interferon release when a cloned cytotoxic T-cell line meets its correct influenza-infected target cell. Nature295:150–152.

44.Moskophidis, D., M. Battegay, M. A. Bruendler, E. Laine, I. Gresser, and R. M. Zinkernagel.1994. Resistance of lymphocytic choriomeningitis virus to alpha/beta interferon and to gamma interferon. J. Virol.68:1951–1955. 45.Moskophidis, D., F. Lechner, H. Pircher, and R. M. Zinkernagel.1993. Virus

persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells. Nature362:758–761.

46.Mullbacher, A., R. T. Hla, C. Museteanu, and M. M. Simon.1999. Perforin is essential for control of ectromelia virus but not related poxviruses in mice. J. Virol.73:1665–1667.

47.Mullbacher, A., M. Regner, Y. Wang, E. Lee, M. Lobigs, and M. Simon.2004. Can. we really learn from model pathogens? Trends Immunol.25:524–528. 48.Muller, C., D. Kagi, T. Aebischer, B. Odermatt, W. Held, E. R. Podack, R. M. Zinkernagel, and H. Hengartner.1989. Detection of perforin and granzyme A mRNA in infiltrating cells during infection of mice with lymphocytic choriomeningitis virus. Eur. J. Immunol.19:1253–1259.

49.Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, and R. Ahmed.1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immu-nity8:177–187.

50.Nansen, A., J. P. Christensen, C. Ropke, O. Marker, A. Scheynius, and A. R. Thomsen.1998. Role of interferon-gamma in the pathogenesis of LCMV-induced meningitis: unimpaired leucocyte recruitment, but deficient macro-phage activation in interferon-gamma knock-out mice. J. Neuroimmunol. 86:202–212.

51.Nansen, A., T. Jensen, J. P. Christensen, S. O. Andreasen, C. Ropke, O. Marker, and A. R. Thomsen.1999. Compromised virus control and aug-mented perforin-mediated immunopathology in IFN-gamma-deficient mice infected with lymphocytic choriomeningitis virus. J. Immunol.163:6114– 6122.

52.Oldstone, M. B., J. L. Whitton, H. Lewicki, and A. Tishon. 1988. Fine dissection of a nine amino acid glycoprotein epitope, a major determinant recognized by lymphocytic choriomeningitis virus-specific class I-restricted H-2Db cytotoxic T lymphocytes. J. Exp. Med.168:559–570.

53.Ramshaw, I. A., A. J. Ramsay, G. Karupiah, M. S. Rolph, S. Mahalingam, and J. C. Ruby.1997. Cytokines and immunity to viral infections. Immunol. Rev.159:119–135.

54.Riera, L., M. Gariglio, G. Valente, A. Mullbacher, C. Museteanu, S. Land-olfo, and M. M. Simon.2000. Murine cytomegalovirus replication in salivary

on November 8, 2019 by guest

http://jvi.asm.org/

(9)

glands is controlled by both perforin and granzymes during acute infection. Eur. J. Immunol.30:1350–1355.

55.Sad, S., R. Marcotte, and T. R. Mosmann.1995. Cytokine-induced differ-entiation of precursor mouse CD8⫹T cells into cytotoxic CD8⫹T cells secreting Th1 or Th2 cytokines. Immunity2:271–279.

56.Sandberg, K., P. Kemper, A. Stalder, J. Zhang, M. V. Hobbs, J. L. Whitton, and I. L. Campbell.1994. Altered tissue distribution of viral replication and T cell spreading is pivotal in the protection against fatal lymphocytic cho-riomeningitis in mice after neutralization of IFN-alpha/beta. J. Immunol. 153:220–231.

57.Schwendemann, G., J. Lohler, and F. Lehmann-Grube.1983. Evidence for cytotoxic T-lymphocyte-target cell interaction in brains of mice infected intracerebrally with lymphocytic choriomeningitis virus. Acta Neuropathol. (Berlin)61:183–195.

58.Sun, D., J. N. Whitaker, Z. Huang, D. Liu, C. Coleclough, H. Wekerle, and C. S. Raine.2001. Myelin antigen-specific CD8⫹T cells are encephalito-genic and produce severe disease in C57BL/6 mice. J. Immunol.166:7579– 7587.

59.Topham, D. J., R. A. Tripp, and P. C. Doherty.1997. CD8⫹T cells clear influenza virus by perforin or Fas-dependent processes. J. Immunol.159: 5197–5200.

60.Utermohlen, O., A. Dangel, A. Tarnok, and F. Lehmann-Grube.1996. Mod-ulation by gamma interferon of antiviral cell-mediated immune responses in vivo. J. Virol.70:1521–1526.

61.von Herrath, M., B. Coon, D. Homann, T. Wolfe, and L. G. Guidotti.1999. Thymic tolerance to only one viral protein reduces lymphocytic choriomen-ingitis virus-induced immunopathology and increases survival in perforin-deficient mice. J. Virol.73:5918–5925.

62.Walsh, C. M., M. Matloubian, C. C. Liu, R. Ueda, C. G. Kurahara, J. L. Christensen, M. T. Huang, J. D. Young, R. Ahmed, and W. R. Clark.1994. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA91:10854–10858.

63.Wille, A., A. Gessner, H. Lother, and F. Lehmann-Grube.1989. Mechanism of recovery from acute virus infection. VIII. Treatment of lymphocytic cho-riomeningitis virus-infected mice with anti-interferon-gamma monoclonal antibody blocks generation of virus-specific cytotoxic T lymphocytes and virus elimination. Eur. J. Immunol.19:1283–1288.

64.Wodarz, D., J. P. Christensen, and A. R. Thomsen.2002. The importance of lytic and nonlytic immune responses in viral infections. Trends Immunol. 23:194–200.

65.Zajac, A. J., D. G. Quinn, P. L. Cohen, and J. A. Frelinger.1996. Fas-dependent CD4⫹cytotoxic T-cell-mediated pathogenesis during virus infec-tion. Proc. Natl. Acad. Sci. USA93:14730–14735.

66.Zhou, S., R. Ou, L. Huang, and D. Moskophidis.2002. Critical role for perforin-, Fas/FasL-, and TNFR1-mediated cytotoxic pathways in down-regulation of antigen-specific T cells during persistent viral infection. J. Vi-rol.76:829–840.

on November 8, 2019 by guest

http://jvi.asm.org/

Figure

FIG. 1. Mice deficient in perforin succumb to CD8�mice/group). (B) Mice received CD8-depleting antibodies or PBS in-traperitoneally on daysated brain disease
FIG. 3. Spontaneous cytokine synthesis in Pfp�values represent the percentage of CD44inversely with the capacity for continued production
Fig. 5B, we could not detect any difference between the geno-
FIG. 5. (A) Pfppresent in these organ sites were calculated. Medianswith 200 PFU LCMV Armstrong, and 6 days later, lymphocytes wereisolated from the liver and lungs

References

Related documents

Duration Immediate 1 week 2 week 1 month 2 months 4 months 6 months Mobility of Implant Swelling Pain Maxillary Sinusitis Soft tissue healing Anaesthesia/

This is to certify that this dissertation titled, EFFECTIVENESS OF SELF INSTRUCTIONAL MODULE ON PREVENTION OF WORM INFESTATION AMONG MOTHERS OF UNDER FIVE CHILDREN IN

RV-infected SCID mice were reconstituted with unseparated peritoneal exudate cells, sorted B1 cells, or B1 cells plus CD4 ⫹ T cells from BALB/c mice.. At 2 to 3 months

No S protein or HE was coimmunoprecipitated with the antibody to N protein (Fig. 1, lane 4), indicating that stable complexes consisting of only the glycoproteins are present

Boulton, Multicenter study of the incidence of and predictive risk factors for diabetic neuropathic foot ulceration diabetes care, July 1998; volume 21, number 7. Carvalho CB,

2C, D, and E, mutants lacking carboxy-terminal amino acids (d1, dS, dH, and d10) bound poly(U), suggesting that the C terminus of NP is not required for RNA binding. This suggestion

The results of these studies demonstrate that the intrinsic tyrosine kinase activity of this receptor is absolutely required for tissue-specific transformation mediated by v-erbB,

Killer toxin-secreting strains of the yeasts Hanseniaspora uvarum and Zygosaccharomyces bailii were shown to contain linear double-stranded RNAs (dsRNAs) that persist within