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Journal of Virology, February 2006, p. 1222-1230, Vol. 80, No. 3
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


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ABSTRACT
 
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 CD8+ T cells is unimpaired in perforin-deficient mice. However, despite the fact that the virus-specific CD8+ effector 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, CD8+ effector 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 CD8+ T cells can induce fatal CNS pathology despite the absence of perforin-mediated lysis and reduced capacity to produce several key cytokines.


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INTRODUCTION
 
CD8+ T cells are key mediators in the immune response to many viral infections. Following activation in the regional secondary 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 interferon (IFN-{gamma}) and tumor necrosis factor alpha (TNF-{alpha}) (15, 33, 43, 49, 55). The relative importance of these effector pathways 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 depends on a number of factors, such as viral tropism, the intrinsic 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 dichotomous 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-{gamma} 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 immunopathology, 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 predominantly 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 molecules, which allows the initial recruitment of relevant effector cells to the CNS. The recruited cells then reinforce the inflammatory response through the production of proinflammatory 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, various possibilities have been suggested (1, 37, 48, 57): cell contact-dependent killing of virus-infected cells, production of proinflammatory cytokines that could induce progressive cerebral 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. infection 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 conditions, effector T cells might initially be sequestered in extraneural 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 parenchyma (10) correlates better with mortalility than overall cell influx into the CNS, including meningeal infiltration. This finding 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. Surprisingly, 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 cytokines in addition to the defect in contact-dependent cell killing.


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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-{gamma} were generated as previously described (51) and bred at the Panum Institute, University of Copenhagen. WT (C57BL/6) mice were purchased from Taconic M&B (Ry, Denmark). All mice were housed under specific pathogen-free conditions, and sentinels were tested regularly for unwanted infections according to Federation of European Laboratory Animal Science Associations 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 CD8+ T cells. The {alpha}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. Cerebrospinal 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/µl 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, Germany).

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 (CD3{varepsilon}, CD4, CD11b, CD8ß, F4/80) were detected by the RiboQuant multiprobe RNase protection assay system (Pharmingen). The template set also included templates for the murine housekeeping genes L-32 (a ribosomal protein) and glyceraldehyde-3-phosphate dehydrogenase (GADPH) to serve as loading controls. The RNase protection assay was performed according to the manufacturer's instructions. Briefly, [{alpha}-32P]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-polyacrylamide 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 (Amersham Pharmacia Biotech), and the data were subsequently analyzed using ImageMaster TotalLab software (Amersham Pharmacia Biotech).

Quantification of IFN-{gamma}. IFN-{gamma} 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 antibodies 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)-conjugated anti-IFN-{gamma} (XMG1.2, IgG1), PE-conjugated anti-TNF-{alpha} (MP6-XT22), and PE-conjugated IgG1 isotype control (R3-34)

Detection of antigen-specific CD8+ T cells by major histocompatibility complex (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% CO2 with a combination of GP33-41 and NP396-404 peptides (both at 0.1 µg/ml) in the presence of monensin (3 µM, 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 intracellularly with anti-IFN-{gamma}, anti-TNF-{alpha}, 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).

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


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RESULTS
 
Lethal CD8+ T-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 determining 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 expiration, 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 replication 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.



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  		FIG. 1. Mice deficient in perforin succumb to CD8+ T-cell-mediated 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 intraperitoneally on days –1, 0, 2, 5, and 9 relative to i.c. infection with 200 PFU LCMV Armstrong (n = 5 mice/group).

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 ß2-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 intraperitoneally 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 CD8+ T 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 conditions 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-2b mice) was determined 6 and 8 days postinfection. 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-{gamma} staining was significantly lower in cells from mice lacking perforin expression (Fig. 2C), indicating a reduced capacity of the individual effector cell to synthesize this cytokine. Furthermore, the ability of virus-specific cells from Pfp–/– mice to coproduce TNF-{alpha} was markedly impaired compared to WT mice on both days examined (Fig. 2D).



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  		FIG. 2. Virus-induced CD8+ T-cell expansion is unimpaired, but cytokine production is reduced in mice deficient in perforin. For evaluation 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 dextramer+) (A) and IFN-{gamma}-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 depicted. There were no significant differences between Pfp–/– and WT mice in any of the measured parameters. (C) Mean fluorescence intensity of IFN-{gamma} staining of gated IFN-{gamma}+ CD8+ T cells is shown. Note that only analyses carried out on the same day can be directly compared. (D) Fraction of IFN-{gamma}-producing CD8+ T cells coproducing TNF-{alpha} 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. *, P < 0.05.

Since a reduced capacity to produce critical proinflammatory 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 functional 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 evaluated 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 peptide 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 cytokine-producing CD8+ T cells did not differ much between peptide 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 conditions and have a limited capacity for continued cytokine synthesis. Nevertheless, these cells still suffice for the induction of lethal CNS disease in mice infected i.c. with LCMV.



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  		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 cytokine staining. Representative plots of gated CD8+ T cells are depicted; values represent the percentage of CD44highCD8+ T cells that produce IFN-{gamma} (upper right quadrant) and mean fluorescence intensity of the staining. Averages ± standard deviations of four mice/group are presented.

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



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  		FIG. 4. Delayed recruitment of effector cells to the CNS in the absence of perforin is IFN-{gamma} independent. Pfp–/–, IFN-{gamma}/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-{gamma} 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. *, P < 0.05.

Delayed inflammation of the CNS is not a result of competition between the CNS and extraneural organ sites for inflammatory 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 clearance 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 CD8ß 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).



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  		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. *, 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 frequencies of CD8+ T cells binding these dextramers were determined using a flow cytometer. From this frequency and the total numbers of mononuclear 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-specific CD8+ T cells (producing IFN-{gamma} 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.

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 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 genotypes 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 adhesion molecule VLA-4 play an important role in targeting effector 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 expression of VLA-4 than effector T cells from WT mice. However, despite this, the frequency of virus-specific cells with an expression level matching that of effector cells from WT mice (Fig. 5C, upper right quadrant) did not fall below the frequency 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-{gamma} independent. To determine the role of IFN-{gamma} in mediating the delayed local inflammatory response in Pfp–/– mice, we infected Pfp–/– and WT mice and followed the production of IFN-{gamma} 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-{gamma} 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-{gamma} 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-{gamma}/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-{gamma}, and qualitative analysis of the inflammatory exudate revealed a similar cellular composition. Thus, factors other than IFN-{gamma} 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-{gamma}/Pfp–/– mice died following LCMV infection (data not shown).


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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 infection 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 impaired 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, alternative effector systems clearly suffice for the induction of fatal disease, questioning the pivotal role of cell lysis in the pathogenesis 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. However, we did not find evidence supporting this possibility. Alternatively, 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 possibility. Interestingly, confirming and expanding on recent findings 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 functional 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 correlates with a decreased capacity for prolonged cytokine synthesis in vitro, indicating that the cells become exhausted through chronic stimulation in vivo. The latter interpretation is consistent 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-{gamma} after antigen 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 impaired production of IFN-{gamma} 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-{gamma} is not essential for the induction of lethal disease in otherwise immunologically intact (i.e., Pfp+/+) mice infected with LCMV Armstrong (50), the possibility that IFN-{gamma} is redundant only in the presence of an intact Pfp pathway and in high concentrations would also induce fatal disease cannot be ruled out. However, following i.c. challenge of IFN-{gamma}/Pfp–/– mice, these double knockout mice develop meningitis with the same kinetics as Pfp–/– mice, demonstrating that severe CNS inflammation can be induced in the absence of both perforin and IFN-{gamma}. Unfortunately i.v. infection of IFN-{gamma}/Pfp–/– mice also results in lethal disease, which prevents us from being able to formally exclude IFN-{gamma} as an alternative mediator of lethal CNS disease in i.c. infected Pfp–/– mice. It is quite interesting that LCMV-infected IFN-{gamma}/Pfp–/– mice die even following i.v. infection. Several studies have indicated that neutralization of IFN-{gamma} 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 previous studies, we here used a low dose of a slowly invasive LCMV strain under which conditions the elimination of IFN-{gamma} 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 cytokines, once accumulated in sufficient numbers, virus-specific CD8+ T cells are capable of and responsible for the development 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 possibility 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 current thinking, the present results underscore that perforin is not pivotal; thus, once again, experimental analysis has revealed that extensive redundancy exists when it comes to key processes in antimicrobial host responses.


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ACKNOWLEDGMENTS
 
We thank Christina Jespersgaard and Jørgen Schøller (DakoCytomation, Glostrup, Denmark) for generously providing the MHC/peptide dextramers used in this study.

This work was supported in part by the Danish Medical Research Council, the Lundbeck Foundation, and the Novo Nordisk Foundation. 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.


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


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Journal of Virology, February 2006, p. 1222-1230, Vol. 80, No. 3
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.3.1222-1230.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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