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-Mediated MHC Up-Regulation1
Departments of
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Neurology,
Pathology, and
Molecular Biology, Keck School of Medicine, University of California, Los Angeles, CA 90033
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Abstract |
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uncovered distinct, nonredundant roles for these antiviral mechanisms. To separately evaluate each effector function specifically in the context of CD8+ T cells, pathogenesis was analyzed in mice deficient in both perforin and IFN- (PKO/GKO) or selectively reconstituted for each function by transfer of CD8+ T cells. Untreated PKO/GKO mice were unable to control the infection and died of lethal encephalomyelitis within 16 days, despite substantially higher CD8+ T cell accumulation in the CNS compared with controls. Uncontrolled infection was associated with limited MHC class I up-regulation and an absence of class II expression on microglia, coinciding with decreased CD4+ T cells in CNS infiltrates. CD8+ T cells from perforin-deficient and wild-type donors reduced virus replication in PKO/GKO recipients. By contrast, IFN--deficient donor CD8+ T cells did not affect virus replication. The inability of perforin-mediated mechanisms to control virus in the absence of IFN- coincided with reduced class I expression. These data not only confirm direct antiviral activity of IFN- within the CNS but also demonstrate IFN--dependent MHC surface expression to guarantee local T cell effector function in tissues inherently low in MHC expression. The data further imply that IFN- plays a crucial role in pathogenesis by regulating the balance between virus replication in oligodendrocytes, CD8+ T cell effector function, and demyelination. |
Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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, TNF, and selected chemokines (1, 2, 3, 4, 5, 6). Cytokines are especially crucial because they exert antiviral actions in multiple ways involving induction of an antiviral state, direct cytolysis, and MHC up-regulation (3, 6, 7, 8, 9). The extent to which distinct effector mechanisms contribute to viral clearance are also determined by Ag recognition in the context of the appropriate MHC molecules. The execution of some, but not all, effector functions by activated CD8+ T cells is strongly influenced by direct TCR interaction with class I-presenting cognate Ag (6, 10). For example, continued IFN- production is strictly dependent on Ag recognition and continued TCR signaling with both rapid on and off response times (10). By contrast, TNF-
production by T cells is more stringently regulated, involving more rapid, Ag-independent off cycling (11). Thus, although secreted IFN- can act on non-MHG-expressing cells, initial and ongoing secretion by CD8+ T cells requires direct MHC/TCR contact. This restriction is not an impediment during T cell activation or during infections involving lymphoid organs. However, the strict dependence on MHC recognition is particularly relevant for the execution of CD8+ T cell effector function in tissues characterized by inherently low levels of MHC expression (12, 13). During many viral infections, this dilemma may be resolved by the early infiltration of IFN--secreting NK cells (7).
Contributions of individual CD8+ T cell-specific effector mechanisms during viral infections have been examined extensively in gene-deficient mice. However, these studies are limited in that many effector mechanisms used by CD8+ T cells are also used by other immune effectors (1, 2, 3, 4, 6, 8). It has been suggested that CD8+ T cell-mediated cytolysis is required for protection against nonlytic viruses, but not for resistance to lytic viruses (2, 14, 15). This correlates preferential expression of lytic or nonlytic effector mechanisms with viral cytopathogenicity. However, infections of perforin-deficient (PKO)4 mice with more virulent viruses suggest that perforin-mediated cytolysis is also critical for resistance to some lytic viral infections in which CD8+ T cells are the predominant protective effectors (16, 17). By contrast, nonlytic mechanisms, especially IFN-, have been suggested to predominate during resolution of viral infections of the liver and CNS (8, 18, 19, 20).
The roles of distinct CD8+ T cell antiviral effector mechanisms during acute CNS infection were examined in mice deficient in either perforin or IFN- to gain insights into the inability of the immune system to prevent viral persistence and chronic demyelination. Acute CNS infection of wild-type (wt) mice by the neurotropic JHM strain of mouse hepatitis virus (JHMV) is accompanied by an extensive infiltration of mononuclear cells, including neutrophils, NK cells, macrophages, B cells, and CD4+ and CD8+ T cells (21, 22, 23, 24). Both NK cells and B cells appear redundant in controlling acute JHMV replication within the CNS (25, 26, 27). By contrast, virus-specific CD8+ and CD4+ T cells both participate in pathogenesis and resolution of the acute disease (22, 23, 28). CD8+ T cells use cytolytic activity as well as IFN- secretion to reduce viral replication (29, 30). JHMV replication in astrocytes and microglia is controlled via a cytolytic mechanism involving perforin, but not Fas/Fas ligand or TNF- (29, 31). By contrast, IFN- controls replication in oligodendrocytes, the cell type that synthesizes and maintains myelin (30). Despite these concerted CD8+ T cell effector mechanisms, sterile immunity is not achieved, resulting in a persistent CNS infection associated with chronic demyelination (23). This outcome is partially attributed to the rapid loss of cytolytic activity coincident with viral clearance (22, 23, 27), although CD8+ T cells persist in the CNS during chronic infection as long as viral Ag is present (32). By contrast, IFN- secretion in the CNS, initiated during acute disease, continues during viral persistence and is increased during JHMV reactivation in B cell-deficient mice (26, 33). The balance between loss of cytolytic activity, but continued cytokine secretion, may reflect an attempt to control infection while reducing CNS immunopathology.
In other viral models of CNS infections, CD4+ T cells are critical to controlling viral replication via cytokine secretion (34) and are key mediators of the pathological changes associated with encephalitis and demyelination (35, 36). The precise roles of CD4+ T cells as direct antiviral mediators during JHMV infection are less well understood, especially their potential contribution to antiviral IFN-. They nevertheless provide crucial accessory functions by enhancing expansion of virus-specific CD8+ T cells and maintaining CD8+ T cell viability within the infected CNS (37, 38). Although it is well established that JHMV-induced demyelination is correlated with T cell infiltration (39), the mechanisms by which the CD4+ and/or CD8+ T cell subsets contribute to macrophage/microglia-mediated demyelination are controversial (40, 41, 42, 43, 44). In wt mice, increased demyelination has been associated with CD4+ T cell influx (41). Furthermore, mice deficient in either perforin or IFN- exhibited severity of demyelination similar to that of wt mice (29, 30), suggesting that these effector molecules do not play a major role in wt mice. By contrast, in adoptive transfer studies using immunodeficient recipients, both CD4+ and CD8+ T cells have been implicated in contributing to demyelination as well as the severity of clinical disease (42, 43, 44). The latter studies suggest that CD8+ T cells enhance demyelination in a IFN--dependent manner (43), whereas CD4+ T cell-mediated demyelination is enhanced in the absence of IFN- (44).
This report examines JHMV-induced CNS pathogenesis in mice deficient in both perforin-mediated cytolysis and IFN- secretion (PKO/GKO) to evaluate the efficiency of each effector function in the presence of endogenous NK and CD4+ T cells devoid of these functions. The role of IFN- vs perforin secretion by CD8+ T cells was examined by analysis of PKO/GKO mice reconstituted with JHMV-specific memory CD8+ T cell populations derived from immune wt, PKO-deficient, or IFN- deficient (GKO) donors. Whereas PKO/GKO mice succumbed to JHMV infection, transfer of CD8+ T cells from wt immune donors cleared infectious virus from the CNS at the expense of increased demyelination. The data support the notion that IFN- is more important for viral clearance from the CNS than perforin-mediated cytolysis. However, analysis of MHC expression on resident CNS microglia in the absence of IFN- during acute infection revealed suboptimal class I expression and the total absence of class II induction. The data thus clearly establish a crucial role for IFN- in both MHC class I and class II expression, thereby potentially facilitating perforin-mediated cytolysis by CD8+ T cells and enhancing CD4+ T cell-mediated help. IFN--dependent virus clearance within the CNS thus appears to involve enhanced Ag/MHG-mediated T cell functions within the CNS, in addition to the establishment of a classical IFN--induced antiviral state.
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Material and Methods |
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BALB/c (H-2d) mice were obtained from Frederick Cancer Research Facility, National Cancer Institute (Frederick, MD). Homozygous BALB/c-Thy-1.1 mice, provided by Dr. J. Harty (University of Iowa, Iowa City, IA), and GKO BALB/c mice, provided by Dr. R. Coffman (DNAX Research, Palo Alto, CA), were bred locally at University of Southern California (Los Angeles, CA). Dr. W. Clark (University of California, Los Angeles, CA) provided homozygous PKO mice on the 129 (H-2b) background (45). PKO mice were backcrossed nine times onto the BALB/c background and then intercrossed to produce homozygous H-2d PKO mice. PKO/GKO mice deficient in both IFN- secretion- and perforin-mediated cytolysis were generated by crossing GKO (H-2d) x PKO (129, H-2d). Because both the genes encoding IFN- and perforin are located on chromosome 10, F1 mice were backcrossed to BALB/c GKO mice to facilitate homologous recombination. GKO/P+/- were selected by PCR and subsequently backcrossed for nine generations (N9) with GKO BALB/c mice and then intercrossed to produce homozygous PKO/GKO mice. Primers for detecting mutated and wt perforin and IFN- genes were: Perforin wt: 5'-primer TGG CCT AGG GTT CAC ATC CAG; 3'-primer CGT GAG AGG TCA GCA TCC TTC (P17); PKO, the 5'-wt primer and 3'-primer ATA TTG GCT GCA GGG TCG CTC within the neomycin insert. IFN- wt: 5'-primer AGA AGT AAG TGG AAG GGC CCA GAA G; 3'-primer AGG GAA ACT GGG AGA GGA GAA ATA T; GKO, neomycin 5'-primer TCA GCG CAG GGG CGC CCG GTT CTT T and neomycin 3'-primer ATC GAC AAG ACC GGC TTC CAT CCG A. Amplification was conducted with 
1 µg of DNA using a single initial denaturation step at 94°C for 4 min followed by 30 cycles of denaturation at 94°C (1 min), primer annealing at 60°C (1 min), extension at 72°C (2.5 min) and concluded by a final extension step for 7 min. Examination of naive single-deficient GKO, PKO, and naive double-deficient mice PKO/GKO mice showed normal CD4+ and CD8+ T cell ratios in spleen compared with wt BALB/c mice. All mice were seronegative for JHMV by ELISA (29, 30). Immunodeficient mice were bred and maintained under sterile conditions.
Virus
The JHMV-neutralizing mAb variant designated 2.2v-1 (46) was used for intracerebral infection. Virus was propagated and plaque assayed on monolayers of DBT cells, a murine astrocytoma as previously described (46). Mice were injected in the left hemisphere with 500 PFU of JHMV diluted in endotoxin-free Dulbecco's PBS in a 30-µl volume or with an equal volume of PBS. The severity of JHMV-induced clinical disease was graded as previously described (29, 30, 46): 0, healthy; 1, hunched back; 2, partial hind limb paralysis or inability to the upright position; 3, complete hind limb paralysis; 4, moribund or dead. CNS virus titers were determined by plaque assay on monolayers of DBT cells as previously described (29, 30, 46). Briefly, one-half of the brains were homogenized in RPMI containing 25 mM HEPES, pH 7.2, using TenBroeck tissue homogenizers. After clarification by centrifugation at 500 x g for 7 min, homogenates were either assayed directly or stored at -70°C.
T cell purification and adoptive transfer
CD8+ T cells for adoptive transfer were prepared from immunized donors. BALB/c wt, BALB/c Thy 1.1, PKO, and GKO donors were immunized by i.p. injection with 1–2 x 106 PFU of JHMV and sacrificed 6 to 14 weeks postimmunization. Approximately 5–15% of splenic CD8+ T cells were virus specific as determined by flow cytometry using MHC class I tetramers (22). Spleen cells were partially depleted of B cells by adsorption onto 150-mm plates coated with goat anti-mouse Ig. CD8+ T cells were purified by positive selection using anti-Lyt-2-coated beads as directed by the supplier (Miltenyi Biotec, Auburn, CA). CD8+ T cells were enriched to 
96% in all experiments as assessed by flow cytometry using FITC anti-CD8+ (clone 53-6.7; BD PharMingen, San Diego, CA). Purified CD8+ T cells (5–10 x 106) were injected i.v. 1 day before intracranial infection.
CNS mononuclear cell (CMC) populations
CMC populations were isolated by centrifugation on Percoll gradients as previously described (22, 24, 27, 32). Either the cell pellets obtained after clarification at 500 x g for 7 min or CNS tissues directly homogenized in RPMI supplemented with 25 mM HEPES, pH 7.2, were adjusted to 30% Percoll (Pharmacia, Uppsala, Sweden), and a 1.0-ml underlay of 70% Percoll was added before centrifugation at 800 x g for 20 min at 4°C. Cells were recovered from the 30%/70% interface and washed in RPMI before analysis.
Flow cytometry
CMC, splenocytes, and cervical lymph node (CLN) single-cell suspensions were blocked with anti-mouse CD16/CD32 (2.4G2; BD PharMingen) for 15 min before staining. For three-color flow cytometric analysis, cells were stained with FITC-, PE-, or Cy-Chrome C-conjugated mAb at 4°C for 30 min in PBS containing 0.1% BSA. Expression of surface molecules was characterized using the following mAb (all from BD PharMingen except where indicated): anti-CD8 (53-6.7); anti-CD4 (GK1.5); anti-CD19 (1D3); anti-Pan-NK (clone DX5); and anti-Thy-1.1 (OX-7). Neutrophils were identified with anti-Ly-6G (RB6-8C5). Virus-specific CD8+ T cells were detected by staining with FITC-labeled anti-CD8 and PE-labeled Ld MHC class I tetramer associated with pN318–326 peptide (LdN318; 0.1–0.2 µg/0.5–1.0 x 106 cells). PE or Cy-Chrome C anti-CD45 (Ly-5) and anti-F4/80 (Serotec, Oxford, U.K.) or in some experiments anti-CD11b (M1/70) was used to distinguish microglia (CD45lowF4/80+; CD45lowCD11b+) from infiltrating/perivascular macrophages (CD45highCD11b+; CD45highF4/80+ (47). MHC expression on microglia was determined using anti-H2Dd specific mAb (34-2-12) and anti-I-A/I-E (2G9), or I-Ad (39-10-8). Stained samples were analyzed on a FACStar or FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). Forward and side scatter signals obtained in linear mode were used to establish a gate containing live lymphocytes, macrophages, and neutrophils, while excluding dead cells and tissue debris. A minimum of 2.5 x 105 viable cells were stained and 5 x 104–1 x 105 events per sample analyzed.
Synthesis of intracellular IFN- in response to stimulation with the dominant pN318–326 epitope was determined by incubating 0.8–1.2 x 106 CMC with 3.0 x 105 J774.1 feeder cells in 200 µl of RPMI complete supplemented with 10% FCS, 1 µM peptide, and 1 µl/ml Golgistop (BD PharMingen) for 5 h at 37°C (38). Peptide was omitted in negative control samples. Cells were left at 4°C overnight and stained 14–16 h later using the Cytofix/Cytosperm Kit (BD PharMingen). After surface CD8 staining, cells were fixed, permeabilized, and stained with mAb specific for IFN- (XMG1.2) as recommended by the supplier (BD PharMingen).
Histopathological analysis
Brains and spinal cords were removed, fixed with Clark's solution (75% ethanol and 25% glacial acetic acid), and embedded in paraffin. Paraffin sections were stained with either H&E or Luxol fast blue as described (29, 30, 46). Distribution of viral Ag was determined by immunoperoxidase staining (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) using the anti-JHMV mAb J.3.3 specific for the C terminus of the viral nucleocapsid protein as the primary Ab and horse anti-mouse as secondary Ab (Vector Laboratories). Sections were scored in a blinded manner for inflammation, viral Ag, and demyelination. Representative fields were identified based on the average score of all sections in each experimental group.
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Results |
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JHMV-infected PKO/GKO mice succumb within 16 days postinfection (p.i.) in contrast to a mortality rate of 
15% in infected wt mice (data not shown). Virus replication within the CNS peaked in both infected wt and PKO/GKO mice between days 3 and 5 p.i. (Fig. 1). Infectious virus was rapidly cleared from the CNS of wt mice, resulting in the absence of detectable infectious virus by day 14 p.i. By contrast, infectious virus was only slightly reduced within the CNS of infected PKO/GKO mice between days 5 and 10 p.i. and was still detectable at 14 day p.i. (Fig. 1). Virus titers remained constant between days 14 and 16 p.i. in the declining number of survivors analyzed (data not shown). An inability to clear infectious virus and 100% mortality was also observed in PKO/GKO mice on the C57BL/6 (H-2b) genetic background (data not shown). The absence of survival past day 16 p.i. and delayed clearance of JHMV from the CNS in infected PKO/GKO mice contrasts with both the delayed mortality despite inefficient virus clearance in GKO mice (30) and the delayed but complete viral clearance and absence of mortality in PKO mice (29).
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thus allows uncontrolled virus replication, albeit without increasing myelin loss.
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(7), were noticeably reduced within CMC of infected PKO/GKO mice compared with wt mice. CD8+ T cells comprised a similar percentage of the infiltrates up to day 8 p.i., but increased considerably in PKO/GKO cells by day 10 p.i. By contrast, the relative percentage of CD4+ T cells was reduced in CNS infiltrates of PKO/GKO mice compared with wt mice from days 6 to 10 p.i. Virus-specific CD8+ T cells specific for the dominant nucleocapsid epitope were visualized by class I tetramer staining (22). Presentation of the percentages of total CD8+ T cell vs tetramer+ CD8+ T cells in the entire CMC population clearly demonstrates that both total and tetramer+CD8+ T cells were increased at least 2- to 3-fold in PKO/GKO-derived cells at days 8–10 p.i. (Fig. 3A, bottom). Thus, both CD8+ T cells of unknown specificity and the virus-specific subset preferentially accumulate in the CNS of PKO/GKO mice. These data suggest that the inability of CD8+ T cell effector functions to effectively inhibit viral replication results in preferential recruitment and/or survival of Ly-6G+ cells and CD8+ T cells, but not CD4+ T cells, in the infected PKO/GKO CNS.
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and perforin both participate in the homeostatic regulation of primed CD8+ T cells (4, 48). Whereas perforin controls expansion, IFN- regulates the death phase (4, 48). In JHMV-infected wt mice, tetramer+CD8+ T cells are only transiently detected in peripheral lymphoid organs (27, 38). The kinetics of virus specific T cell expansion was compared in wt and PKO/GKO mice to assess the potential impact of IFN- and perforin on activation and survival of virus-specific CD8+ T cells within peripheral lymphoid organs during viral-induced encephalomyelitis (Fig. 4). In CLN derived from wt mice, tetramer+CD8+ T cells were below detection throughout infection. By contrast, virus-specific CD8+ T cells were detected at day 6 p.i. in the CLN of infected PKO/GKO mice, comprising 8% of the CD8 population, and remained detectable to day 10 p.i. Total CD8+ T cell percentages remained constant in both groups. However, CLN in infected PKO/GKO mice had significantly increased cellularity between days 4 and 8 p.i., reaching a maximum of 5- to 8-fold higher cell numbers at days 6 and 7 (e.g., day 6 p.i., 45 x 106 vs 5 x 106). In contrast to CLN, spleens from both groups did not differ considerably in cellularity during infection. Total CD8+ T cell percentages were slightly increased in spleens of wt mice and tetramer+CD8+ T cells were only transiently detected at day 6 p.i., comprising 3% of CD8+ T cells. By contrast, in infected PKO/GKO mice, they constituted 15% of splenic CD8+ T cells at day 6 p.i. and increased further to 30% at days 8 and 10 p.i. These data suggested that increased CLN cellularity in infected PKO/GKO mice is not due to preferential expansion of a particular T cell subset but to an overall increase in activation. Similarly, no evidence for skewing of T cell subsets within the spleen was detected. The major difference between infected wt and PKO/GKO mice was increased and prolonged maintenance of tetramer+CD8+ T cells within the infected CNS and spleen, similar to results in PKO/GKO mice after infection with Listeria monocytogenes (48, 49, 50). Thus, early elevated levels of virus-specific CD8+ T cells in the periphery and delayed elevation at the site of infection, confirm enhanced expansion and/or possible prolonged survival of virus-specific CD8+ T cells in the absence of IFN- and perforin, although inefficient clearance of viral Ag in PKO/GKO mice may contribute to prolonged tetramer+CD8+ T cells detection.
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enhances MHC class I expression and induces MHC class II expression within the CNS
In addition to its role as a potent antiviral mediator, IFN- enhances both MHC class I and class II expression and Ag presentation (8, 9, 12, 51). The absence of IFN- may thus significantly impede MHC up-regulation by CNS cells, which are inherently limited in MHC expression in the quiescent state and dependent on activation for effective Ag presentation (12, 13). Class I and class II expression was analyzed on microglia and infiltrating CD45high cells to evaluate the contribution of IFN- in regulating MHC expression within the CNS microenvironment (Fig. 5). Microglia from naive wt mice express no detectable MHC class I (data not shown); however, class I was rapidly up-regulated by day 4 p.i. in infected wt mice. By day 6 p.i. increased class I expression was detected on the majority of microglia. These levels were sustained at day 8 p.i. Microglia derived from the CNS of infected PKO/GKO mice exhibited class I expression patterns similar to those of wt mice at day 4 p.i. However, in contrast to wt mice, no increase in either the percentage of microglia expressing class I or relative expression levels was noted after day 4 p.i. These results indicated that class I expression is up-regulated independent of IFN- early during infection but significantly enhanced after infiltration of IFN- secreting lymphocytes.
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within the CNS was more detrimental to MHC class II expression (Fig. 5). In infected wt mice, microglia expressing class II were rare at day 4 p.i., approximating the percentage detected in naive mice (data not shown). By day 6 p.i., the frequency of class II+ microglia increased to 74%, and class II was up-regulated further by day 8 p.i. In stark contrast, microglia derived from the CNS of infected PKO/GKO mice remained devoid of MHC class II expression throughout infection. These data imply that both the class I and class II Ag presentation potential within the infected CNS is significantly reduced in PKO/GKO mice.
IFN- is critical for viral clearance
To evaluate the relative contributions of perforin-mediated cytolysis and IFN- secretion by CD8+ effectors to viral clearance, CD8+ T cells from immunized wt, GKO, or PKO donors were adoptively transferred into infected PKO/GKO recipients. Memory, rather than activated, CD8+ T cells were chosen to minimize detrimental effects associated with highly activated cells, i.e., trafficking to other organs, specifically the liver (52), increased rates of apoptosis following recognition of cognate Ag (53), and limited IFN- secretion (54). CD8+ T cells derived from wt donors reduced virus replication in the CNS of PKO/GKO recipients to levels of detection by day 10 p.i. (Fig. 6). CD8+ T cells from PKO donors, capable of secreting IFN-, but not perforin, partially controlled JHMV replication. By contrast, CD8+ T cells derived from GKO donors, which retain cytolytic activity (30), exhibited minimal antiviral activity at day 8 p.i. within the CNS of PKO/GKO recipients, but no detectable antiviral activity at day 10 p.i. compared with untreated PKO/GKO-infected mice (Fig. 6).
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secretion in the absence of perforin provided selective clearance from any CNS cell type.
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-mediated up-regulation of adhesion molecules does not play a major role in infiltrate distribution. Although the differences were not striking, these data suggest that perforin expression correlates with the ability of CD8+ T cells to mediate the loss of myelin in this model.
CMC were isolated at day 8 p.i. from infected PKO/GKO CD8+ T cell recipients and examined for the frequency of CD45high, Ly-6G+, CD8+, tetramer+CD8+, and CD4+ cells to determine the effects of donor cells on the composition of CNS infiltrating cells (Fig. 8A). Recipients of all donor populations had reduced yields of CNS cells, consistent with a 50% reduction in the percentage of CD45high infiltrating cells. The Ly-6G+ population was affected most strongly, decreasing to <5% in all recipients groups. Similarly, the CD8+ T cell population was reduced by 30–40% compared with untreated PKO/GKO mice. Overall, the CD8+ population in all recipient groups constituted the vast majority of the CD45high cells compared with the untreated group. Furthermore, all recipients exhibited a high frequency of virus-specific CD8+ T cells. CD4+ T cell percentages remained constant at 6–8% in all groups (data not shown). Although adoptive transfer of immunocompetent effectors resulted in a decrease in total CNS CD8+ T cells, tetramer+CD8+ populations within the CNS were not significantly altered and thus comprised a large portion of all infiltrating cells. Adoptive transfer of CD8+ T cells from immunized Thy-1.1 donors was examined to assess the relative proportion of donor (Thy-1.1+) vs endogenous (Thy-1.2+) CD8+ T cells recruited to the CNS (Fig. 8B). The majority of CD8+ T cells within the CNS were donor derived (Thy-1.1+) at both days 7 and 10 p.i. (70 and 83%, respectively). Furthermore, 90% of tetramer+ cells within the CNS of both Thy-1.1 and PKO donor cell recipients secrete IFN- in response to pN peptide stimulation, confirming that the majority of CNS-infiltrating CD8+ T cells derived from memory populations are donor derived.
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(Fig. 8C). MHC class I expressing microglia increased from 50% in untreated infected mice to 80% after transfer of memory CD8+ T cells derived from wt donors. In addition to an increased frequency, the level of class I expression also increased. A similar effect was achieved by IFN--competent CD8+ T cells derived from PKO donors, providing indirect evidence for IFN- secretion within the CNS parenchyma of PKO/GKO recipients during JHMV infection. This is strongly supported by the abundance of IFN- producing CD8+ T cell after in vitro stimulation (Fig. 8B). Although histological and flow cytometry suggested that CD8+ T cells derived from GKO donors infiltrated the CNS of infected PKO/GKO recipients, their presence had no effect on microglial class I expression. In contrast to class I expression, only a very modest increase in the frequency of MHC class II+ microglia was achieved by either wt or PKO donor CD8+ T cells (Fig. 8C). An explanation for the vast discrepancy between class II expression in wt mice (Fig. 4) compared with recipients of IFN--secreting virus-specific CD8+ T cells (Fig. 8) may reside in the additional contribution of infiltrating NK and CD4+ T cells to IFN- secretion in wt mice. These data suggest that class I expression is more sensitive than class II expression to CD8+ T cell-secreted IFN-. The inability to detect a role for perforin-mediated cytolysis may thus reside in insufficient class I Ag presentation to trigger TCR recognition of infected resident cell types. |
Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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The neurotropic JHMV used in these studies infects a variety of CNS cell types, including ependymal cells, astrocytes, microglia/macrophages, and oligodendroglia (46, 57). Infection of neurons is a rare event, detected primarily after infection of immunodeficient mice. Nonredundant roles for perforin-mediated cytolysis and IFN- as distinct antiviral effector mechanisms were previously established by studies in mice selectively deficient for either perforin or IFN-. Higher mortality rates and decreased viral clearance in GKO mice implicated a prominent role for IFN-. Furthermore, GKO mice were unable to control JHMV infection of oligodendroglia (30). By contrast, in the absence of perforin, the predominant cell types infected were astrocytes and microglia/macrophages, with relative absence of virus replication in oligodendroglia (29). PKO/GKO mice were examined as recipients of CD8+ T cells to circumvent effects of IFN- and perforin expression by recipient NK cells (7). PKO/GKO recipients also have the advantage of an endogenous CD4+ T cell population capable of providing IFN--independent accessory functions to donor CD8+ T cells.
Analysis of JHMV pathogenesis in untreated infected PKO/GKO mice revealed several differences compared to wt mice. First, uncontrolled CNS virus replication confirmed the necessity for perforin and IFN- in protection (29, 30). An absence of antiviral control was supported by increased and diverse Ag distribution in all CNS cell types within the brain and prominent Ag in spinal cord oligodendroglia. Although histological examination suggested that inflammatory cells remained predominantly perivascular in the brain of both groups of mice, a slight increase in CNS parenchyma inflammation in PKO/GKO mice compared with wt mice coincided with increased Ly-6G+ cells detected by flow cytometry and cells with neutrophil morphology within the CNS (data not shown). These data suggest that the inability to inhibit virus replication was not associated with a paucity of parenchymal infiltration into the brain in the absence of IFN-. By contrast, infiltration into spinal cords was equally extensive in both groups, suggesting differential recruitment into the parenchyma of the brain vs the spinal cord. A basis for this may reside in altered endothelium, chemokine, and/or metalloprotease components (24, 41). Second, demyelination was reduced in infected PKO/GKO mice relative to wt mice, suggesting that increased virus replication may not directly contribute to the extent of myelin loss, especially in the absence of CD8+ T cell function. Third, even despite the vast accumulation of virus-specific CD8+ T cells within the CNS of PKO/GKO mice, virus replication was not contained, confirming that neither function is compensated for by alternative mechanisms. Whether increased accumulation of tetramer+CD8+ T cells within the CNS reflects increased viral Ag, recruitment from an increased peripheral pool, and/or enhanced survival is unclear. The selective increase of both total CD8+ and tetramer+ T cells, but not CD4+ T cells, after day 7 p.i. may be explained by increased expansion and survival of Ag-specific CD8+ T cells in PKO/GKO mice (4, 48, 49, 50). Increased frequencies of virus-specific CD8+ T cells in CLN and spleens of infected PKO/GKO mice relative to wt mice are consistent with this notion. Although more prevalent Ag cannot be excluded as a mechanism, reemerging virus replication in the absence of Ab-mediated protection is not associated with increased total CD8+ or tetramer+ T cells in either the CNS or periphery (26, 27). The transient, nevertheless striking increase in CLN cellularity supports the concept that priming and expansion of virus-specific CD8+ T cells occur in CLN during CNS infection (55, 58). Furthermore, enhanced frequencies and prolonged accumulation of virus-specific CD8+ T cells in the spleen are reminiscent of dysregulated homeostatic control of activated/memory CD8+ T cells in the absence of perforin and IFN- (4, 48, 49, 50), although suboptimal viral clearance in PKO/GKO mice may play an additional role in increased and sustained detection of peripheral virus-specific CD8+ T cells.
Despite a substantial increase in Ly-6G+ cells in the CNS of PKO/GKO mice compared with wt mice, similar virus titers early in infection suggest the absence of direct antiviral function. Furthermore, the paucity of NK cells within the CNS of PKO/GKO mice had no apparent influence on retention and/or recruitment of CD8+ T cells, suggesting that NK cell function is not critical for T cell recruitment into the inflamed CNS. Nevertheless, NK cells in wt mice comprise an early source of IFN- (7), potentially enhancing local MHC expression. The observation that both the frequency and level of class I expression on microglia were similar in both groups before T cell infiltration, independent of IFN-, implicates possible regulation by IFN-
(7). Sufficient target structures were thus initially available to activate CD8+ T cell effector function. However, although class I was detected on the majority of microglia from wt mice concomitant with lymphocyte infiltration, it remained low on microglia derived from infected PKO/GKO mice. These data suggest a strong influence of IFN- released by infiltrating T cells on class I expression by resident CNS cells. The inability to detect class II expression on microglia derived from PKO/GKO mice further demonstrated that in contrast to class I up-regulation, class II expression is strictly dependent on IFN- during CNS inflammation. Reduced CD4+ T cells infiltration into the CNS of PKO/GKO mice may thus be a consequence of limited class II expression on resident cells.
JHMV-specific memory CD8+ T cells derived from wt donors eliminated virus from the CNS of PKO/GKO recipients, confirming their recruitment and functional viability within the CNS. Furthermore, PKO donor CD8+ T cells only partially reduced virus replication in PKO/GKO mice, supporting a contribution of perforin, established in PKO mice (29). By contrast, the apparent lack of perforin-mediated antiviral function by GKO donors (30) in the absence of IFN- contradicted a role for perforin. Enhanced distribution of infiltrating cells in parenchymal tissue, compared with untreated PKO/GKO mice, suggested that the inability to affect virus replication was not attributed to restricted donor cell access. These contradictory data can be reconciled by considering the low levels of class I expression on microglia achieved after infection of GKO recipients compared with wt and PKO recipients. These results support the notion that IFN- derived from CD8+ T cells not only is critical in direct control of viral replication but also enhances perforin-mediated cytolysis by up-regulating class I on both infected and noninfected cells. Although perforin expression is not as tightly regulated by MHC/TCR contact as IFN- (6, 10), TCR engagement is required for targeted cytolysis. The effects of donor cells on viral replication were largely confirmed by histological analysis. However, due to the effective clearance by wt and PKO donor CD8+ T cells, preferential inhibition of virus replication in distinct cell types could not be determined in either the brain or spinal cord. The lack of viral clearance by GKO CD8+ T cells correlated with increased viral Ag in glial cells with a similar distribution to those in untreated infected PKO/GKO mice. It was thus not possible to discern perforin-mediated effects, consistent with impaired class I expression.
The mechanism(s) of demyelination induced by JHMV infection is not clear (23, 40, 41, 42, 43, 44); however, CD8+ T cells have been suggested to both increase (42, 43) and decrease demyelination (56). Similar to infection of GKO and PKO mice (29, 30), demyelination in infected PKO/GKO mice was only slightly reduced compared with wt mice. Furthermore, CD8+ T cells from wt and GKO donors, but not PKO donors, increased demyelination to the levels found in wt mice. Although the changes noted were not substantial, these data suggest that perforin and IFN- acting together make discrete contributions to the demyelination process. In contrast to these data, no clear antiviral effects by wt CD8+ T cells or IFN- were observed after transfer of highly activated T cells into infected RAG1-/- recipients (42, 43). Despite the inability to affect viral clearance, donor T cells had small but distinct effects on the extent of demyelination (42). In this model, activated CD8+ T cells enhanced macrophage-induced demyelination via IFN-, but not perforin or TNF- (43), in contrast to our results. The apparent inability of recently in vivo activated effectors to influence virus replication in the latter studies may be due to activation-induced apoptosis and/or the lack of CD4+ T cell help (37, 53). Nevertheless, in vitro activated CD8+ T cells from wt donors displayed similar antiviral effector function as resting memory populations in both H-2d and H-2b PKO/GKO recipients (data not shown). Increased demyelination in GKO CD8+ T cell recipients, in the absence of an apparent antiviral effect concomitant with the presence of a vast number of infected oligodendrocytes, is consistent with a role for IFN- in facilitating perforin-mediated effects via optimizing class I expression. Higher virus load and diversity of infection may trigger distinct defense functions involving responses not necessarily reflected in viral clearance, e.g., secretion of macrophage-activating chemokines. TNF- is an unlikely candidate because continued secretion is rapidly lost (11) and inhibition of TNF- in infected wt mice (61), or transfer of activated CD8+ T cells from TNF--deficient donors into infected RAG1-/- recipients (43), did not alter demyelination. These results suggest that demyelination is a net consequence of the balance between virus replication in predominantly oligodendrocytes on one side and CD8+ T cell immune effector function on the other. These different outcomes regarding both demyelination and virus clearance using distinct models thus may reside in either the distinct populations of T cells transferred, the time points analyzed, or the underlying immunodeficiencies of the recipients. Nevertheless, these data demonstrate the complexity of interactions between adaptive immunity, the infected CNS, and the pathological outcomes of these interactions.
In summary, these results demonstrate three crucial findings: 1) memory CD8+ T cells from immune wt donors infiltrate the infected CNS and effectively clear replicating virus, even in an environment in which the NK and CD4+ T compartments are unable to secrete IFN-; 2) partial clearance by CD8+ T cells from PKO donors indicates that perforin and IFN- synergize in optimizing viral clearance, consistent with the delayed viral clearance from the CNS of infected PKO mice (29); 3) the apparent lack of perforin-mediated antiviral function in the absence IFN- suggests that IFN- is crucial to optimize perforin-mediated CD8+ T cell effector function within the CNS. Among the multiple functions elicited by IFN-, up-regulation of MHC and adhesion molecules is likely to be most critical for enhancing CD8+ T cell-mediated clearance. The balance and kinetics of the individual antiviral effects appear to have distinct pathological consequences, including demyelination.
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2 Current address: Department of Microbiology, Universidad del Valle, Cali, Colombia.
3 Address correspondence and reprint requests to: Dr. Stephen Stohlman, 1333 San Pablo Street, MCH 142, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033. E-mail address: stohlman{at}usc.edu
4 Abbreviations used in this paper: PKO, perforin deficient; CLN, cervical lymph nodes; CMC, CNS-derived mononuclear cells; JHMV, the JHM strain of mouse hepatitis virus; GKO, IFN- deficient; PKO/GKO, perforin and IFN- deficient; p.i., postinfection; wt, wild type.
Received for publication September 26, 2002. Accepted for publication January 15, 2003.
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, wt cells; 
, PKO/GKO cells. Middle row, relative percentages of Ly-6G+, NK, CD4+, and CD8+ cells within the infiltrating CD45high population. Data are calculated based on setting the total CD45high population to 100%. Bottom row, percentage of total CD8+ (left) and tetramer+CD8+ (right) cells within the total CMC population. Numbers on the columns depict the relative percentage of tetramer+ cells within the respective CD8 population. Data represent one of three similar experiments. B, Top row, forward (FSC) and side light scatter characteristics and the R1 region used for analysis of cell subsets; R1 typically comprised 65–75% of total events. Middle row, flow cytometry density plots showing expression of CD45 (y-axis) in the gated R1 population at day 8 p.i. Gates were set on CD45high and CD45low cells to distinguish infiltrating bone marrow-derived cells (R2) from resident microglia (R3) and to set quadrants. This analysis is representative for data derivation shown in A. Bottom row, density plots showing expression of CD45 (y-axis) vs Ly-6G (x-axis) or no Ab control (co) in the gated R1 population. The highlighted population, R4, exhibits higher side scatter characteristics than lymphocytes, consistent with neutrophils (data not shown). Numbers represent percentages of positive cells in the R4 gate relative to total CMC. The data reveal a significantly higher percentage of CD45high cells with a prominent Ly-6G+ population in the CNS of PKO/GKO compared with wt mice at day 8 p.i. FL, fluorescence.
