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Departments of
*
Immunology and
Neurology, Mayo Clinic, Rochester, MN 55905; and
Department of Neurology, University of Chicago Medical Center, Chicago, IL 66906
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
serine proteinases, lymphotoxin, and other cytolytic molecules (4, 5, 6, 7, 8, 9).
Resistance vs susceptibility to wild-type DA (wt-DA)-induced
demyelination maps genetically to the MHC class I H-2D region (1, 3).
Although CNS cells do not normally express MHC Ags (10), both class I
and class II molecules are induced on microglia, astrocytes, and
endothelial cells following TMEV infection (11, 12). Viral Ags are
expressed on oligodendrocytes, astrocytes, microglia, and macrophages
during acute disease, thus serving as potential targets for a class
I-restricted immune response (13, 14, 15). These observations suggest that
virus-infected MHC-expressing CNS cells present viral peptides to T
cells, which are activated to either lyse CNS targets or secrete
soluble lymphokines. A number of previous lines of evidence indicate that class I-restricted CTLs are critical in clearing virus from the CNS of resistant genetic strains. In resistant B10 mice, depletion of T cell subsets with mAbs directed at CD4+ or CD8+ T cells results in an increased frequency of paralysis and death due to acute encephalitis (16). More extensive CNS abnormalities resulted following treatment with mAbs to CD3 or combined treatment with mAbs to CD4+ or CD8+ T cells. B6x129 ß2m-/- mice (deficient in ß2m) of a resistant H-2b haplotype express low levels of MHC class I, have few CD8+ T cells, and develop virus persistence and severe demyelination following wt-DA-infection (17, 18, 19). At 7 days postinfection (d.p.i.), CNS-infiltrating lymphocytes (CNS-ILs) isolated from resistant B10 mice are primarily CD8+ T cells (20). In contrast, few CD8+ T cells are isolated from the CNS of susceptible SJL/J (H-2s) mice at this time point. The wt-DA-specific CD8+ CTLs are generated in the CNS of infected resistant B10 (H-2b) mice and presumably play an important role in viral clearance, whereas no virus-specific cytotoxicity is demonstrated in the CNS of susceptible B10.S (H-2s) and B10.Q (H-2q) mice (21). A 10-residue peptide VP2121–130 has been identified as the H-2Db-restricted target of the cytotoxicity in resistant B10 mice (22, 23). These data are consistent with the conclusion that the class I-restricted immune response plays a critical role in determining resistance to wt-DA-induced demyelination and virus persistence.
Picornaviruses generally synthesize a long polyprotein with one open reading frame. However, the DA strain of TMEV translates another protein, L*, which is out of frame with the polyprotein and is initiated 13 nucleotides downstream from the AUG used to initiate the polyprotein. Other members of the TO subgroup besides DA have an AUG at this location, while members of the G DVII subgroup, which do not demyelinate or persist, have an ACG and therefore do not synthesize L*; this observation suggested the importance of L* to the late TO subgroup demyelinating disease. DA virus with a mutation at the L* initiation codon (designed DAL*-1) fails to synthesize L* and has decreased demyelinating activity in the CNS of susceptible SJL/J mice (24). We considered whether DAL*-1 fails to demyelinate because it fails to persist, raising the question of whether DAL*-1 infection might induce a protective virus-specific CTL response in susceptible mice. In this report we demonstrate that susceptible mice infected with DAL*-1 do mount CTL responses in the CNS that clear virus infection. The experiments suggest that the presence of L* inhibits the generation of H-2K-restricted virus-specific CTLs in the CNS of susceptible strains of mice, resulting in the failure to clear virus and thus predisposing to chronic inflammatory demyelination similar to that in human multiple sclerosis. To our knowledge, this is the first demonstration of a viral protein inhibiting a CTL response in a class I locus-specific manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The wt-DA and DAL*-1 viruses were used in all experiments (2, 24). DAL*-1 virus was generated by making a point mutation at nucleotide 1080 of a full-length infectious wt-DA cDNA clone (DAFL3), changing the L* initiation codon from AUG to ACG. Virus with this mutation does not change the amino acid sequence (which is in a different reading frame) of the polyprotein, but fails to synthesize L*. The viruses were propagated in BHK-21 cells.
Animals
Four- to eight-week-old B10 (H-2b), B6 (H-2b), SJL/J (H-2s), and B10.S (H-2s) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B6x129 ß2m-/- (ß2m-deficient H-2b) mice were obtained from Mayo Clinic Immunogenetic Mouse Colony (C. David, Director; Rochester, MN).
Pathologic analysis
Forty-five d.p.i., mice were anesthetized with pentobarbital and perfused by intracardiac puncture with Trump’s fixative (phosphate-buffered 4% paraformaldehyde and 1% glutaraldehyde, pH 7.2). This time point after infection was chosen for analysis because it allows differentiation of resistance vs susceptibility to virus persistence and demyelination (3). Spinal cords were removed, sectioned coronally and serially into 15–20 blocks, osmicated, and embedded in 2-hydroxyethyl methacrylate (JB-4 system from Polysciences, Warrington, PA). Using a modified erichrome method with a cresyl violet counterstain (25), 2-µm sections were stained to detect demyelination and inflammation. Detailed morphologic analysis was performed by examining each quadrant from 15–20 spinal cord coronal sections from each mouse for the presence or the absence of demyelination, white matter inflammation, and gray matter inflammation (15). The presence or the absence of the pathologic abnormality was determined in every spinal cord quadrant. The total score was expressed as the percentage of spinal cord quadrants with the specific abnormality, such that a maximum score of 100 represents the presence of disease in every quadrant of every spinal cord section examined. Analysis of variance (and Bonferroni adjustment t tests) was used to evaluate significant differences in pathological scores between mice infected with different viruses.
In situ hybridization
Brain and spinal cords of mice were embedded with paraffin and sectioned. The tissue sections were treated with xylene and ethanol followed by hybridization overnight with a 35S-labeled probe complementary to the coding region of VP1 (nucleotides 3053–3305) (26). After extensive washing, slides were exposed in NBT-2 emulsion (Eastman Kodak, Rochester, NY) for 48 h as described previously (27). Slides were counterstained with hematoxylin.
Virus-specific Ab determination
Anti-TMEV Abs in the sera of infected mice were determined by ELISA using purified virus Ag as described previously (27).
Cells
BHK-21, baby hamster kidney cells, were obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 with 5% FCS. The SV40-transformed fibroblast cell lines C57SV (Kb, Db), KSSV (Ks, Ds), PSJLSV (Ks, Ds), KHTTSV (Ks, Dd), and KB10.DASV (Kq, Ds) were obtained from Barbara Knowles (The Jackson Laboratory) and were cultured in RPMI 1640 with 5% FCS. Transfected C57SV/LP cells (expressing wt-DA leader, VP4, VP2, and VP3) were cultured in RPMI 1640 supplemented with 5% FCS and 300 µg/ml G418 (28). Transfected fibroblast NABB (Kk, I-Ab, Dk) cells were obtained from Ned Braunstein (Columbia University, New York, NY). Transfected fibroblast L/Kb (Kk/b, Dk) and L/Db (Kk, Dk/b) cells described previously (21). NABB, L/Kb, and L/Db cells were cultured with 10% FCS and HAT medium (0.0136% hypoxanthine, 0.0044% aminopterin, and 0.0072% thymidine). Expression of MHC molecules on these cell lines was determined by FACS with class I-specific Abs as described previously (28). For infected targets, fibroblast C57SV, KSSV, PSJLSV, KHTTSV, KB10.DASV, NABB, L/Kb, or L/Db cells were infected with either DA or DAL*-1 at 10 PFU/cell 1 day before assay. On the day of assay, target cells were trypsinized from the flasks, labeled with sodium chromate (51Cr) at 100 mCi/106 cells for 1 h, washed with RPMI 1640, and resuspended to 2 x 104/ml in RPMI 1640 with 5% FCS. The efficiency of virus infection of all the cell lines used in these experiments was determined by immunohistochemistry staining with polyclonal rabbit Ab against DA. The results indicated that all the cells were infected to a similar extent with either DA or DAL*-1.
Preparation of CNS-ILs from virus-infected mice
Mice were inoculated intracerebrally with 2 x 105 PFU of wt-DA or DAL*-1 virus in a 10-µl volume. For each experiment a minimum of five animals were pooled by experimental group. At 7 d.p.i. CNS-ILs were isolated by Percoll gradient as described previously (28) to serve as effectors for the cytotoxicity assay.
Preparation of spleen effector cells from DA-infected mice
Mice were intradermally inoculated on days 0 and 14 with 5 x 106 PFU of virus with CFA in 0.3 ml (0.15 ml of virus in PBS buffer mixed with 0.15 ml of CFA). On day 28, splenocytes were isolated and stimulated in vitro with irradiated virus-infected cells as described previously (21).
Cytotoxicity assay
The cytotoxicity assay was performed as described previously (21, 28). Mean radioactivity values were calculated from triplicate wells, and results were expressed as the percent specific lysis according to the formula [(experimental counts - spontaneous counts)/(maximum counts - spontaneous counts)] x 100%. The SEM was determined from the results obtained from pooled lymphocyte samples in triplicate wells. Statistical comparisons were made using unpaired Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The wt-DA synthesizes both the viral polyprotein as well as L* protein, which is initiated at an alternative AUG in infected cells. The wt-DA induces chronic demyelination in the spinal cords of susceptible SJL/J (H-2s) and B10.S (H-2s) mice following intracerebral inoculation (2, 3, 24). In contrast, DAL*-1 virus, which was generated from wt-DA cDNA by making a point mutation at the L* initiation codon, synthesizes DA polyprotein (which is cleared into viral structural and nonstructural proteins), but not L*, in infected cells. DAL*-1 fails to induce demyelination in the spinal cords of SJL/J mice (24).
We first tested whether DAL*-1 would induce chronic demyelination in
the spinal cords of another wt-DA-susceptible mouse strain (B10.S) with
the same MHC as SJL/J but different background genes. Forty-five days
following intracerebral inoculation with DAL*-1 or wt-DA viruses, B10.S
and SJL/J mice were perfused with Trump’s fixative, and the spinal
cords were removed, sectioned, and stained to detect myelin and
inflammatory cells. No demyelination was observed in the spinal cords
of five SJL/J and five B10.S mice inoculated with DAL*-1 virus (Table I
). In contrast, prominent demyelination
was observed in the spinal cords of B10.S and SJL/J mice following
intracerebral infection with wt-DA (Table I). Minimal white matter
inflammation was observed in the spinal cords of B10.S or SJL/J mice
following infection with DAL*-1 (p > 0.05
between them), whereas prominent white matter inflammation was observed
in mice infected with wt-DA (p < 0.01 in
comparison with mice infected with DAL*-1; Table I). These results
confirmed the critical role of L* in the late demyelinating disease
phenotype induced by DA in the CNS of B10.S and SJL/J mice.
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). Clinically, B10.S mice infected with DAL*-1
showed rear leg stiffness at 45 d.p.i., whereas SJL/J mice did
not. B10.S and SJL/J mice infected with wt-DA did not show paralysis at
this time point. Therefore, the disease phenotype induced by DAL*-1 was
altered in B10.S compared with SJL/J mice, as evidenced by clinical
deficits and the degree of neuronal vacuolation. This indicated that
differences in background genes outside the MHC were contributing to
the phenotype. L* plays a critical role in virus persistence in the CNS of wt-DA-infected mice
To determine whether the inability of DAL*-1 to induce
demyelination was due to its failure to persist in the CNS, B10.S and
SJL/J mice were infected intracerebrally with DAL*-1 or wt-DA. At
45 d.p.i. mice were perfused, and spinal cords were embedded in
paraffin. The viral RNA genome was demonstrated by in situ
hybridization in the spinal cords of each SJL/J mouse infected with
wt-DA, but not in the CNS of B10.S and SJL/J mice infected with DAL*-1
(Table I). We conclude that the synthesis of L* protein prevents
clearance of virus from the CNS of mice infected with wt-DA.
Wt-DA and DAL*-1 stimulated production of a similar level of virus-specific Abs
We considered whether a difference in the humoral immune response
could explain the markedly different phenotypes induced by these
viruses. We collected sera from B10.S and SJL/J mice 45 d.p.i.
with wt-DA or DAL*-1. B10.S and SJL/J mice infected with either wt-DA
or DAL*-1 produced similar high levels of virus-specific Abs (Fig. 1). This finding indicates that these
viruses replicated in the CNS of B10.S and SJL/J mice and induced a
specific humoral immune response. In addition, the data suggest that
differential clearance of virus in susceptible strains inoculated with
DAL*-1 vs wt-DA is not a function of the humoral immune response.
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CNS-ILs isolated from resistant B6 (H-2b) mice
infected with DAL*-1 for 7 days and a wt-DA LP-specific CTL line
generated from the spleens of wt-DA-infected B6 mice served as
effectors for CTL assays. The wt-DA- or DAL*-1-infected C57SV cells and
transfected C57SV/LP cells (expressing leader, VP4, VP3, and VP2)
served as targets. CNS-ILs from DAL*-1-infected B6 mice and the CTL
line directed against wt-DA LP lysed DAL*-1- or wt-DA-infected C57SV
cells, and C57SV/LP cells; however, there was no killing against
uninfected C57SV cells (Fig. 2,
A and B). The results demonstrate that
DAL*-1-specific CTLs and wt-DA LP-specific CTLs can recognize the class
I-presented antigenic peptide(s) from either DAL*-1- or wt-DA-infected
C57SV cells, suggesting that L* does not inhibit the binding of viral
peptides with class I molecules or the presentation of viral peptides
to specific CTLs. Therefore, L* does not inhibit the generation of
H-2Db-restricted virus-specific CTLs.
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CNS-ILs isolated from B10 mice 7 days following infection with
DAL*-1 or wt-DA were used as effectors against DAL*-1- or
wt-DA-infected and uninfected L/Kb (Kk/b,
Dk) and L/Db (Kk, Dk/b)
cells. CNS-ILs from DAL*-1-infected mice lysed DAL*-1-infected
L/Db and L/Kb cells but not uninfected cells
(Fig. 3A). The
H-2Db-restricted virus-specific cytotoxicity was higher
than the H-2Kb-restricted response. Therefore,
virus-specific cytotoxicity in the CNS of B10 mice during acute DAL*-1
infection is both H-2Kb and H-2Db restricted.
In contrast, virus-specific cytotoxicity in the CNS of B10 mice
infected with wt-DA was restricted to only H-2Db, but not
H-2Kb (Fig. 3B) (21, 28). Therefore, L* inhibits
the generation of H-2Kb-restricted cytotoxicity in the CNS
of resistant B10 mice, but does not inhibit the generation of
H-2Db-restricted virus-specific cytotoxicity. The
virus-specific cytotoxicity in the CNS of resistant B10 mice at 45
d.p.i. could not be examined because wt-DA is cleared from the CNS,
expression of class I Ags in the CNS is decreased, and few if any
lymphocytes are present in the CNS at that time.
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B6x129 ß2m-/- (H-2b) mice deficient in ß2m have a low level of MHC class I expression and have few functional CD8+ T cells. We used these mice to study whether class II-restricted virus-specific cytotoxicity is present in the CNS of infected mice. Seven days postintracerebral DAL*-1 infection, the isolated CNS-ILs did not lyse uninfected (1.8 ± 0.5%), wt-DA-infected (0 ± 1.2%) or DAL*-1-infected NABB (Kk, IAb, Dk) cells (0 ± 4.8%). Previous experiments also failed to demonstrate class II restricted virus-specific cytotoxicity in CNS-ILs using B10 mice infected with wild-type DA virus (28). NABB cells express the appropriate class II allele (I-Ab) but irrelevant class I alleles (Kk, Dk). Thus, intracerebral DAL*-1 inoculation of mice of an H-2b haplotype does not induce generation of class II-restricted virus-specific CTLs.
Class I-restricted virus-specific cytotoxicity is present in the CNS of B10.S (H-2s) and SJL/J (H-2s) mice infected with DAL*-1, but not with wt-DA
B10.S and SJL/J mice infected with wt-DA develop chronic
demyelination and virus persistence, whereas mice infected with DAL*-1
do not. Failure to generate class I-restricted virus-specific CTLs in
the CNS of B10.S and SJL/J mice infected with wt-DA could result in a
failure to clear virus and predispose the mouse to subsequent
immune-mediated chronic demyelination. To determine whether this was
the case, CNS-ILs isolated on 7 d.p.i. from B10.S or SJL/J mice
infected with either wt-DA or DAL*-1 were used as effectors in a CTL
assay. Infected and uninfected KSSV (Ks, Ds) or
PSJLSV (Ks, Ds) cells served as targets,
testing for H-2s-restricted virus-specific cytotoxicity.
CNS-ILs isolated from B10.S or SJL/J mice infected with DAL*-1 lysed
either DAL*-1- or wt-DA-infected KSSV or PSJLSV cells, but not
uninfected KSSV or PSJLSV cells (Fig. 4,
A and B). No killing of MHC-mismatched SQSV
(Kq, Dq) targets was observed using CNS-ILs
isolated from the CNS of B10.S mice infected with DAL*-1 whether the
targets were infected with wt-DA (1.9 ± 1.4%) or DAL*-1
(3.6 ± 4.6%) or were uninfected (2.1 ± 2.1%). Therefore,
the cytolytic activity of the T cells was H-2 restricted. In contrast,
CNS-ILs isolated from B10.S or SJL/J mice infected with wt-DA were not
lytic against wt-DA- or DAL*-1-infected targets. As shown in Fig. 4, A and B, the CNS-ILs from B10.S mice infected
with wt-DA did not lyse wt-DA-infected (2.7 ± 1.2%) or
DAL*-1-infected (3.0 ± 2.2%) PSJLSV cells, nor did CNS-ILs from
SJL/J mice infected with wt-DA lyse wt-DA-infected (2.7 ± 1.2%)
or DAL*-1-infected (3.0 ± 2.2%) KSSV cells. Because cells
infected with wt-DA virus serve as effective targets for CTL activated
by DAL*-1, we conclude that L* does not inhibit the binding of
antigenic peptides with class I molecules or Ag presentation by
infected target cells. However, L* appears to inhibit the in vivo
generation of class I-restricted virus-specific CTLs in the CNS of
B10.S and SJL/J mice.
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Following DAL*-1 infection, virus-specific cytotoxicity by CNS-ILs
directly assayed without in vitro stimulation showed significant, but
low level, killing of infected targets. To amplify this response,
CNS-ILs isolated from B10.S and SJL/J mice infected with DAL*-1 were
stimulated in vitro with irradiated DAL*-1-infected KSSV cells for 13
days to serve as effectors for the CTL assay. DAL*-1- or wt-DA-infected
and uninfected KSSV (Ks, Ds), PSJLSV
(Ks, Ds), NHTTSV (Ks,
Dd), and KB10.DASV (Kq, Ds) cells
served as targets. In independent experiments the effectors from both
strains showed prominent lysis of either DAL*-1- or wt-DA-infected
KSSV, PSJLSV, and NHTTSV, but not KB10.DASV, cells at an E:T cell ratio
of 30:1 (Fig. 5, A and
B). Therefore, we conclude that virus-specific cytotoxicity
in the CNS of B10.S and SJL/J mice infected with DAL*-1 is restricted
to H-2Ks but not H-2Ds. Vaccinia-infected
B10.DASV target cells were lysed readily by vaccinia-specific CTL
(41 ± 6% for infected cells vs 0 ± 3% for uninfected
cells, using a CTL:target ratio of 100:1), indicating that their
inability to be recognized by DAL*-1 virus-specific CTL was not a
result of the inability of the targets to present Ags in the context of
Ds.
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Having established that virus-specific CTLs can be demonstrated from the CNS of susceptible mice infected with DAL*-1, we asked whether class I-restricted virus-specific CTLs can be generated in the spleens of mice normally susceptible to demyelination. B10.S mice were intradermally inoculated with DAL*-1 or wt-DA with CFA twice, separated by a 2-wk interval. Two weeks after the second inoculation, splenocytes were isolated and stimulated in vitro with irradiated DAL*-1- or wt-DA-infected KSSV cells for 5 days to serve as effectors. The wt-DA-infected and uninfected KSSV cells were used as targets. Splenocytes isolated from DAL*-1-infected B10.S mice lysed wt-DA-infected KSSV cells (31.5 ± 4.0%), but did not significantly lyse uninfected KSSV cells (7.0 ± 1.9%). Splenocytes isolated from wt-DA-infected mice did not lyse either uninfected (0 ± 5.2%) or infected KSSV cells (0 ± 2.8%). Therefore, infection with DAL*-1 does result in the generation of virus-specific CTLs in the spleens of susceptible mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The reasons why the Ks and Ds Ag-presenting molecules fail to elicit an antiviral CTL response in susceptible SJL/J and B10.S susceptible mice is not known, but could be the result of their inability to bind and present viral peptides, their inability to positively select TCRs capable of recognizing viral peptides, their failure to be expressed in the correct cellular context during the development of the immune response, or viral interference with the recognition of Ags in the context of K molecules. The present data begin to address the reasons why anti-viral CTL are not elicited in susceptible strains of mice infected with the DAV strain of TMEV. We demonstrate that the TMEV mutant virus DAL*-1 elicits CTL responses restricted by the Ks molecules, but not by Ds, in the genetically defined susceptible H-2s lines. Recognition of viral Ags presented in the context of K is also observed in the resistant B10 (H-2b) strain. Of significant interest is the finding that CTL generated against DAL*-1 recognize cells infected with the wild-type or mutant virus in the context of Kb, Db, and Ks and that CTL generated against wt-DAV also recognize either the wild-type or mutant viruses, but only in the context of Db. Since the only difference between these two viruses is the absence of the protein product encoded by the alternative reading frame, L*, we conclude that this protein interferes with the development of K-restricted CTL during intracranial wt-DAV infection. These findings allow us to address a number of the hypotheses regarding the restricted nature of the class I-directed response against wt-DAV in resistant and susceptible mice. The data clearly demonstrate that Ag-presenting molecules previously associated with nonprotective MHC haplotypes are capable of presenting viral peptides to T cells and that CTL precursors bearing anti-viral TCR are present in sufficient frequency to support an immune response in the adult repertoire of both resistant and susceptible mice. Therefore, the reason why no response ensues against wt-DAV in susceptible mouse strains must lie elsewhere.
The L* protein could influence the class I-restricted immune response by interacting with host cells at a number of different levels. The protein could influence a broad range of virus-host interactions, such as the tropism of the virus, virus growth properties within cells, recruitment of T cells to the site of infection, or host proteins that comprise the Ag-presenting machinery of professional APCs. An increased utilization of the L* AUG by ribosomes and decreased initiation of translation at the polyprotein’s AUG may limit the production of capsid protein in certain cell types, such as microglia, an important reservoir of virus during the persistent CNS infection (32). This decreased synthesis of capsid proteins may prevent the generation of a cytolytic T cell response and thus favor restricted virus expression. Changes in the tropism and growth properties of a virus could influence both the route of sensitization and the amount of Ag encountered by the immune system. As little is known about the factors that govern the development of a CTL response in the CNS, it is difficult to assess precisely how tropism or growth would influence CTL generation in this case.
There are a number of mechanisms by which viruses can modulate or counteract the effect of the protective immune response of their hosts (33). There are several theoretical possibilities by which L* may inhibit the generation of Kb- and Ks-restricted virus-specific CTLs. L* may interfere with the expression of MHC class I molecules in the CNS. Some viral proteins appear to target MHC class I heavy chain proteins, as has been reported for human CMV, adenovirus, murine CMV, and herpes simplex virus (34, 35, 36, 37, 38, 39, 40). Other viral proteins target the peptide transporter functions of TAP molecules (41). Viral mechanisms that involve direct interactions between viral proteins and the MHC molecules present attractive hypotheses, as these physical interactions could take on a locus-specific character, accounting for why the K locus, but not the D locus, effects are noted. However, the fact that CNS-ILs from susceptible mouse strains infected with DAL*-L are able to mount an antiviral CTL response against wt-DA-infected KSSV (Ks, Ds) or PSJLSV (Ks, Ds) suggests that L* (which is synthesized in the wt-DA infection) does not interfere with Ag presentation or expression of MHC proteins. Of interest, during acute intracranial infection mice with wt-DA, the expression of both D- and K-specific loci is up-regulated in the CNS (11). However, this does not rule out the possibility that they are differentially expressed in professional APCs responsible for the activation of CTL precursors.
Other anti-inflammatory properties have been noted that could conceivably account for the activity of L*. The protein may inhibit the activation or proliferation of T cells with specific TCR or that recognize specific loci or alleles, as has been observed during measles infection of dendritic cells, where there is an interference with mitogen-dependent proliferation of PBLs (42). L* may stimulate the secretion of cytokines that, in turn, inhibit the activation or proliferation of H-2Kb- or H-2Ks-restricted virus-specific CTLs. For example, TNF can mediate the death of CD8+ T cells (43), IL-10 may down-regulate the cellular immune response (44, 45), and TGF-ß1 has potent immunosuppressive activity and is up-regulated in human astrocytes following infection (46). By induction of these factors, L* may inhibit the generation or proliferation of class I-restricted virus-specific CTLs. Alternatively, L* may induce the expression of Fas ligand in susceptible mice, which down-regulates virus-specific CTLs, and, in turn, may silence CTLs in vivo (47, 48). Finally, L* could inhibit the generation of virus-specific CTLs by interfering with apoptosis. GDVII virus, which fails to synthesize L*, induces more apoptosis than wt-DA in vitro (49) and in vivo (50). Furthermore, L* has been demonstrated to have anti-apoptotic activity in the macrophage cell line, P388D1, but not in the hamster kidney cell line, BHK. A potential problem with these proposed mechanisms is difficulty understanding how such generalized effects could inhibit K-restricted responses but not D-restricted responses.
A novel aspect of this paper is the differential influence of a viral protein on the generation of K vs D immune responses in the CNS. Deciphering the mechanism by which this protein specifically inhibits K-restricted CTL responses will provide new insights into how viruses persist by subverting immune detection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Moses Rodriguez, Department of Neurology and Immunology, Mayo Clinic, Rochester, MN 55905. E-mail address:
3 Abbreviations used in this paper: DA, Daniel’s strain; TMEV, Theiler’s murine encephalomyelitis virus; CNS, central nervous system; wt, wild type; d.p.i., days postinfection; CNS-ILs, CNS-infiltrating lymphocytes; PFU, plaque-forming unit.
Received for publication June 18, 1998. Accepted for publication August 31, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to activate HIV-1 from latently and acutely infected cells of monocyte/macrophage lineage. J. Immunol. 156:841.[Abstract]
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 P. D. Murray, D. B. McGavern, S. Sathornsumetee, and M. Rodriguez Spontaneous remyelination following extensive demyelination is associated with improved neurological function in a viral model of multiple sclerosis Brain, July 1, 2001; 124(7): 1403 - 1416. [Abstract] [Full Text] [PDF]
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 S. B. Deitz, D. A. Dodd, S. Cooper, P. Parham, and K. Kirkegaard MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A PNAS, November 22, 2000; (2000) 250483097. [Abstract] [Full Text]
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O. van Eyll and T. Michiels Influence of the Theiler's Virus L* Protein on Macrophage Infection, Viral Persistence, and Neurovirulence J. Virol., October 1, 2000; 74(19): 9071 - 9077. [Abstract] [Full Text]
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M. Obuchi, J. Yamamoto, T. Odagiri, M. N. Uddin, H. Iizuka, and Y. Ohara L* Protein of Theiler's Murine Encephalomyelitis Virus Is Required for Virus Growth in a Murine Macrophage-Like Cell Line J. Virol., May 15, 2000; 74(10): 4898 - 4901. [Abstract] [Full Text]
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K. Yamasaki, C. C. Weihl, and R. P. Roos Alternative Translation Initiation of Theiler's Murine Encephalomyelitis Virus J. Virol., October 1, 1999; 73(10): 8519 - 8526. [Abstract] [Full Text] [PDF]
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A. J. Johnson, M. K. Njenga, M. J. Hansen, S. T. Kuhns, L. Chen, M. Rodriguez, and L. R. Pease Prevalent Class I-Restricted T-Cell Response to the Theiler's Virus Epitope Db:VP2121-130 in the Absence of Endogenous CD4 Help, Tumor Necrosis Factor Alpha, Gamma Interferon, Perforin, or Costimulation through CD28 J. Virol., May 1, 1999; 73(5): 3702 - 3708. [Abstract] [Full Text]
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S. B. Deitz, D. A. Dodd, S. Cooper, P. Parham, and K. Kirkegaard MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A PNAS, December 5, 2000; 97(25): 13790 - 13795. [Abstract] [Full Text] [PDF]
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