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Jun 29, 1992 - Sci. 532:238-255. 22. Leo, O., M. Foo, D. H. Sachs, L. E. Samuelson, and J. A. Bluestone. 1987. Identification of a monoclonal antibodyspecific.
Vol. 67, No. 1

JOURNAL OF VIROLOGY, Jan. 1993, p. 75-81

0022-538X/93/010075-07$02.00/0 Copyright 0 1993, American Society for Microbiology

Susceptibility to Measles Virus-Induced Encephalitis in Mice Correlates with Impaired Antigen Presentation to Cytotoxic T Lymphocytes STEFAN NIEWIESK,t* UTE BRINCKMANN, BETTINA BANKAMP, SILVIA SIRAK, UWE G. LIEBERT, AND VOLKER TER MEULEN Institut fuir Virologie und Immunbiologie, Versbacher Strasse 7, 8700 Wiirzburg, Germany Received 29 June 1992/Accepted 15 October 1992

In measles virus (MV) infection in humans, meninigitis and encephalitis are important complications. However, little is known of the pathogenesis of MV encephalitis, in particular about the role of the immune response. We have examined the role of cytotoxic T lymphocytes (CTL) in a mouse model of MV-induced encephalitis. We report here that the resistance of inbred strains of mice to MV-induced encephalitis correlated with the major histocompatibility complex (MHC) haplotype and that only resistant mouse strains mounted an effective CTL response to MV. Mice with low susceptibility to MV infection, such as the BALB/c strain (H-2"), generated CTL, whereas the highly susceptible strains, C3H (H-2k) and C57BV6 (H-2?), revealed very poor CTL responses. MV-induced CTL were usually CD8+, and the generation of these cells was independent of the route of inoculation or the time postinfection. CD4+ T cells were generally only weakly Iytic. The nudeocapsid protein was the major target antigen for CTL in BALB/c mice, although in some experiments the hemagglutinin was also recognized. CTL from C3H and C57BIJ6 mice did not lyse MV-infected target cells. However, targets infected with vaccinia virus recombinants expressing the nucleocapsid protein or bemagglutinin were lysed, but levels of cytotoxicity were still low. Experiments using target cells transfected with single MHC class I genes suggested inefficient antigen presentation of MV proteins by the MHC molecules of the H-2 and H-2? haplotypes.

bility complex (MHC) class I molecules and cannot be recognized by CTL (20). In our present study, we observed clear differences among strains in levels of both morbidity and mortality after intracerebral inoculation of mice with MV. CD8+ CTL were found to be an important component of the immune response to MV, and the ability to generate CTL appears to correlate with the resistance or susceptibility of mice to MV-induced encephalitis. Mice with low susceptibility, such as the BALB/c and DBA/2 strains (H-2d), generated good CTL responses, whereas the highly susceptible strains, C3H, CBA (H-2), and C57BL/6 (H-? ), produced very poor CTL responses. Our experiments suggest that this poor CTL induction is due to an impaired presentation of MV antigen by the H-2 molecule.

Acute measles virus (MV) infection is among the primary causes of infant death in the third world, and sporadic outbreaks of acute measles still occur in industrialized countries despite vaccination (7). MV is also associated with three central nervous system diseases known as postinfectious MV encephalitis (19), MV inclusion body encephalitis and subacute sclerosing panencephalitis (39). In the latter two diseases, MV persistence provides the basis for the development of disease, which is characterized by a restricted expression of MV envelope proteins (2, 6, 23) on the membranes of brain cells, as a result of which the humoral immune response is incapable of influencing the persistent

infection. In other viral infections, clearance of infected tissue is dominated by cytotoxic T lymphocytes (CTL) of the CD8+ phenotype (1, 21, 49). CTL can clear not only infected organs at the periphery but also viral infections in the central nervous system (30, 31). In MV infection, the role of this defense mechanism has been controversial and evidence for CD4+ as well as CD8+ T lymphocytes as CTL has been presented (18, 43, 44). However, regardless of which CTL phenotype is important in MV infection, the following question remains: why is the cellular immune response not able to clear virus from brain cells expressing internal viral proteins? It is possible that MV antigen processing or presentation is impaired and that therefore the generation of CIL is limited, as is seen with other viruses (5, 11, 13, 27). Alternatively, it is also conceivable that the target cells in the central nervous system do not express major histocompati*

MATERIALS AND METHODS Cells. Adherent cells were cultured in Dulbecco modified essential medium-10% fetal calf serum, and suspension cells were cultured in RPMI 1640 containing 10% fetal calf serum, 1% nonessential amino acids, 1% sodium pyruvate, 5 x 10-5 mol of P-mercaptoethanol per liter, 2 mmol of glutamine per liter, 50 IU of penicillin per liter, and 50 ,ug of streptomycin per liter (referred to as RPMI/10). The following target cells were used: P815; A20 (kindly provided by T. Hunig, Wurzburg, Germany); L(MTK-) (kindly provided by E. Serfling, Wurzburg); L929; EL4 (obtained from ECACC, Porton Down, Salisbury, United Kingdom); 145 2cll (antiCD3 hybridoma); YAC1 (kindly provided by A. Schimpl, Wurzburg); L-Kd, L-Dd, and L-Ld (transfectants kindly provided by U. H. Koszinowski, Ulm, Germany); L-Kd (transfected with Kd and ICAMl; kindly provided by J.-P. Abbastado, Paris); and P-Kb and P-Db (transfected with Kb

Corresponding author.

t Present address: Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom. 75

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and Db; kindly provided by H.-J. Wallny, Tubingen, Germany). Virus. The rodent-adapted neurotropic MV strain CAM/ RBH was grown and used as described previously (25). The MV Edmonston strain was grown in Vero cells, and for the stimulation of CD4+ T cells it was purified and UV inactivated as described previously (24). Vaccinia virus recombinants (VVR) expressing single MV genes were constructed and grown as reported previously (3, 4). Influenza A virus of the PR8 strain and the VVR expressing the nucleoprotein of influenza A virus were kindly provided by L. Stitz (Giessen, Germany). Mice. BALB/cJ, DBA/2J, C57BL/6J, and C3H/He mice were purchased from Zentralinstitut fuir Versuchstierzucht (Hannover, Germany), BALB/k, BALB/b, and B1OD2/N mice were purchased from Harlan Olac (Bicester, United Kingdom), and CBA/J Crl BR mice were purchased from WIGA/Charles River (Sulzfeld, Germany); all mice were certified pathogen-free by the respective companies. Every 6 to 8 months, the animals were checked for pathogens by serological examination. Animals were kept in a barrier system with light negative pressure (150 mPA) and a 12-h day (artificial light) and were fed and watered ad libitum. The room temperature (22°C + 2°C) and the humidity (50% 5%) were regulated by air conditioning. Mice between the ages of 6 and 18 weeks were used. Infection of mice. Mice were infected intracerebrally with 5 x 103 PFU of MV (strain CAMIRBH) in a 25-,ul volume or intraperitoneally with 107 PFU of the MV Edmonston strain in a 1-ml volume. Unless otherwise indicated, spleens were removed 1 to 3 months after infection. Intracerebrally infected mice were weighed and checked for clinical symptoms daily. T cells. For the generation of CD8+ T cells, spleen cells from mice were irradiated (300 Gy) and infected with MV (Edmonston strain) at a multiplicity of infection of 1 at 37°C for 2 h. Infected stimulators (3 x 106) were cultivated in an upright 50-ml flask containing 15 ml of RPMI/10 and 1.5 x 107 spleen cells from infected animals. After 7 days, living cells were separated on a percoll gradient (1.083 g/ml) and plated at a density of 106/ml in tissue culture plates. Infected spleen cells (10 /ml) (see above) and 2% rat spleen concanavalin A supernatant were added. For the growth of CD4+ T cells, 3 x 107 to 4 x 107 spleen cells from primed animals were cultured in an upright 50-ml flask in 15 ml of RPMI/10 with 60 ,g of UV-inactivated MV for 3 days. Blast cells were obtained on a Percoll gradient (1.077 g/ml) and cultured in RPMI/10-2% rat spleen concanavalin A supernatant at a density of 6 x 105/ml. For restimulation, 3 x 106 to 4 x 106 T cells were mixed with 3 X 107 to 4 x 107 irradiated spleen cells and 60 p,g of UV-inactivated MV. Unless otherwise stated, T cells were tested for CTL activity 5 days after the second stimulation. Cytotoxic assay. Target cells were infected for 5 h with VVR (multiplicity of infection = 5) or MV (multiplicity of infection = 10), and 106 cells were labeled with 3.7 MBq of Na51CrO4 (DuPont) for 80 min at 37°C and washed twice. Labeled target cells (104) in a volume of 50 pl were added to various numbers of T cells in 100-,ul volumes in U-bottomed microtiter plates. After 5 h (20 h for CD4 T cells) of incubation at 37°C, 75 ,ul of supematant was harvested for counting. The percentage of lysis was calculated as 100 x [(experimental spontaneous release)/(total spontaneous release)]. Whenever extra T cells were available, YAC1 cells were included in the assay as a control for natural killer cells. -

-

TABLE 1. Susceptibility of different inbred strains of mice to MV induced encephalitisa Inbred strain

Mortality (%)

No. animals infected dead/no,

Haplotype

of

0

0/27

H-2d

BALB/c B1OD2/N

10 10

6/60 2/20

H-2d H-2d

C57BL/6 BALB/b

52 57

23/44 12/21

H-2b H-2b

C3H CBA

75 69

55/73 40/58

H-2k H-2k

BALB/k

58

11/19

DBA/2

H-2k

a Mice were infected intracerebrally on day 56 after birth with 5 x 103 PFU of the rodent-adapted MV strain CAM/RBH. Diseased animals showed clinical symptoms such as weight loss, ruffled fur, and cramps. Mice of susceptible strains died between days 8 and 16.

RESULTS

Susceptibility of mice to MV-induced encephalitis is correlated with MHC. Different inbred strains of mice were infected intracerebrally with MV. Susceptible strains exhibited clinical signs of encephalitis and died between days 8 and 16 postinfection (Table 1). BALB/c and DBA/2 mice (H-2Y) demonstrated a low level of susceptibility to MVinduced encephalitis, whereas CBA, C3H (H-2k ), and C57BL/6 (H-2") mice are highly susceptible. The susceptibility of mice to infection could be shown to correlate with the MHC by infecting congenic inbred strains such as BALB/k (H-2) and BALB/b (H-2') (both highly susceptible) and B1OD2/N (H-2d) (poorly susceptible). Neutralizing antibodies did not seem to influence disease, because a significant titer could be demonstrated only from day 20 postinfection (data not shown). These differences in susceptibility to MV-induced encephalitis between MHC haplotypes are statistically significant. H-2" strains are more susceptible than H-2d strains (P < 0.001), and H-2! strains are in turn more susceptible than H-2' strains (P < 0.05; chi-square test with correction for continuity). Generation of MV-specific CD4+ and CD8+ T cells. The correlation between the susceptibility to MV-induced encephalitis in mice and the CTL activity of the different strains was investigated. Following viral infection, spleens from BALB/c mice were removed and stimulated with irradiated spleen cells, which acted as antigen-presenting cells for inactivated MV. After a 7-day period of incubation, CD4+ T cells were obtained. CD8+ T cells could be grown by stimulation with MV-infected spleen cells (data not shown). CD4+ as well as CD8+ T cells could be demonstrated in intracerebrally or intraperitoneally inoculated animals from 1 week after infection and up to 3 months after infection (data not shown). As shown in Fig. 1, CD8+ T cells were able to lyse MV-infected target cells in a short-term assay in an MHC class I-restricted manner, whereas the CD4+ T cells demonstrated a significant lysis in an overnight assay alone. Nucleocapsid protein is the major antigen for MV-specific CTL. The protein specificity of MV-specific BALB/c CD8+ T cells was evaluated with VVR, which express single MV genes (Fig. 2). The major target antigen for MV-induced CTL (MV-CTL) is the nucleocapsid protein, while the hemagglutinin is only a minor antigen for the CTL response and is not always recognized. Targets infected with VVR

VOL. 67, 1993

SUSCEPTIBILITY TO MEASLES VIRUS-INDUCED ENCEPHALITIS Z:T 30:1 *

TABLE 2. Induction of CTL by VVR in BALB/c micea

20:1

E:T

VacWT

MV or VVR used to infect P815 cells

* Nv

60

MV

Lysis

40

20

VacN VacH VacF VacM VacP

-

-

n

U

I

CD8

I __ T cells CD4 T

I cells

CD4

T

5 h

cells

20 h

FIG. 1. Cytotoxicity of MV-induced T cells. The cytolytic capacity of MV-specific CD4 and CD8 T cells was tested in 5- and 20-h chromium release assays. Targets for CD8 T cells were P815 (H-2", MHC class I), and those for CD4 T cells were A20 (H-2d, MHC classes I and II). For the 5-h chromium release assay, MV-infected targets were used; for the 20-h assay, 10 ,ug of UV-inactivated MV per well was added.

expressing the matrix protein, fusion protein, or phosphoprotein were not lysed more than control targets. To evaluate the importance of the different MV proteins for CTL induction, BALB/c mice were infected with VVR and checked for generation of CTL. Only the VVR expressing the nucleocapsid protein (VacN) induced a CT'L response to MV (Table 2), which confirmed the importance of nucleocapsid protein as a target antigen. The VVR expressing hemagglutinin and fusion protein (VacH and VacF) induced CTL, which could be stimulated by MV-infected stimulator cells. These CTL lysed only weakly with VacH- or VacFinfected target cells and did not kill MV-infected ones at all. VVR expressing matrix protein and phosphoprotein (VacM and VacP) did not induce an MV-specific CTL response. Similar CTL data were obtained with DBA/2 mice (H-2d), which share the low level of susceptibility to MV-induced encephalitis with BALB/c mice. C3H mice show an impaired CTL response towards MV. so E:T

U30:1

s0

X15:1

Lysis 40

20

0

VacWT

NV

VacN

VacH

VacP

VacK

77

VacP

FIG. 2. Recognition of MV proteins by BALB/c CTL. The protein specificity of MV-specific BALB/c CIL was investigated by infecting P815 cells with VVR expressing the nucleocapsid (VacN), hemagglutinin (VacH), fusion protein (VacF), matrix protein (VacM), or phosphoprotein (VacP). Cells infected with wild-type vaccinia virus (VacWT) served as the control. Effector/target cell (E:T) ratios of 30:1 and 15:1 were used. No YAC1 lysis was ever observed.

CTL activity in BALB/c mice infected withb: VacN

VacH

VacF

VacM

VacP

+++ +++ -

-

-

-

-

+ -

+

-

-

a BALB/c mice were infected intraperitoneally three times at weekly intervals with 1 x 106 to 3 x 106 PFU of the respective VVR. Four weeks later, spleens were removed and stimulated with MV-infected stimulators. CTL activity was tested on P815 cells infected with the respective VVR or MV. ++ +, 45 to 60% lysis of target cells; +, 20 to 30% lysis; -, no cytolytic response above background.

MV-CiT of C3H mice (H-2k), which are highly susceptible to MV-induced encephalitis, did not recognize MV-infected L929 or L(MTK-) cells (data not shown). To determine whether these mice generated CTL at all, a hybridoma cell line expressing anti-CD3 antibodies was used as a target (22). In our laboratory, this hybridoma is lysed in an antigenindependent manner by CD8+ T cells in a short-term assay and by CD4+ T cells in a long-term one. This hybridoma was lysed efficiently (data not shown). Since the target cell lines L929 and L(MTK-) were originally obtained from C3H/An mice, fibroblasts and macrophages from C3H/He mice were tested to exclude the possibility that lack of recognition might have been due to a substrain-specific difference. C3H/He CTL did not lyse C3H/He cells either (data not

shown). In order to explain this finding we investigated the ability of the target cells to function in a cytotoxic assay. According to FACScan analysis, target cells expressed MHC class I molecules in amounts comparable to those of spleen cells and were infectable by MV (by immunofluorescence). The cells served as targets in a mixed lymphocyte reaction (data not shown). The C3H mice were checked for their ability to produce a normal CTL response. CTL were generated in a mixed lymphocyte reaction and following influenza A virus infection (data not shown). C3H CTL recognize VacN-infected target cells but not MV-infected cells. MV-CTL not recognizing MV-infected cells were tested on targets infected with VVR. Interestingly, only VacN-infected target cells were recognized (Fig. 3). This level of lysis was quite low compared with that obtained with BALB/c CTL (Fig. 2). The antigen-specific recognition could be enhanced by using L cells transfected with ICAM1 (Fig. 3). To exclude the possibility that inefficient CTL induction was due to poor viral replication in infected mice, C3H mice were infected with VacN, since vaccinia virus is known to replicate vigorously in mice. Spleen cells from these mice were stimulated with MVinfected stimulator cells. After two cycles of stimulation, there was still a significant CTL response towards wild-type vaccinia virus (VacWT). These CTL did not lyse MVinfected targets (Fig. 4); however, VacN-infected targets were lysed weakly. These results indicate that even a good expression of nucleocapsid protein results in a poor CTL response in C3H mice. Impaired CTL function is due to antigen presentation. We showed above that susceptibility to MV-induced encephalitis correlates with H-2 haplotype. We now wished to deter-

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NIEWIESK ET AL.

78

40 -

so

30 -X

Targets infected with

Lysis

*

Lysis

E2

ted with

30 -

*

VacWT

ca xv

VacWT

*MV

20-

Targets infec-

40 -

20 -

VacN

10 L

929

L 929

+ ICAI

1

0P

1:T 30:1

FIG. 3. Recognition of nucleocapsid protein by MV-induced C3H CTL is enhanced by ICAML. MV-induced C3H CITL were tested for their cytotoxic activity on L929 cells compared with activity on L929 cells transfected with ICAML. Targets were infected with VacWT, MV, and VacN.

mine whether the inefficient generation of MV-specific CTL in C3H mice is due to poor antigen presentation by MHC class I molecules per se or possibly to defective processing of antigen, since several components of the antigen processing machinery are now known to be encoded in the MHC region of the genome. To address differences in the genetic background, BALB/c and C3H mice were crossbred and the resulting F1 generation was used as a source of MV-CTL and of fibroblasts as target cells. MV-CTL taken from F1 animals lysed only MV-infected P815 (H-2) cells, not L929 (H-2) cells (Fig. 5). F1 fibroblasts were lysed by BALB/c CTL but not by C3H CTL (data not shown). These experiments suggested that impaired antigen presentation rather than defective antigen processing resulted in an inefficient generation of CTL. This view was confirmed by the experimental finding that MV-infected L cells (H-2k) transfected with Ld were readily lysed by BALB/c CTL (Fig. 6). Impaired CTL are seen in other susceptible mouse strains as well. If the impaired antigen presentation played a major role in the susceptibility to MV-induced encephalitis, other susceptible strains should show the same poor CTL response. To test this hypothesis, CBA (H-2'y) and C57BL/6 (H-2b)

815

L 929

3:T 70:1

FIG. 5. F1 animals (BALB/c x C3H) generate only H-2d-restricted MV-CTL. Spleen cells from MV-infected F1 animals (BALB/c x C3H) were stimulated with MV-infected stimulators. CTL activity was tested after one round of stimulation to avoid selection due to in vitro culture. Target cells expressing the parental haplotypes (P815 [H-2d] and L929 [H-2"]) were used.

mice were tested. MV-CTL in CBA mice showed the same poor response as C3H CTL (data not shown). MV-CTL from C57BL/6 mice also did not lyse target cells infected with MV but recognized targets infected with VacH weakly (Fig. 7). This phenomenon could be shown to be independent of the target cell antigen processing machinery with transfected P815 cells. P815-Db cells infected with VacH were lysed weakly by MV-CTL, whereas MV was not recogized (Fig. 7). The lysis was restricted to the Db molecule, since P815-Kb cells were not recognized. The impaired CTL response is also seen in other highly susceptible mouse strains that succumb to MV-induced encephalitis and seems to be a more general mechanism of escape from CTL surveillance by MV.

DISCUSSION The susceptibility of inbred strains of mice to MV-induced encephalitis is shown to be correlated strongly with MHC haplotype (H-21 > H-2" > H-2d). MV infection of BALB/c mice results in a strong CD8+ CIL response to MV, which is mainly directed towards the nucleocapsid protein. Highly susceptible mouse strains (C3H, CBA, and C57BL/6) gener-

Targets inected with * VacWT * MV

*

vacT

Nv

30

e] VeoN rysis Lysis

20

-

10

VacN-CTL

KV-CTL 0 ±

E:T 30:1

FIG. 4. Induction of C3H CTL with VacN compared with induction of CTL with MV. C3H mice were infected with VacN and MV. After two cycles of stimulation with MV-infected stimulators the induction of a CTL response was compared among L929 cells infected with VacWT, MV, and VacN. VacN-induced CTL are shown on the left, and MV-induced ones are shown on the right.

D-d

x-d

I L-d

FIG. 6. Ld is the restriction element for BALB/c CTL. L cells transfected with the three MHC class I molecules of the H-2d haplotype, Kd, Dd, and Ld, were used as target cells for BALB/c CTL. Only L cells transfected with Ld were recognized. CTL were tested after one round of stimulation with an effector/target (E:T) ratio of 50:1.

SUSCEPTIBILITY TO MEASLES VIRUS-INDUCED ENCEPHALITIS

VOL. 67, 1993

40

Targets infected with 30 *

Lysis

*

VacWT NV

0

VacH

*

VacP

20

Vacl *

10 *

ZL4

VacN Vacp

P815-Db

FIG. 7. Recognition of VacH in C57BL/6 mice is restricted by a single MHC class I molecule. The recognition of MV proteins by C57BU6 CTL was tested on ELA cells infected with MV or the various VVR (a), and the allele specificity was confirmed with P815 cells transfected with the Db gene (b) (effector/target ratio, 30:1). P815 cells transfected with K were never recognized (data not shown).

ate a poor CTL response. Unexpectedly, their CTL do not recognize target cells infected with MV but lyse weakly targets infected with VacN (H-21) or VacH (H-21). As shown by experiments with transfectants, the poor CTL response is due to antigen presentation by the MHC molecules. In viral infections, virus-specific CTL play a major role in the clearance of infected tissue and in recovery from disease. In most viral systems, the CTL response is MHC class I restricted (41), but MHC class II restriction has also been reported (26). In concordance with the findings by van Binnendijk (44) and experimental evidence from the influenza A virus system (28), the generation of MV-specific CD4+ or CD8+ T cells could be shown to be determined by the antigen form (infectious virus or soluble viral protein). The CTFL response is mainly directed against a protein forming the nucleocapsid, as has been shown for respiratory syncytial virus (32), a member of the family Paramyxoviridae, as well as for influenza A virus (14, 41) and vesicular stomatitis virus (33). An impaired CTL function has been demonstrated in other viral systems. Adenovirus has been shown to interfere with the processing of MHC class I mRNA and the glycosylation of MHC class I molecules (42). As a consequence, infected cells do not express MHC class I molecules (5) and cannot be recognized by influenza A virus-specific CGL (50). In simian virus 40-transformed fibroblasts, reduced MHC expression is correlated with in vivo resistance to immune surveillance (13). Epstein-Barr virus downregulates MHC expression (27) and the number of the adhesion molecules ICAM1 and LFA3 on the cell surface (15), which resulted in escape from CTL surveillance. But these observations seem to depend on the virus substrain and the experimental conditions, because contradictory findings have been reported (16). An interesting observation of inefficient antigen processing was seen with a VVR expressing the hemagglutinin of influenza A virus (40). The lack of recognition by CTL was believed to be due to either viral proteases, which are expressed late in the vaccinia virus replication cycle, or the stability of the hemagglutinin protein. According to the N end rule, the amino acid at the amino terminus directs the turnover rate of a protein. A VVR expressing a hemagglutinin modified at the amino terminus under the control of an early viral promoter was recognized efficiently by CTL. A very specific impairment of antigen recognition of CTL could be shown in the murine cytomegalovirus system (11).

79

The immunodominant very early protein pp89 is recognized in the very early and late phases of protein expression but not in the early phase despite its expression during the whole replication cycle. The CTL response towards other viral proteins is not impaired, and the MHC class I expression is normal. An undefined mechanism interferes with antigen processing or presentation. To date, there is no evidence that antigen processing is a nonrandom (e.g., antigen-specific) process; it can be mimicked chemically by proteolysis (37) or enzymatically with proteases (46). The putative peptide transporter molecules (8, 38) seem to be able to transport a wide variety of antigenically unrelated peptides into the endoplasmic reticulum (29). After transfection of a certain MHC class I molecule into mouse cell lines of different inbred strains, the appropriate peptide was generated in each cell line (12). Also, mice transgenic for a human MHC class I molecule produce and transport, after influenza virus infection, the same peptide as human cells (47). The ability of the single Ld molecule transfected into L cells (H-2k) to present antigen to BALB/c CTL and the nonrecognition of the P815-Db cells by C57BLV6 CTL clearly argue against a defective antigen processing or transport of MV proteins. While antigen processing and transport appear to be nonspecific, a large amount of available data indicates that the binding and presentation of a certain epitope (peptide) is entirely dependent on the MHC class I molecules. MHC class I molecules choose the appropriate nonameric peptide from a mixture of peptides of different sizes (35, 36), independently of cell type and species (12, 47). In.the murine cytomegalovirus system, it could be shown that a nonameric peptide expressed as a hybrid protein (with an unrelated viral protein) by a WR was recognized by CTL (10). The specific interaction between the peptide and the MHC class I molecule was explained by the finding that peptides binding to a certain allele share a simple binding motif (34). Crucial for the binding are two conserved amino acids at a certain position; the others influence the binding to a much lesser extent. Despite the fact that MHC class I molecules are responsible for the nonrecognition of MV-infected targets by CTL, the following question remains: why is MV able to induce a proliferation of T cells which lyse VacN- or VacH-infected cells but not MV-infected ones? An explanation might be that the affinity of the immunodominant MV-derived peptide is very low and that therefore only few MHC-peptide complexes are expressed at the cell surface. The number of such complexes required for CD4+ T-cell proliferation was shown to be small (a few hundred) (17). The number of MHC class I-peptide complexes on MV-infected cells might be large enough to induce a low level of CTL proliferation in C3H and C57BU6 mice but too small to induce lysis. VVRinfected cells express more MV protein than do MV-infected cells (unpublished observation). As a consequence, at a higher peptide concentration enough occupied MHC class I molecules might be exhibited at the cell surface to induce a low level of lysis. The number of MHC class I molecules that have to be occupied by a certain peptide in order to induce a lysis is not totally clarified. Although a few thousand MHC class I-peptide complexes per cell have been found (45), only a few hundred seem to be sufficient to induce lysis (9, 48). It is possible that the number of MHC class I-peptide complexes needed for lysis varies on the basis of the cell type. Further studies will have to investigate the affinity between the MV-derived peptides and the MHC class I molecules in question.

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Our data show that, in the MV-mouse model, MHC class I-restricted CTL play a prominent role in MV infection and suggest that the susceptibility to MV-induced encephalitis is due to inefficient CTL induction caused by an impaired

J. VIROL.

antigen presentation.

17.

ACKNOWLEDGMENTS We thank Lee Dunster and Charles R. M. Bangham for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium fir Forschung und Technologie. Stefan Niewiesk was supported by a grant from the Studienstiftung des Deutschen Volkes.

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19. 20.

1.

2.

3. 4.

5. 6. 7. 8.

9.

10.

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