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Journal of General Virology (1998), 79, 2583–2591. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Recognition of measles virus-infected cells by CD8MT cells depends on the H-2 molecule Claudia Neumeister and Stefan Niewiesk Institute of Virology and Immunobiology, University of Wu$ rzburg, Versbacher Str. 7, 97078 Wu$ rzburg, Germany H-2d mice are resistant to measles virus-induced encephalitis (MVE) and develop Ld-restricted CD8M T cells which lyse target cells infected with measles virus or with a vaccinia virus recombinant expressing the nucleocapsid protein of measles virus (vvN). In contrast, H-2k mice are susceptible to MVE and generate CD8MT cells which lyse target cells infected with vvN, but not those infected with MV. We were able to demonstrate that this difference is not due to a defect in the antigen processing machinery, but that Kk molecules require 100-fold more peptide to sensitize target cells for lysis by CTL. vvN replicates well in target cells and therefore enhances the level Introduction Infection with measles virus (MV) leads to acute measles which is characterized by fever and the typical rash. Usually, the disease is overcome within two weeks, but often complications such as encephalitis, pneumonia and diarrhoea, as well as secondary infections due to the marked immune suppressive effect of MV arise (for a review, see Katz, 1995). During infection, B and T cell responses arise. Observations in patients with agammaglobulinaemia indicate that B cell responses against MV are dispensable (Bruton, 1953 ; Good & Zak, 1956). In contrast, patients with defects in the cellular immune response are not able to clear the virus (Nahmias et al., 1967). Although CD4+ as well as CD8+ T cells are generated during acute infection, their contribution to overcoming infection remains to be elucidated. The frequency of CD4+ T cells and their highly lytic potential has been emphasized (Long & Jacobson, 1989). MV is lymphotropic and, at least in tissue culture, very cytopathic. Therefore, the generation of CD8+ T cells with MV-infected stimulator cells has been problematic, although with recent technical advances, a number Author for correspondence : Stefan Niewiesk. Fax 49 931 201 3934. e-mail viro025!mail.uni-wuerzburg.de 0001-5677 # 1998 SGM of epitope peptide available for CTL recognition. In contrast, MV infection is abortive in mouse cells and low levels of epitope peptide are produced. As Ld requires 100-fold less peptide than Kk to sensitize target cells for lysis, the low level of epitope peptide is enough to induce lysis by CD8MT cells, whereas for recognition via Kk, increased synthesis of protein is required. We propose that the differences in peptide binding between the two H-2 molecules will have consequences for the kinetics of the generation of CD8M T cells as well as the absolute numbers of CD8MT cells generated. of reports have described CD8+ T cell responses (Nanan et al., 1995 ; Uytdehaag et al., 1994). However, the contribution of CD4+ versus CD8+ T cells in overcoming disease is not yet known. Indirect evidence for the importance of CD8+ T cells is the fact that a high frequency of CD8+ T cells has been found in children recovering from acute measles (van Binnendijk et al., 1990). In the mouse model of MV-induced encephalitis (MVE), susceptibility and resistance of various inbred and H-2 congenic mouse strains depend on their MHC haplotype (Niewiesk et al., 1993). In resistant H-2d mice (like BALB}c), the CD8+ T cell response is restricted by the Ld molecule and in susceptible H-2k mice (like C3H) this response is restricted by the Kk molecule. BALB}c mice generate good CD8+ T cell responses which result in lysis of target cells infected with MV as well as cells infected with a vaccinia virus recombinant expressing the nucleocapsid (N) protein of MV (vvN). In contrast, infection of C3H mice with MV and stimulation of spleen cells with MV-infected stimulators give CD8+ T cells restricted by Kk which do not recognize MV-infected target cells, but readily lyse target cells infected with vvN (Niewiesk et al., 1993). CD8+ T cells from both mouse strains recognize target cells pulsed with the respective peptide epitopes [aa 281–289 on the N protein for Ld (Beauverger et al., 1993) ; aa 52–59 and aa 81–88 for Kk (Beauverger et al., 1994)]. In this Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 28 Apr 2017 21:59:54 CFID C. Neumeister and S. Niewiesk paper, we demonstrate that recognition of MV via Kkrestricted CTL can be achieved if target cells allow replication, thus increasing the number of N protein molecules for processing. In comparison to Ld, Kk requires 100-fold more of its epitope peptide to sensitize target cells for lysis by CD8+ T cells. Methods + Mice. Mice were bought from Harlan Winkelmann (Borchem) and were specific pathogen-free (company specification). Every 6–8 months, the animals were checked for pathogens by serological examination. Animals were kept in a barrier system with light negative pressure (150 Pa) and a 12 h day (artificial light), and were fed and watered ad libitum. The room temperature (24³2 °C) and the humidity (70³5 %) were regulated by air conditioning. Mice were used between the ages of 6–18 weeks. + Infection of mice. For the generation of CTL, mice were infected intraperitoneally (i.p.) with 8¬10' p.f.u. MV (Edmonston strain) or 2¬10' p.f.u. of a vaccinia virus recombinant (vvR) expressing the nucleoprotein (NP) of influenza A virus strain PR8. + Cells, antibodies and peptides. RMA-S cells that were stably transfected with H2-Kk were obtained from Gu$ nther Ha$ mmerling, Heidelberg, Germany and cells that were stably transfected with Ld were from Hans-Georg Rammensee, Tu$ bingen, Germany. P815 cells that were stably transfected with H2-Kk were from Denis Gerlier and Chantale Rabourdin-Combe, Lyon, France, and C1R-Kk cells were from Peter Cresswell, Yale, USA. All cell lines were cultured in RPMI}10 % FCS. L929, L-Ld and NS20Y cells were cultured in MEM}10 % FCS. The MAb 11-4.1, which is specific for the Kk molecule, was purchased from Pharmingen. Hybridoma F227, produced at the Institute of Virology and Immunobiology, secretes MV N protein-specific antibody (Liebert et al., 1990). Peptides LDRLVRLI (from MV Edmonston strain N protein aa 52–59, Kk-restricted), VESPGQLI (MV N protein aa 81–88, Kk-restricted), YPALGLHEF (MV N protein aa 281–289, Ld-restricted) and SDYEGRLI (influenza A virus NP aa 50–57, Kk-restricted) were synthesized and purified (" 90 %) by Jerini Biotools. + Generation and culture of CTL. For the generation of CD8+ T cells, spleen cells from mice were irradiated (300 Gy) and either infected with MV (Edmonston strain) at an m.o.i. of 1 or incubated with peptide (50 µg}10( cells) at 37 °C for 2 h. Infected stimulators (3¬10') were cultivated in an upright 50 ml flask containing 15 ml RPMI}10 % FCS and 1±5¬10( 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 10' cells}ml in tissue culture plates. For restimulation, spleen cells (10' cells}ml) infected with virus or incubated with peptide (see above) and 2 % rat spleen Concanavalin A supernatant were added. T cells were tested for their cytolytic activity 5 days after restimulation. + Cytotoxic assay. Cells (10') were labelled with 3±7 MBq Na&"CrO % (DuPont) for 80 min at 37 °C and washed twice. If used, peptide was added during the labelling step (50 µg}10' cells). Labelled target cells (10%) in a volume of 50 µl were added to various numbers of T cells in 100 µl volumes in U-bottomed microtitre plates. After 5 h incubation at 37 °C, 75 µl supernatant was harvested and counted. The percentage lysis was calculated as follows : 100¬[(experimental®spontaneous release)}(total®spontaneous release)]. If not otherwise indicated, target CFIE cells were infected with MV strain Edmonston (m.o.i. of 10) overnight and with vvR expressing the NP of influenza A virus strain PR8 (vvNP) or the N protein of MV strain Edmonston (vvN) (m.o.i. of 5) for 5 h. + Recombinant vaccinia viruses. Recombinant vaccinia viruses (vvR) were made by transfecting vaccinia virus Copenhagen DNA and the shuttle vectors psc11.30R.2 or psc11L (Elliott et al., 1995) containing the various epitopes into Tk-143 cells infected with vaccinia virus ts7 as described previously (Bankamp et al., 1991). Vaccinia virus was propagated in Tk-143 cells and isolated by standard procedures. Plasmids psc11.30R.2 and psc11L both contain a Kozak sequence and were kindly provided by Tim Elliott, Oxford, UK. In addition, psc11L contains the HA-1 leader sequence to ensure peptide transport independent of the antigen presentation transporter (TAP). Oligonucleotides encoding either LDRLVRLI (from MV Edmonston strain N protein aa 52–59), VESPGQLI (MV N protein aa 81–88) or YPALGLHEF (MV N protein aa 281–289) were constructed with a 5« end NcoI site (encoding Met at position 1) and a BglII site at the 3« end. These were cloned into NcoI}BglII-cut psc11.30R.2 or psc11L plasmids and the sequences confirmed by dideoxynucleotide sequencing. vvN and vvNP have been described previously (Niewiesk et al., 1993). + Competition and sensitization assay. The &"Cr-release assay was used to assess peptide competition as previously described (Cao et al., 1996). RMA-S-Kk target cells (10%) were incubated with various concentrations of competitor peptide for 15 min at room temperature. Target peptide at a concentration of 10-) M was added for another 15 min at room temperature. For the sensitization assay, target peptide, but no competitor peptide, was added in various concentrations. CTL specific for the target peptide were then added at an effector to target (E : T) ratio of 5 : 1 for the competition assay and 20 : 1 for the sensitization assay in a final volume of 100 µl. After 5 h incubation at 37 °C, 50 µl supernatant was harvested and counted. + Western blotting and immune precipitation. Western blotting and immune precipitation were done as previously described (Reich et al., 1992 ; Niewiesk et al., 1997). Briefly, L929 cells were infected with MV Edmonston strain (m.o.i. of 10) overnight and with vvN (m.o.i. of 5) for 5 h. For Western blotting, cell lysates were separated on SDS–PAGE, blotted onto nylon membranes and stained with a human hyperimmune serum. For immune precipitation, proteins from cells labelled with [$&S]methionine and -[$&S]cysteine (Pro-mix ; Amersham) were precipitated using a human hyperimmune serum and separated by SDS–PAGE. + Flow cytometry. Cells were fixed with 3±7 % formaldehyde for 5 min, permeabilized with a Triton X-100 solution (0±25 % in PBS) for 3 min and stained with the N protein-specific antibody F227 and a donkey antimouse serum labelled with FITC (Dianova). Cells were subsequently analysed by flow cytometry (Facscan ; Becton Dickinson). Results General influence on antigen processing ? We have verified that MV-infected H-2k mice generate CD8+ T cells which lyse vvN-infected target cells, but not MV-infected cells (Fig. 2 a). Some large DNA viruses interfere by various mechanisms with the recognition of antigen irrespective of its origin. Adenovirus, for example, inhibits CTL recognition of influenza A virus after co-infection of target cells (Yewdell et al., 1988). We tested whether MV was able to inhibit lysis by influenza A virus-specific CTL after coinfection of target cells with influenza A virus and MV. MV Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 28 Apr 2017 21:59:54 CTL in MV encephalitis (a) (b) Fig. 1. Kk-expression of L929 cells infected with MV or treated with IFN-γ. (a) Cell surface expression of the Kk molecule on L929 cells was determined by flow cytometry. Cells were infected with MV (m.o.i. of 10) for 18 h, treated with 100 µg/ml IFN-γ for 48 h or left untreated and were stained with Kk-specific antibody 11-4.1 or an isotype control. (b) To test whether the Kk molecule was retained during MV infection, peptide 52–59 was added to uninfected cells and cells were infected with vvNP or MV (Edmonston strain). Recognition of target cells was tested using CD8+ T cells specific for the 52–59 peptide. The same results were obtained with peptide 81–88. +, E : T ratio 1 : 1 ; , E : T ratio 30 : 1. infection did not influence the activity of influenza A virusspecific CTL (data not shown) and co-infection did not lead to enhanced recognition of MV by CTL (data not shown). Sometimes, faulty recognition of target cells due to an inhibitory effect of a virus on antigen processing can be overcome by the addition of IFN-γ. This cytokine changes the recruitment of different subunits of the proteasome and enhances the level of expression of the TAP transporters and the MHC class I and II molecules (Hengel et al., 1994 ; Sibille et al., 1992). Incubation of MV-infected cells with IFN-γ led to an enhanced level of Kk expression (Fig. 1 a), but not to recognition of MV-infected cells by CD8+ T cells (data not shown). We investigated whether the Kk molecule might be retained in the cell as a result of MV infection. Staining with the Kk-specific MAb 11-4.1 and subsequent analysis by flow cytometry indicated that MV-infected cells expressed more Kk than non-infected cells (Fig. 1 a). In addition, MV-infected cells could be rendered susceptible to lysis by addition of peptides 52–59 or 81–88 (Fig. 1 b) indicating that there is no retention of the Kk molecule. These data confirm that the nonrecognition of MV-infected target cells by Kk-restricted CD8+ T cells is specific for MV and not an overall effect on the cellular machinery. Impairment of antigen processing or presentation ? The lack of recognition could in principle be due to a defect in antigen presentation (due to Ld or Kk) or processing (due to various genes). In BALB}c mice, which are resistant to MVE, Ld-restricted CD8+ T cells are generated which lyse MV- infected P815 cells well (Niewiesk et al., 1993). To test whether the generation of CD8+ T cells was dependent on the genes of the BALB}c mouse background, we assayed CTL activity in BaC3F1 and BALB}k mice. In BaC3F1 mice, every molecule is encoded by an allele of each parent. In BALB}k mice (congenic to BALB}c), H-2 is of the k haplotype whereas all other molecules are imprinted by BALB}c genes. Interestingly enough, the CTL response seemed to be governed by the H-2 molecule only. Kk-restricted CD8+ T cells (either derived from BALB}k or BaC3F1 mice) lysed target cells (L929) infected with vvN, but did not recognize MV-infected target cells (data not shown). In contrast, Ld-restricted CTL (also derived from BaC3F1 mice) lysed MV-infected cells (P815) (Fig. 2 b). To investigate the importance of cell type on CTL recognition, we tested Ld-restricted CTL (from BALB}c mice) on L929 cells (H-2k) transfected with Ld (L-Ld) (Fig. 2 b). These cells were lysed after infection with MV, although never quite as efficiently as P815 cells. In the reverse experiment, CD8+ T cells from C3H mice specific for either Kk-restricted epitope were tested on P815 cells transfected with Kk. Infection with vvN but not MV led to recognition of target cells (Fig. 2 c). These data indicate that the non-recognition of target cells is either due to differences in presentation by Ld and Kk or differences in processing of the respective epitopes. No defect in antigen processing As the level of protein content might differ between vvNand MV-infected target cells, we estimated the level of protein Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 28 Apr 2017 21:59:54 CFIF C. Neumeister and S. Niewiesk be defined as the cause of the non-recognition of MV-infected cells. (a) Protein synthesis is important for recognition by CD8M T cells So far the only difference in antigen recognition was the difference in lysis between MV- and vvN-infected cells. A major difference between vaccinia virus and MV is that vaccinia virus replicates well in murine cells whereas MV replicates abortively. Although MV is taken up by L929 cells and its presence can be detected by Western blotting, immune precipitation showed that N was produced in large quantities only in vaccinia virus-infected cells (Fig. 3 b). To investigate the effect of MV replication on lysis by CD8+ T cells, we used the semi-permissive mouse neuroblastoma cell line NS20Y and a fully permissive human B cell line (C1R) transfected with the Kk molecule. After infection of NS20Y cells, no more than 15 % of cells expressed the N protein (as measured by flow cytometry) and in persistently infected NS20Y, no more than 45 % were N protein-positive (data not shown). The extent of N protein expression correlated with lysis seen by CD8+ T cells (Fig. 5 a). Similarly, the percentage of N protein-positive C1R-Kk cells after infection correlated with lysis (Fig. 5 b). These data showed that a higher amount of N protein resulted in lysis. (b) (c) Good binding of peptides to the Kk molecule Fig. 2. Expression of the Kk or Ld molecule rather than the nature of the target cell determines recognition of MV-infected cells. (a) CD8+ T cells from C3H mice infected with MV were tested on L929 cells infected with vvN (+), MV (_) and vvNP ( ). (b) CD8+ T cells from BALB/c mice were tested on cells expressing the Ld molecule (P815, E : T ratio 30 : 1 ; L929-Ld, E : T ratio 6 : 1). +, vvNP ; 8, MV ; , vvN. (c) CD8+ T cells from C3H mice were tested on cells expressing the Kk molecule (L929, E : T ratio 30 : 1 ; P815-Kk, E : T ratio 6 : 1). +, vvNP ; 8, MV ; , vvN. present in infected cells. After infection under standard conditions, protein was present in both cell samples (as seen by Western blotting ; Fig. 3 a). Therefore, both samples should be recognized by Kk- and Ld-restricted CD8+ T cells. As Kkrestricted epitopes are not recognized, antigen processing of these epitopes might be impaired. We tried to address specific steps in the antigen processing machinery by expressing the full-length N protein, the Kk-restricted epitopes alone (to circumvent faulty protein degradation by the proteasome), and the epitopes with the HA-1 signal peptide (to circumvent putatively inefficient transport by the TAP molecules) in vvR (Fig. 4). Kk-restricted CD8+ T cells recognized all constructs equally well so that no specific step in antigen processing could CFIG If more protein (and therefore more peptide epitope) is needed to sensitize target cells for lysis then this indicates a low binding affinity of the epitope peptides to the Kk molecule. We tested the ability of MV-derived epitope peptides (52–59 and 81–88) to sensitize RMA-S cells transfected with Kk for lysis by CD8+ T cells in a competition assay (Feltkamp et al., 1995). RMA-S cells are not able to transport peptide from the cytosol to the endoplasmic reticulum due to expression of a truncated TAP 2 molecule (Powis et al., 1992). Therefore, addition of peptide from the outside is the only possibility for stabilization and expression of MHC class I molecules on the cell surface. As a positive control, we chose a Kk-restricted peptide epitope from the NP of influenza A virus (aa 50–57) (Brown et al., 1994). In competition experiments, the two peptides 52–59 and 81–88, derived from MV N protein, bound at least as well as the peptide derived from influenza A virus NP (Fig. 6). In a different approach, we tested the off-rate (i.e. the rate of dissociation of the epitope peptide from the H-2 molecule) of MV-derived peptides. Twenty hours after peptide-pulsing, target cells were compared to freshly pulsed targets in a &"Cr-release assay in order to estimate the stability of peptide binding to Kk. Target cells pulsed with MV peptides 52–59 and 81–88 were killed more efficiently than cells loaded with influenza A virus peptide 50–57 (data not shown). This indicates a low off-rate and good binding of MV epitope peptides to the Kk molecule. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 28 Apr 2017 21:59:54 CTL in MV encephalitis (a) (b) 1 2 3 1 2 3 4 60 kDa 56 kDa 60 kDa Fig. 3. MV N protein is present in MV-infected cells, but protein synthesis occurs after infection with vvN. (a) To test for the presence of MV N protein in L929 cells after infection by Western blotting, cell lysates from uninfected cells (lane 1), cells infected with vvN (lane 2) and MV (lane 3) were blotted onto nylon membranes and stained with a human serum containing MV-specific antibodies. (b) To test for synthesis of MV N protein by immune precipitation, labelled cell lysates of uninfected cells (lane 1), cells infected with vvNP (from influenza A virus, 56 kDa ; lane 2), vvN (from MV, 60 kDa ; lane 3) or MV (lane 4) were precipitated with a human serum containing antibodies specific for MV and influenza A virus and separated by PAGE. Discussion Fig. 4. No difference in recognition of vvR expressing the full-length N protein, the peptide epitope or the epitope bound to the HA-1 leader peptide. L929 cells were infected with vvNP, MV, vvN (expressing the fulllength N protein), vv52–59 (expressing the 52–59 peptide) and vvL52–59 (expressing the 52–59 peptide with a leader peptide). Recognition of the different viruses was determined with CTL specific for the 52–59 peptide. The same set of experiments was performed with the 81–88 peptide and the same results were obtained. +, E : T ratio 3 : 1 ; 8, E : T ratio 30 : 1. Kk requires more peptide than Ld in a sensitization assay As the binding of Kk-restricted peptides seemed to be comparable to that of the influenza A virus epitope (which is well recognized after infection), we compared the ability of the Kk- and Ld-restricted peptides to sensitize their respective target cells for lysis. A 100-fold more Kk-restricted peptide was required to sensitize L929 cells for lysis than Ld-restricted peptide was required for P815 cells (Fig. 7 a). In order to ensure that this effect was not due to differences in cell type, we compared sensitization efficiencies on RMA-S cells transfected with either Kk or Ld. The results were the same and could be reproduced with different T cell lines (Fig. 7 b). In most virus infections, CD8+ T cells play a dominant role in clearing virus infection and protecting against disease (Koszinowski et al., 1991 ; Kagi & Hengartner, 1996). For MVE, it has been reported that, in the resistant BALB}c mouse, depletion of CD4+ T cells leads to breakdown of resistance (Finke & Liebert, 1994). In the light of recent findings, these data have to be re-evaluated. Firstly, it has been shown that without CD4+ T cell help, the clonal burst of CD8+ T cells specific for poorly replicating viruses (like MV in the mouse) is greatly diminished (Zimmermann et al., 1997) and secondly, even primed CD8+ T cells are not able to clear virus from the brain without CD4+ T cell help (Stohlman et al., 1998). In MVE, CTL activity correlates with resistance and susceptibility of different mouse strains (Niewiesk et al., 1993). Ld-restricted CD8+ T cells (from resistant BALB}c mice) recognize both target cells infected with MV and target cells infected with vvN. Kk-restricted CTL (from susceptible C3H mice) recognize the N protein after expression via a vvR but not after infection of target cells with MV. Infection with adenovirus leads to non-recognition of target cells co-infected with influenza A virus (Yewdell et al., 1988). After co-infection of target cells with influenza A virus and MV, recognition by influenza A virus-specific CTL is not impaired. In addition, IFN-γ, which helps to overcome inefficient antigen processing in cells infected with murine cytomegalovirus (Hengel et al., 1994), does not affect the recognition of MV-infected cells. These data indicate that there is no general effect of MV gene products on the antigen processing machinery of the cell. As CD8+ T cells restricted by Ld lyse MV-infected cells [L-Ld, P815 (Niewiesk et al., 1993) or C1R-Ld (C. Neumeister & S. Niewiesk unpublished)], this phenomenon seems to be due to either the Kk-molecule itself or to the processing of the Kk Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 28 Apr 2017 21:59:54 CFIH C. Neumeister and S. Niewiesk (b) (a) 1 2 3 4 Fig. 5. The level of recognition correlates with the number of N protein-synthesizing cells. (a) Uninfected (1), persistently infected (2) and NS20Y cells infected with MV (m.o.i. of 5) for 24 (3) or 48 h (4) were tested for recognition by peptide 81–88-specific CD8+ T cells in a 51Cr-release assay. Similar data were obtained for peptide 52–59-specific CD8+ T cells. E : T ratio was 20 : 1. (b) C1R-Kk cells were infected with MV (m.o.i. of 0±1) for 24 or 48 h. The percentage of N protein-positive cells was determined by flow cytometry using the N protein-specific antibody F227. The percentage of specific lysis was measured in a 51Cr-release assay using CD8+ T cells specific for the 52–59 peptide and CD8+ T cells specific for the 81–88 peptide. E : T ratio was 40 : 1. E, N protein-expressing C1R-Kk cells ; *, 52–59 peptide-specific CD8+ T cells ; ^, 81–88 peptide-specific CD8+ T cells. (b) (a) Fig. 6. Efficient binding of 52–59 and 81–88 peptides to Kk. A competition assay was performed using RMA-S-Kk cells. CTL specific for the 81–88 (a) and 52–59 (b) peptides were used as effector cells (E : T ratio 5 : 1). Peptide 50–57 from the NP of influenza A virus strain PR8 or the other MV-derived peptide were used as competitor peptides. (a) E, Peptide 50–57 from influenza A virus ; ^, 52–59 peptide. (b) E, Peptide 50–57 from influenza A virus ; *, 81–88 peptide. lg, Log scale. peptide epitopes. In contrast to herpesvirus (Hill et al., 1995 ; Del Val et al., 1992 ; Wiertz et al., 1996 ; Ahn et al., 1996) and adenovirus (Jeffries & Burgert, 1990) infection, our investigations have shown that the transport of peptide and binding to the Kk molecule is not impaired and that the Kk molecule is not retained within the cell after MV infection. Some viruses, e.g. adenovirus 12, impair the proteasome activity and the TAP-dependent transport of peptides (Rotem-Yehudar et al., 1996). However, if the MV N protein is expressed by a vvR it is recognized by Kk-restricted CTL. The recognition is the same whether the full-length protein, an epitope or an epitope with a leader peptide enabling transport into the endoplasmic reticulum is expressed. This means that there is no specific CFII defect in antigen processing. The important difference between MV and vvN infection of mouse cells is the poor replication of MV. It is known that MV usually does not replicate in murine cells (Horvat et al., 1996 ; Niewiesk et al., 1997). Also primary mouse B cells, which express the human MV receptor CD46 via a transgene, replicate MV in vitro after mitogen activation but not in vivo (Horvat et al., 1996). In agreement with these data, we found that infection of L929 cells with MV leads to uptake of virus (as detected by Western blot), but not to replication (as shown by immune precipitation). In contrast, vvN does replicate in L929 cells and is recognized by CD8+ T cells. In the MV semi-permissive NS20Y mouse neuroblastoma cell line, CTL recognition is partly restored. In human C1R cells Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 28 Apr 2017 21:59:54 CTL in MV encephalitis (b) (a) Fig. 7. Kk requires 100-fold more peptide than Ld to sensitize target cells for lysis. Target cells were incubated with different peptide concentrations and the percentage of specific lysis was measured in a 51Cr-release assay using peptide-specific CD8+ T cells. (a) P815 cells (naturally expressing the Ld molecule) were pulsed with the Ld-restricted 281–289 peptide (*), L929 cells (naturally expressing the Kk molecule) were pulsed with the Kk-restricted 52–59 (E) or the 81–88 (_) peptides. (b) RMA-S cells, transfected with the Ld molecule, were pulsed with the 281–289 peptide (*), RMA-S cells, transfected with the Kk molecule, were pulsed with the 52–59 (E) or the 81–88 (_) peptides. lg, Log scale. transfected with Kk, MV replicates well and these cells are readily lysed by CTL. It therefore seems that the amount of newly synthesized protein (and therefore epitope peptide produced), rather than the processing, limits CTL recognition. This does not completely rule out variation in the processing efficiency of the Kk and Ld epitopes as has been described for other pathogens (Anto! n et al., 1997 ; Sijts et al., 1996). Our data demonstrate that the binding affinity of the Kk-restricted epitopes is at least as good as the affinity of the Kk-restricted influenza A virus epitope. In addition, the amount of peptide required to sensitize Ld-expressing cells correlates with binding studies of peptide to Ld (Kageyama et al., 1995). However, in comparison the amount of epitope peptide required to sensitize cells expressing Ld for CTL recognition is 100-fold less than the amount required to sensitize cells expressing Kk. It should be noted that, although the number of MHC–peptide complexes as well as T cell receptor affinity may influence the competition and sensitization assay, these systems seem to correlate well with peptide binding (Feltkamp et al., 1995). In mice, MV replicates in brain tissue after intracerebral (i.c.) infection, whereas i.p. administration leads to abortive replication. Still, CD8+ T cells develop after i.c. as well as i.p. infection and can be stimulated with MV-infected stimulator cells in vitro (Niewiesk et al., 1993). MV replicates in brain tissue of H-2k mice (Fennelly et al., 1995) and therefore CD8+ T cells should be able to recognize it and protect against encephalitis. In a previous report, it has been demonstrated that the lytic ability of CD8+ T cells in vitro correlates with their protective capacity in vivo (Del Val et al., 1991). Similarly, we find that Kk-restricted CD8+ T cells have poor lytic ability and this might explain their lack of protection in vivo. Another important point might be the kinetics of infection and development of CD8+ T cells. C3H mice die after i.c. infection with MV after 5–9 days. In the lymphocytic chorio-meningitis virus system, it has been shown that CD8+ T cells against a major epitope develop after 6 days with peak activity on day 8, whereas CD8+ T cells against a minor epitope were seen on day 8 with peak activity on day 10 (Weidt et al., 1998). Therefore, a delayed CD8+ T cell response might occur too late to exert a protective effect. In summary, we have shown that the difference in the CTL response in C3H mice susceptible to MVE and BALB}c mice resistant to MVE depends on the amount of newly synthesized viral protein and the binding abilities of the H-2 molecules. We are grateful for the generous gift of cells from Gu$ nther Ha$ mmerling (Heidelberg, Germany), Hans-Georg Rammensee (Tu$ bingen, Germany), Chantale Rabourdin-Combe (Lyon, France) and Peter Cresswell (Yale, USA) and plasmids from Tim Elliott (Oxford, UK). S. N. was supported by Stipendienprogramm Infektionsforschung (Bundesministerium fu$ r Bildung, Wissenschaft, Forschung und Technologie). This work was in part supported by Deutsche Forschungsgemeinschaft, Bundesministerium fu$ r Bildung, Wissenschaft, Forschung und Technologie, Pfleger-Stiftung and the World Health Organization. References Ahn, K., Angulo, A., Ghazal, P., Peterson, P. A., Yang, Y. & Fruh, K. (1996). 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