INTRODUCTION

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CD8 T cells play a crucial role in the clearing of viral infections. Consequently, viruses have evolved multiple strategies to evade these CD8 T-cell responses, including modulation of surface major histocompatibility complex (MHC) expression and interference with peptide transport or cytokine production (2, 21). In addition, many viruses display considerable sequence heterogeneity, which favors the selection of virus variants that carry mutations in T- and/or B-cell epitopes. In the context of T-cell responses, even subtle changes can result in prevention of peptide processing, failure to bind MHC molecules, or a lack of recognition of the MHC-peptide complex by the T-cell antigen receptor (TCR). The emergence of such cytotoxic T lymphocyte (CTL) escape variants has been documented during several chronic viral infections in humans, such as human immunodeficiency virus type 1, hepatitis B and C viruses, and Epstein-Barr virus (3, 6, 8, 18).

CD8 T-cell responses are highly effective at controlling lymphocytic choriomeningitis virus (LCMV) infection (7). Depletion of CD8 T cells, either genetically or by antibody treatment, results in a failure to control acute LCMV infection (14). Given the importance of the CD8 T-cell response, there is strong selective pressure for the emergence of variants that carry mutated CTL epitopes (13, 15, 20, 29). In C57BL/6 (B6) mice, several D^b- and K^b-restricted CTL epitopes have been mapped in the LCMV nucleoprotein (NP) and glycoprotein (GP), including the D^b-restricted NP396-404, GP276-286, and GP92-101 epitopes and the K^b-restricted NP205-212 epitope (9, 22, 27). In addition, the viral glycoprotein also encodes two overlapping epitopes (11). The GP33-43 (KAVYNFATCGI) epitope (9) is presented by D^b, and the GP34-43 epitope (AVYNFATCGI) is K^b restricted (11). Both are immunodominant epitopes and, along with the NP396-404 epitope, comprise the three dominant LCMV CTL responses elicited during acute infection in H-2b mice.

Transgenic mice expressing a TCR specific for the D^b-restricted GP33-43 epitope have been developed (19). A large percentage (75 to 90%) of T cells in these mice express the transgenic TCR, allowing rapid control of low doses of LCMV (20). However, when these mice are infected with high doses of virus, there is an initial decline in virus titers followed by an increase, at which point CTL escape variants appear (20). We have used this system to generate LCMV variants with mutations in the GP33-43 epitope. This region (amino acids 33 to 43) of the LCMV glycoprotein contains the two overlapping D^b- and K^b-restricted epitopes (10). The development of MHC class I tetramers (4, 16) and the availability of D^b and K^b knockout mice (17) has allowed us to comprehensively dissect how mutations within these overlapping epitopes differentially affect the presentation and induction of responses to each individual T-cell epitope.

Here we present a detailed study of how substitutions within this overlapping epitope affect both the D^b-restricted GP33-43-specific response and the K^b-restricted GP34-43-specific response.

	MATERIALS AND METHODS

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Mice and infections. B6 mice and B6 D2-TgN^TCRLCMV 327sdz transgenic (P14) mice were obtained from Jackson Laboratories (Bar Harbor, Maine). The P14 mice express an LCMV GP33-41-specific H-2D^b-restricted TCR (19, 20) and were back-crossed onto a B6 background. H-2D^b (D^b) or H-2K^b (K^b) knockout mice (17) were also constructed on a B6 background. For analysis of D^b and K^b responses, mice were inoculated intraperitoneally (i.p.) with 10³ PFU (unless otherwise indicated) of either wild-type LCMV (strain A22.2b) or variant LCMV and sacrificed 8 days after infection.

Isolation of LCMV variant viruses. TCR-transgenic P14 mice were infected i.p. with 2 × 10⁶ PFU of LCMV strain A22.2b. Mice were bled retroorbitally at 29 days after infection. Virus in serum samples was titrated using Vero cell monolayers, and isolated plaques were picked from the plates. Virus stocks were propagated by growth in BHK-21 cells. Throughout the text, the parental A22.2b strain will be referred to as the wild type and the CTL escape variant (the L1 virus) will be referred to as the variant virus.

Preparation of viral cDNA and sequence analysis. BHK-21 cells were infected with plaque-purified virus at a multiplicity of infection (MOI) of 0.2 and allowed to adsorb for 1 h at 37°C. Cultures were incubated for 2 days before cells were lysed in solution containing 4.0 M guanidine isothiocyanate, 0.1 M Tris-Cl [pH 7.5], 1% -mercaptoethanol, and sodium dodecyl sulfate 0.5% and layered over a CsCl-EDTA density cushion. Total RNA was harvested from each cushion after centrifugation overnight at 30,000 rpm in an SW41 rotor (4°C). Between 5 and 6 µg of total RNA was used to prepare cDNA using reverse transcriptase (Boehringer Mannheim), according to the manufacturer's instructions. Subsequently, 2 µl of this reverse transcription reaction was used as the template for PCR amplification. PCR products (0.4 kb) were purified from an agarose gel before being sequenced (ABI systems). Approximately 300 nucleotides from the PCR DNA were analyzed. Oligonucleotide primer sequences were as follows: RT, 5'-GCTCGAAACTATACTCATGA-3'; PCR#1, 5'-TTCCTCTAGATCAACTGGGTGTCA-3'; PCR#2, 5'-GCAGAGGTCAGATTGCAAAAGTTG-3'; and SEQ, 5'-AATGTTTGAGGCTCTGCCTC-3'.

Ex vivo CTL assay. Total splenocytes were harvested from infected mice and used in chromium release CTL assays as previously described (1).

Intracellular cytokine staining. Detection of CD8 T cells producing gamma interferon (IFN-) in response to stimulation by virus-specific peptides in vitro was done as previously described (16, 25). Briefly, 10⁶ splenocytes were cultured with interleukin-2, the LCMV-specific epitope (0.1 µg/ml unless otherwise indicated), and brefeldin A for 5 h at 37°C. After this period, cell surface staining for CD8 was performed, followed by intracellular staining for IFN- with the Cytoperm/Cytofix kit (PharMingen, San Diego, Calif.) as described by the manufacturer.

Tetramer staining. MHC class I tetramers specific for the D^b NP396-404, D^b GP33-43, and K^b GP34-43 epitopes were made as described (4, 16, 25). Tetramer staining was performed at 4°C.

Plaque assays. Infectious virus was quantified by plaque assay as previously described. Briefly, 7.5 × 10⁵ Vero cells were grown overnight to confluency. Samples to be titrated were added in a 200-µl volume. After adsorption at 37°C for 1 h, cells were overlaid with agarose, and plates were incubated for 4 days at 37°C. Plaques were counted after overnight neutral red staining.

Peptide-MHC class I binding assay. H-2K^b and H-2D^b peptide-binding assays were done as previously described (23, 27, 28). The radiolabeled probes used were a Pro-to-Tyr (position 7) analog of the adenovirus E1A epitope (SGPSNTYPEI) for D^b and the vesicular stomatitis virus (VSV) NP52-59 epitope (RGYVFQGL) for K^b. The 50% inhibitory concentrations (IC₅₀s) were 4.4 nM for the E1A epitope and 3.1 nM for the VSV NP52-59 epitope.

	RESULTS

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Selection of LCMV CTL escape variants in vivo. Infection of B6 mice with a high dose of LCMV strain A22.2b (which will be referred to as the wild-type virus) resulted in a high-grade viremia during the first 2 weeks, followed by a gradual resolution of the infection (Fig. 1A). In contrast, TCR-transgenic mice initially controlled the infection, and virus titers in the serum were approximately 100-fold lower at day 8 postinfection than in nontransgenic B6 mice. Subsequently, virus titers increased, suggesting a loss of immune-mediated virus control. To determine whether this reemergence of virus was due to the selection of CTL escape variants, virus was isolated from the serum of infected mice at 29 days after infection. The nucleotide sequence spanning the D^b-restricted GP33-43 epitope and its flanking sequences was determined for seven plaque-purified viral isolates and also for the parental A22.2b strain. The nucleotide sequence analysis revealed that all seven isolates harbored the same nucleotide substitution (U to C at position 181). This mutation resulted in a change from valine to alanine at amino acid position 35 of the glycoprotein, yielding the variant epitope KAAYNFATCGI instead of the wild-type sequence KAVYNFATCGI. We did not find any other mutations in the region of the glycoprotein sequenced. For the purpose of these studies, one isolate (which will be referred to as the variant virus) was chosen for all subsequent experiments.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   (A) P14 transgenic mice fail to control LCMV infection. Transgenic mice were infected with 2 × 106 PFU of A22.2b virus i.p. (n = 2, open squares). B6 mice were infected with 106 PFU of A22.2b virus i.p. (n = 3, solid circles). Serum was taken for titration at several time points. (B) TCR-transgenic GP33-specific CTL do not recognize LCMV L1-infected cells. Splenocytes were harvested from transgenic mice infected with A22.2b (LCMV wild type, left panel) or LCMV-L1 (LCMV variant, right panel), pooled, and measured for their ability to kill virus-infected target cells in direct ex vivo CTL assays. E:T, effector-to-target cell ratio.

Immune response to LCMV L1 variant virus in B6 mice. We next wanted to determine the effect of this mutation on a polyclonal response, which would include the K^b-restricted GP34-42-specific response. To test this, B6 mice were infected with the variant virus. As a control, we infected transgenic mice with the same variant virus. Splenocytes were harvested on day 8 postinfection and tested for their capacity to generate IFN- in response to stimulation with variant GP33-43 peptides (KAAYNFATCGI). CD8 T cells from variant-virus-infected transgenic mice failed to respond to the variant GP33 peptide (Fig. 2A). However, CD8 cells from nontransgenic B6 mice infected with the same virus did respond to the variant GP33 peptide (Fig. 2B). The observation that mice infected with the escape variant can still mount a GP33-specific response is in apparent contradiction to the results shown in Fig. 1. However, the GP33-43 peptide can be presented by both H-2D^b and H-2K^b MHC class I molecules (11). Whereas the transgenic mice used in the generation of this escape variant contain only T cells carrying TCRs that are H-2D^b restricted, infection of normal mice should yield D^b- and K^b-restricted responses. As such, it is possible that the variant virus has escaped only the H-2D^b-restricted response, leaving the H-2K^b response intact. Alternatively, it is possible that the polyclonal response in nontransgenic mice includes additional fine specificities of D^b-restricted CD8 T cells that recognize the variant epitope.

View larger version (38K): [in this window] [in a new window]   FIG. 2.   (A) Splenocytes were harvested from P14 transgenic mice 8 days after infection with either the parental A22.2b strain (wild type) or the L1 variant strain (variant). Cells were stimulated with the variant GP33-43 peptide (KAAYNFATCGI) and stained for intracellular IFN- production (n = 2 for each group). The percentage of CD8 T cells producing IFN- is shown in the top right corner of each plot. (B) Splenocytes from B6 mice infected with A22.2b or LCMV L1 were harvested 8 days postinfection and stimulated with the variant GP33-43 peptide before staining for cytokine production (n = 6 for each group). The percentage of CD8 T cells producing cytokine is listed in the top right corner of each plot.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   LCMV L1-primed B6 mice lack Db GP33-specific CD8 T cells but retain the ability to generate a Kb-restricted GP34 response. (A) Splenocytes from mice infected with A22-2b (wild type) or LCMV L1 (variant) were harvested 8 days postinfection and stained with tetramers specific for Db NP396, Db GP33, and Kb GP34 epitopes. All cells were gated on the CD8-positive population before being analyzed for tetramer versus CD44 expression. The percentage of CD8 T cells that are tetramer positive is listed in the top right corner of each plot. (B) Enumeration of LCMV-specific CD8 T cells from wild-type (WT)- and variant-infected mice (n = 4 to 7 mice).

Immune response to variant virus in H-2D^b and H-2K^b knockout mice. To further investigate how the V-to-A change at residue GP35 affects CD8 T-cell responses, we infected D^b and K^b knockout mice as well as B6 mice with the wild-type and variant strains. T-cell responses were monitored by measuring IFN- production after peptide stimulation.

View larger version (62K): [in this window] [in a new window]   FIG. 4.   Escape mutation affects epitope recognition by Db- and Kb-restricted CTL and the induction of the Db-restricted CTL response. (a) Splenocytes from B6 (+/+), Kb knockout (Kb /), or Db knockout (Db /) mice infected with LCMV A22-2b (WT) or LCMV L1 (variant) were harvested 8 days postinfection and stimulated with wild-type (WT) GP33 or variant GP33-43 peptide before staining for IFN- production. The percentage of CD8 T cells producing cytokine is given in the top right corner of each plot. (B) Intracellular IFN- staining results from wild-type- and variant-infected mice (n = 3 to 5 mice).

Effect of mutation on MHC class I binding. To determine whether the V-to-A substitution affects the MHC class I binding of the epitope, we performed two experiments. First, the ability of the peptides to bind and stabilize MHC class I complexes was measured directly in an in vitro peptide-MHC class I binding assay. This assay is based on competition for binding of the peptide of interest with a radiolabeled standard peptide that binds the class I molecules in question with high affinity (23, 28). The concentration necessary to inhibit binding of the standard peptide to solubilized class I molecules by 50%, the IC₅₀, is measured and approximates the affinity of a given peptide-MHC interaction.

View larger version (23K): [in this window] [in a new window]   FIG. 5.   Binding of wild-type and variant peptides to the MHC class I Db molecule. Kb knockout mice were infected with LCMV strain Armstrong (2 × 105 PFU i.p.), and splenocytes were harvested at 8 days postinfection. Splenocytes were stimulated with 1 to 105 µg of the wild-type and variant 9-mer and 11-mer peptides per ml. Responses were quantitated by intracellular IFN- staining. Peptide sequences are shown.

	DISCUSSION

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This study describes a CTL escape mutant that carries a single-amino-acid substitution in the viral glycoprotein (valine to alanine at position 35). This LCMV variant was selected in a transgenic mouse expressing the TCR specific for the D^b-restricted GP33-43 epitope after infection with the LCMV A22.2b strain (wild type). The mutation changes the sequence of the D^b-restricted epitope from KAVYNFATCGI to KAAYNFATCGI and has a very strong negative effect on the D^b-binding affinity of this peptide. The surprising result of our study is that this mutation has a much weaker effect on class I binding of the overlapping K^b-restricted epitope (GP34-43), which changed from AVYNFATCGI to AAYNFATCGI. In neither case does the substitution affect the primary or auxiliary anchor residues (22), and the dramatic effect on D^b binding was therefore unexpected. Clearly, the amino acid residue at position 3 plays a role in MHC binding. This conclusion is consistent with the recent determination of the H-2D^b-GP33 crystal structure, in which the position 3 side chain is buried in the H-2D^b binding cleft (26).

The phenotypic effects that we have observed correspond very well with the binding data. The very low binding affinity of the variant epitope explains why the variant virus escaped the monoclonal CD8 T-cell response in the transgenic mice and why target cells infected with the variant virus are not lysed by transgenic D^b-restricted effector cells. This very low binding affinity also accounts for the inability of K^b knockout mice to elicit a D^b-restricted, GP33-specific response following infection with the L1 variant. In apparent contrast to these data, GP33-specific, D^b-restricted effectors (for instance, from the K^b knockout mice) do respond to the variant peptide, as detected by IFN- production. This indicates that their TCRs do recognize the variant peptide and that exogenously added peptide is apparently presented to these T cells. The peptide dilution experiments provide an explanation for this by showing that IFN- production in response to the variant peptide is a function of peptide concentration. IFN- production is apparent if stimulator cells are pulsed with high concentrations of peptide but is no longer observed at lower peptide concentrations. This result confirms our conclusion that the major defect of the variant epitope is its impaired D^b binding. Pircher and coworkers have also generated a CTL escape mutant containing a substitution at position 35 (valine to leucine) (20), and it is possible that their mutation may have effects on D^b and K^b responses similar to those described here.

A complicating factor in the interpretation of our data is that many studies have been done with a 9-mer peptide in which the natural cysteine residue at position 41 was changed to a methionine (i.e., KAVYNFATM instead of KAVYNFATC) (5, 12, 26). This cysteine-to-methionine substitution prevents the formation of cysteine dimers by the synthetic peptides. Our data show that the cysteine-to-methionine substitution also increases the apparent MHC class I binding: the IC₅₀ for the KAVYNFATM peptide is 21 nM, whereas the 9-mer KAVYNFATC peptide has an IC₅₀ of >5,000 nM (27). The result of this binding stabilization is that the variant 9-mer peptide (KAAYNFATM) still bound to D^b molecules with intermediate affinity (IC₅₀ = 329 nM). Introduction of the mutation in the 11-mer peptide (which carries the C at position 41) decreases the binding affinity from 395 to 17,089 nM.

In our experiments, we observed the same recognition specificity for the 9-mer peptides as for the 11-mer peptides, although the differences in cytokine production by CD8 T cells stimulated with the wild-type peptide versus variant-peptide-stimulated CD8 T cells was less exaggerated. The strong phenotypic effect of the V-to-A mutation on in vivo T-cell responses shows that this conservative amino acid change has a remarkable effect on this overlapping epitope. Although a previous study (10) has eluted this epitope from virus-infected cells, it is clear that, given our findings, the direct sequencing of the naturally processed epitope is warranted. Moreover, we believe that data regarding binding affinities and the fine specificity of the GP33-specific response, obtained using methionine-substituted peptides (5, 12, 26), should be interpreted with caution.