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Antiviral Cytotoxic T Cells Cross-Reactively Recognize Disparate Peptide Determinants from Related Viruses but Ignore More Similar Self- and Foreign Determinants

Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia

	Abstract

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We have investigated the reactivities of cytotoxic T (Tc) cells against the two immunodominant, H-2K^k-restricted determinants from the Flavivirus Murray Valley encephalitis virus (MVE), MVE₁₇₈₅ (REHSGNEI) and MVE₁₉₇₁ (DEGEGRVI). The respective Tc cell populations cross-reactively lysed target cells pulsed with determinants from the MVE₁₇₈₅- and MVE₁₉₇₁-corresponding positions of six other flaviviruses, despite low sequence homology in some cases. Notably, anti-MVE₁₇₈₅ Tc cells recognized a determinant (TDGEERVI) that shares with the determinant used for stimulation only the carboxyl-terminal amino acid residue, one of two H-2K^k anchor residues. These reactivity patterns were also observed in peptide-dependent IFN- production and the requirements for in vitro restimulation of memory Tc cells. However, the broad cross-reactivity appeared to be limited to flavivirus-derived determinants, as none of a range of determinants from endogenous mouse-derived sequences, similar to the MVE-determinants, were recognized. Neither were cells infected with a number of unrelated viruses recognized. These results raise the paradox that virus-immune Tc cell responses, which are mostly directed against only a few "immunodominant" viral determinants, are remarkably peptide cross-reactive.

	Introduction

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The TCR of CD8⁺ T lymphocytes interfaces with an antigenic structure comprising a short peptide, normally 8–10 aa in length, embedded in a MHC class I molecule. Conserved interactions by the class I helices and the complementarity-determining region 1 of the TCR provide a framework in which the TCR lies diagonally across the peptide-binding cleft, whereas the complementarity-determining region 3 contacts the center of the bound peptide (1). Most of the surface area of the peptide, particularly the N and C termini, is buried within the cleft of the peptide-binding region of class I molecules and is thus inaccessible for direct contact with the TCR (2, 3, 4). Peptide discrimination by the TCR has been suggested to be mediated by one, or a few, central amino acid side chains pointing toward the TCR, thus providing the "primary TCR contact" residues (3, 5), whereas a TCR can often tolerate substantial differences in the remainder of the peptide and still deliver activation signals (6, 7, 8, 9, 10, 11), and in some cases T cell clones have been shown to recognize determinants even without primary sequence homology with the cognate determinant (12, 13, 14, 15). The limited direct interaction and the generally poor structural complementarity of the TCR-peptide-MHC interface may provide an explanation for the observed degeneracy of T cell recognition (16). Alternatively, it has been suggested that the TCR may recognize conformational changes in the class I molecule itself as a consequence of diverse peptide binding (17).

Most of the studies demonstrating the degeneracy of T cell recognition relied on T cell clones, and its relevance in vivo is still unclear. The number of different clones participating in an immune response and their relative clonal frequencies are likely to have a large impact on the combined reactivity of the response (18).

Cross-reactive T cell responses may have medical importance in sequential infections with closely related viruses. Flaviviruses may be an example for this. They are a family of arthropod-borne, positive-strand RNA viruses, including Yellow Fever (YF),³ Dengue (DEN), Japanese Encephalitis (JE), West Nile virus (WNV), and Murray Valley Encephalitis (MVE) virus. The recent outbreak of WNV in New York City (19, 20) and the emergence of JE in northern Australia (21) are evidence that flaviviruses can spread to hitherto nonendemic regions, and may infect a population immune to endemic flaviviruses, thus harboring potentially cross-reactive lymphocytes.

Ab responses to flaviviruses are usually not cross-protective. In fact, the presence of cross-reactive, non-neutralizing Abs may contribute to DEN hemorrhagic fever and DEN shock syndrome (22). This has raised interest in a possible protective role for cytotoxic T (Tc) cells to avoid Ab-mediated immune enhancement (23). Tc cell responses against many flaviviruses are flavivirus-cross-reactive (24, 25, 26, 27, 28) and are predominantly directed against determinants from the viral nonstructural (NS) 3 protein (25, 26, 27, 28, 29, 30). Here we analyze the cross-reactivity patterns of Tc cells to homologous NS3 peptide determinants from different flaviviruses.

	Materials and Methods

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Mice

Specific pathogen-free CBA/H (H-2^k) mice were obtained from the Animal Breeding Facility at the John Curtin School of Medical Research (Canberra, Australia). Mice were immunized with virus at the age of 6 wk or older.

Viruses

Working stocks of MVE (prototype strain MVE-1-51), WNV (Sarafend strain), kunjin (KUN) (MRM16), and JE (Nakayama strain) were 10% suckling mouse brain homogenates in HBSS (pH 8.0) supplemented with 0.2% BSA (31). Virus stocks were titrated by plaque formation on Vero cell monolayers (32). Vaccinia virus (VV) recombinant, VV-C.NP_50–57/1785, encodes peptides NP_50–57 (from influenza A virus nucleoprotein) and MVE₁₇₈₅ downstream from the signal peptidase cleavage site of the membrane-anchored form of the MVE capsid protein (33). This results in targeting of the tandem peptide into the lumen of the endoplasmic reticulum and efficient presentation of peptide MVE₁₇₈₅ due to its COOH-terminal location (33). Crude VV stocks were prepared from infected CV1 cell lysates (34). Adenovirus type 5 (35), Semliki Forest virus (SFV) (36), the influenza viruses A/Pr8 and B/Lee (37), and Sendai virus (38) were grown and titrated as has been described.

Cells

T2 cells (defective in TAP) stably transfected with the mouse H-2K^k heavy chain (T2.K^k) (39) (provided by P. Cresswell, Yale University, New Haven, CT) were maintained in RPMI 1640, supplemented with 10% FCS. Mouse 2R cells (fibrosarcoma; H-2K^k, D^b) were maintained in Eagle’s minimal essential medium supplemented with 5% FCS.

Synthetic peptides

The amino acid sequences of synthetic peptides used in this study are shown in Tables I and IV and were synthesized by the Biomolecular Resource Facility in the John Curtin School of Medical Research. Peptides were dissolved at 10^-3 M in 0.1 M HEPES buffer (pH 7.4) containing 5% DMSO. The final DMSO concentration used did not increase spontaneous ⁵¹Cr release in cytotoxicity assays (data not shown). A database search was performed for mouse sequences conforming to the sequence motifs x(ED)Hx(GA)x(ED)(VI) and x(ED)(GA)xxx(VAI)(VI), using the PeptideSearch tool (http://www.mann.embl-heidelberg.de/Services/PeptideSearch/PeptideSearchIntro.html). The peptides shown in Table IV were purchased as PepSet from Chiron Technologies (Melbourne, Australia). Individual peptides were dissolved based on the estimated yield as above. Single letter amino acid codes are used in sequence comparisons.

The MHC class I stabilization assays were performed essentially as described (40). T2.K^k cells were cultured overnight at 26°C, then incubated in the presence of indicated concentrations of peptide for another 12 h at 26°C. Cells were washed in PBS/BSA (1%) and treated with H-2K^k-specific mAb TIB-95 (American Type Culture Collection (ATCC), Manassas, VA) at saturating concentrations for 30 min at 4°C. After washing twice, the cells were stained with FITC sheep anti-mouse mAb (Silenus, Melbourne, Australia) for 30 min at 4°C. Analysis of the surface fluorescence of live cells was performed on a Becton Dickinson FACScan (Becton Dickinson, Franklin Lakes, NJ). Fluorescence index (FI) was calculated as (mean fluorescence with peptide)/(mean fluorescence without peptide). FI₅₀ is given as the peptide concentration that yields 50% optimal K^k up-regulation.

For the determination of half-lives of peptide-K^k complexes, T2.K^k cells were loaded with peptide (10^-4 M) as described above, washed twice, and then chased at 37°C. At different time points (0–9 h), aliquots were removed and kept on ice until the end of the assay, when they were labeled for K^k as above. During storage on ice, no increase of constitutive surface K^k expression was observed (data not shown).

Generation of effector Tc cells

CBA/H mice were immunized i.p. with flavivirus (5 x 10⁶ PFU). Spleens from primed mice were harvested 6–8 days or 2–10 weeks (for memory Tc cells) later, and single cell suspensions were prepared. For restimulation with virus, one-fifth of the spleen cell suspensions were infected with flavivirus for 1 h at a multiplicity of 5 PFU/cell, washed, and cultured with the rest of the splenocytes for 5 day in Eagle’s minimal essential medium supplemented with 10% FCS and 10^-4 M 2-ME (culture medium). For restimulation with peptide, one-fifth of the spleen cell suspensions were pulsed for 1 h with 10^-4 M peptide, washed, and cultured with the rest of the splenocytes for 5 day. MVE-primed splenocytes restimulated in vitro with virus or peptide are designated anti-virus or anti-peptide Tc cells. Also, the two groups of flavivirus-derived peptides are designated 1785 peptides (those with sequences corresponding to MVE_1785–1792) and 1971 peptides (those with sequences corresponding to MVE_1971–1979).

⁵¹Cr release cytotoxicity assay

2R target cells were infected with flavivirus for 24 h, or incubated in medium only (mock), using 50 PFU/cell and labeled with ⁵¹Cr for 1 h. T2.K^k target cells were labeled with ⁵¹Cr for 1 h followed by treatment with peptide (10^-4 M) for 1 h. Targets were washed three times with culture medium and cocultured with titrated numbers of effector cells for 6 h. All assays were performed in triplicate in 96-well plates; SEM was never greater than 5% and medium release was never greater than 22%. Target cells were infected with adenovirus type 5 (35), SFV (36), the influenza viruses A/Pr8 and B/Lee (37), and Sendai virus (38) and labeled with ⁵¹Cr as has been described.

Staining for intracellular IFN-

MVE-primed mice (5 x 10⁶ PFU) were challenged 5 wk later with 5 x 10⁷ PFU VV-C.NP_50–57/1785. Four days after challenge, splenocytes were isolated and red cell-depleted, using a Ficoll gradient. Cell populations were incubated at 5 x 10⁵/200 µl in 96-well plates with or without peptide (5 x 10^-5 M) for 6 h, in culture medium supplemented with 10% supernatant from the IL-2-secreting cell line 6310 (ATCC) and 10 µg/ml brefeldin A (BFA; Sigma, St. Louis, MO). After culture, cells were washed twice in ice-cold PBS containing BFA (10 µg/ml) and stained with rat anti-mouse CD8-PE mAb (PharMingen, San Diego, CA) for 30 min at 4°C. After washing in PBS/BFA, cells were fixed in 2% paraformaldehyde in PBS for 30 min at 4°C. Paraformaldehyde was then washed off and cells were permeabilized in PBS with 0.5% saponin (PBS/saponin; Sigma) for 10 min at room temperature and labeled with rat anti-mouse IFN--FITC mAb (PharMingen) in PBS/saponin for 30 min at 4°C. Analysis was performed on a Becton Dickinson FACScan. Lymphocyte populations were gated for CD8⁺ cells and assessed for FITC fluorescence. CD8⁺ cells from naive animals incubated with peptide always stained <0.5% positive for IFN-.

	Results

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Flavivirus-immune Tc cells are broadly flavivirus cross-reactive

Tc cell populations raised against one flavivirus are cross-reactive against targets infected by a number of other flaviviruses (24, 25, 26, 27, 28). Fig. 1 shows an example lysis of target cells infected with the homologous or heterologous flaviviruses by secondary in vitro flavivirus-immune Tc cells from CBA/H mice (H-2^k). All four viruses used are members of the JE-serocomplex (41). MVE and WNV generated the strongest Tc cell responses in vitro and displayed a similar hierarchy of lytic activity against target cells infected with heterologous flaviviruses: MVE- and WNV-infected targets were lysed most efficiently, followed by KUN- and JE-infected targets. Mock-infected target cells were also lysed, particularly by anti-MVE and anti-KUN Tc cells (42). Lysis of target cells infected with the virus used for priming was not necessarily the most efficient, possibly because of the differential infectivity of target cells by the various virus preparations. Anti-JE Tc cells gave only weak lysis of flavivirus-infected target cells (25) and were least discriminatory in the recognition of heterologous viruses. The only Tc cell population that did not recognize all of the tested viruses were anti-KUN effectors, which only lysed MVE-, WNV-, and KUN-, but not JE-infected targets above the level of mock-infected target cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Cross-reactivity of anti-flavivirus Tc cells. Secondary in vitro flavivirus-immune Tc cells were tested for lysis on 2R target cells infected with homologous or heterologous viruses. Targets infected with MVE (), WNV (), KUN (), or JE (), or mock-infected ().

We have identified two peptide determinants (MVE₁₇₈₅ and MVE₁₉₇₁) as dominant targets of the H-2K^k-restricted Tc cell response against MVE (Ref. 29 ; M. Regner, A. Mulbacher, R. Blanden, and M. Lobigs, unpublished data). To test whether the extensive cross-reactivity of flavivirus-induced Tc cells is based on cross-recognition of similar MHC class I-restricted peptide determinants, we used the corresponding (based on amino acid sequence alignments; Ref. 43) octameric peptides from six different flaviviruses, four from the JE-serocomplex (MVE, WNV, KUN, and JE), and YF and DEN (Table I). Both determinants are derived from the flaviviral NS3 protein. All but one of the peptides contain the anchor residues constituting the K^k binding motif: Glu or Asp at position 2 and Ile or Val at the C terminus (44). YF₁₇₈₅ is an exception with the acidic Glu of MVE₁₇₈₅ at position 2 substituted by a large nonpolar His, a residue likely to interfere with binding to K^k. Consistent with their antigenic and genetic relatedness, the peptides from viruses of the JE serocomplex have higher homology to MVE₁₇₈₅ and MVE₁₉₇₁ (two or fewer amino acid differences) than YF and DEN (three or more amino acid differences). Also shown in Table I is the sequence of an H-2K^k-restricted Tc cell determinant from influenza virus nucleoprotein (NP_50–57) (45), which was used as a control peptide.

Peptide affinity for K^k of peptide-K^k complexes

To evaluate the relative affinities of the peptides for H-2K^k, MHC class I stabilization assays were performed using human T2 cells stably transfected with H-2K^k (39). This cell line has low MHC class I cell surface expression (46) due to a deletion of the peptide transporter genes required for efficient MHC class I Ag presentation (47). Exogenously added K^k-binding peptides can increase K^k cell surface expression, which can be measured by flow cytometry.

The ability to stabilize K^k varied substantially between the peptides tested (Table I). YF₁₇₈₅ was the peptide with the lowest affinity, which is likely due to the nonconservative amino acid substitution from Glu to His at the K^k anchor at position 2. DEN₁₉₇₁ also had low affinity for K^k. The strongest binding peptide was KUN₁₉₇₁. WNV₁₉₇₁ and JE₁₉₇₁ also had high affinity, whereas the other six peptides were of intermediate affinity. At 10^-4 M all peptides induced optimal up-regulation (FI = 11–12), except YF₁₇₈₅ (FI = 4.8) and DEN₁₉₇₁ (FI = 6.2). A concentration of 10^-4 M was used for peptide loading of target cells in cytotoxicity assays throughout this study, unless stated otherwise.

Because the off-rate of the peptide from the restriction element is thought to provide a better estimate of its immunogenicity than affinity (48), we also chased peptide-pulsed T2.K^k cells at 37°C to assess the stability of the peptide-K^k complexes on the cell surface. According to this criterion, the peptides can be divided into three groups: two peptides, YF₁₉₇₁ and DEN₁₉₇₁ have very low peptide-K^k half-lives (<30 min), and the DEN₁₉₇₁ peptide also had a weak overall affinity for K^k. Half-lives of medium duration (2.9–5.4 h) were found for YF₁₇₈₅ and for the two MVE-derived peptides, whereas all other peptides gave half-lives in excess of 8 h. Surprisingly, YF₁₇₈₅-K^k complexes were found to have a half-life of medium duration (3.1 h), indicating a comparatively slow off-rate of this peptide lacking the binding motif for H-2K^k.

Flavivirus-immune Tc cells recognize homologous peptide determinants

To analyze the molecular basis of cross-reactivities in the flavivirus-immune K^k-restricted Tc cell response, 2R target cells were treated with MVE₁₇₈₅, MVE₁₉₇₁, or the corresponding homologous flavivirus-derived peptides. Recognition of peptide-pulsed target cells by three secondary in vitro virus-stimulated (8 day postinfection) Tc cell effectors (anti-MVE, anti-WNV, and anti-KUN) was tested (Table II). Anti-MVE and anti-WNV Tc cells showed an almost identical recognition pattern for target cells treated with the 1785 peptides, with high lysis against MVE₁₇₈₅, KUN₁₇₈₅, and JE₁₇₈₅, and some recognition of DEN₁₇₈₅ and YF₁₇₈₅ (which binds only weakly to K^k). This recognition pattern is consistent with the sequence identity of the 1785 peptides from MVE and WNV and suggests that the 1785 peptide is also an important determinant in the K^k-restricted Tc cell response against WNV. Anti-KUN Tc cells gave only marginal lysis of targets treated with the 1785 peptides.

Anti-WNV Tc cells recognize the 1971 peptides less efficiently than anti-MVE Tc cells. A broad cross-recognition pattern, despite peptide variability, was again observed. Anti-KUN Tc cells showed only marginal lysis of 1971 peptide-pulsed targets. Although KUN₁₉₇₁ had the highest affinity for K^k of all peptides tested, it appears that this determinant is not highly immunogenic in the K^k-restricted Tc cell response against this flavivirus.

Cross-reactivity patterns of flavivirus peptide-reactive Tc cells

Because the great majority of MVE-reactive Tc cells in the MVE-primed spleen recognize either MVE₁₇₈₅- or MVE₁₉₇₁-treated K^k-bearing targets (29) we anticipated that the recognition of other flavivirus-derived peptides must be mediated by either, or both, of these MVE-reactive subsets. Therefore, we used the panel of peptides, shown to be recognized by anti-MVE Tc cells, to pulse target cells and tested them for lysis by secondary Tc cells generated by restimulation in vitro with MVE₁₇₈₅ or MVE₁₉₇₁ 8 day after priming with MVE. T2.K^k cells were used as target cells in all subsequent cytotoxicity assays, enabling us to compare the efficiency of lysis of peptide-pulsed targets directly with the level of K^k surface-expression induced by the peptides in the MHC class I stabilization assay.

Effectors stimulated with MVE₁₇₈₅ lysed targets pulsed with MVE₁₇₈₅, KUN₁₇₈₅, and JE₁₇₈₅ with high efficiency, and DEN₁₇₈₅- and YF₁₇₈₅-pulsed targets less efficiently (Table III). Thus, all 1785 peptides sensitized targets for lysis by anti-MVE₁₇₈₅ Tc cells, albeit with different efficiencies. The degree of target cell lysis by anti-MVE₁₇₈₅ Tc cells correlated with the degree of amino acid homology of the peptides with MVE₁₇₈₅ (Table I). No lysis was observed on NP_50–57-treated target cells. Neither did MVE-primed splenocytes stimulated in vitro with NP_50–57 lyse any of the targets (data not shown).

MVE-primed splenocytes were also restimulated in vitro with each of the heterologous peptides and tested for lysis of targets pulsed with each peptide. Restimulation with KUN₁₇₈₅ and JE₁₇₈₅ yielded effectors with a reactivity pattern very similar to that obtained with MVE₁₇₈₅ (Table III). DEN₁₇₈₅ stimulation gave effectors that also efficiently recognized all 1785 peptides, but with less clear hierarchy. Notably, these Tc cells were the most cross-reactive and the only effectors studied that lysed YF₁₇₈₅-pulsed targets as efficiently as other 1785 peptide-pulsed targets. This demonstrates that YF₁₇₈₅, despite its low affinity for K^k, is present at sufficient density to sensitize target cells for lysis by appropriate Tc cells, despite its low affinity for K^k. In contrast, anti-YF₁₇₈₅ effectors displayed only very weak cytolytic activity against any of the targets used. All 1785 peptides, except the generally weak stimulator YF₁₇₈₅, generated effectors capable of lysing DEN₁₉₇₁-pulsed targets. These are striking data because in most cases only the C-terminal anchor residue is shared (Table I). For anti-KUN₁₇₈₅ and anti-JE₁₇₈₅ effectors the specific lysis of DEN₁₉₇₁-pulsed targets was similar to, or higher than, lysis of YF₁₇₈₅-pulsed targets.

Restimulation with the 1971 peptides generated reactivity patterns similar to MVE₁₉₇₁ restimulation, with several notable exceptions: 1) only anti-WNV₁₉₇₁ and anti-YF₁₉₇₁ effectors lysed WNV₁₉₇₁-pulsed targets with high efficiency; 2) anti-JE₁₉₇₁ effectors clearly showed better recognition of JE₁₉₇₁ and MVE₁₉₇₁ than of the other 1971 peptides; 3) anti-JE₁₉₇₁ Tc cells failed to recognize DEN₁₉₇₁ despite sharing five residues; and 4) DEN₁₉₇₁ was unable to generate efficient effectors in vitro. Also, anti-WNV₁₉₇₁, anti-KUN₁₉₇₁, and anti-YF₁₉₇₁ effectors were cross-reactive against most 1785 peptides, with DEN₁₇₈₅ being recognized most efficiently in all cases. Of these observations, the strong cross-reactivity between YF₁₉₇₁ and DEN₁₇₈₅ was most surprising because only the anchor residues are shared. However, NP_50–57 was never recognized, despite sharing up to four amino acids with peptides from the 1971 group.

We also found a nonreciprocal antigenicity of JE₁₉₇₁ compared with other 1971 peptides. Although peptides WNV₁₉₇₁, KUN₁₉₇₁, and YF₁₉₇₁ were efficient in generating JE₁₉₇₁-reactive Tc cells, the converse was not the case; thus, JE₁₉₇₁-stimulated Tc cells only weakly lysed targets pulsed with WNV₁₉₇₁, KUN₁₉₇₁, and YF₁₉₇₁ although efficiently lysing MVE₁₉₇₁- and JE₁₉₇₁-pulsed targets. This may suggest an antagonistic effect of JE₁₉₇₁ in these instances. However, restimulation with mixtures of titrated peptides, such as JE₁₉₇₁, with any of the other 1971 peptides failed to provide evidence for such an antagonistic effect (data not shown).

Thus, in vitro restimulation of MVE-primed splenocytes was achieved with a range of peptides with varying degrees of sequence homology to the corresponding MVE-derived Tc cell determinants. The effectors generated were broadly peptide cross-reactive, but there were many instances where peptide sequence homology did not correlate with cross-reactivity. Only YF₁₇₈₅ and DEN₁₉₇₁ failed to restimulate strong cytolytic activity against any of the targets tested. This suggests that their affinity for H-2K^k (Table I) is too low to permit efficient in vitro restimulation. However, they could sensitize target cells for lysis, indicating that triggering effector function in Tc cells is less demanding than stimulation of clonal expansion over 5 days in vitro. Thus, the affinity of the peptides for K^k seems to provide a better correlate for its immunogenicity (YF₁₇₈₅ and DEN₁₉₇₁ have the lowest affinity) than peptide-K^k complex stability.

Anti-MVE memory Tc cells are peptide cross-reactive ex vivo

To guard against the possibility that the observed cross-reactivity was an artifact of the in vitro culture system, we used IFN- production as a functional readout for in vivo-generated effector cells. To prevent high anti-1971 peptide reactivity generated by MVE infection masking the possible cross-reactivity of anti-1785 Tc cells against 1971 peptides, we boosted MVE-primed mice (5 wk postinfection) with VV-C.NP_50–57/1785, a recombinant VV encoding the MVE₁₇₈₅ peptide at the C-terminal position of an endoplasmic reticulum-targeted tandem peptide. Infection with this virus results in efficient, TAP-independent presentation of the MVE₁₇₈₅ determinant (33). Four days after challenge, freshly isolated splenocytes were incubated for 6 h with the panel of flavivirus-derived peptides. This protocol selectively stimulates MVE₁₇₈₅-reactive, but not MVE₁₉₇₁-reactive memory Tc cells (Fig. 2 and data not shown). There was a clear, peptide-dependent hierarchy in the number of CD8⁺ lymphocytes induced to produce IFN- (Fig. 2), similar to that of lysis of peptide-pulsed target cells by MVE-primed splenocytes stimulated in vitro with MVE₁₇₈₅ (Table III). All peptides that sensitized target cells for lysis by in vitro anti-MVE₁₇₈₅ Tc cells also induced IFN- production in memory Tc cells ex vivo, with 4 and 5% of MVE₁₇₈₅-reactive CD8⁺ T cells also recognizing YF₁₇₈₅ and DEN₁₉₇₁, respectively. The number of CD8⁺ lymphocytes producing IFN- in response to peptide was strongly dose-dependent, indicative of a range of avidities present in the secondary Tc cell pool against MVE₁₇₈₅.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. IFN- production by MVE-immune memory Tc cells in response to peptide stimulation. MVE-primed mice were challenged 5 wk after priming with VV-C. NP50–57/1785. Four days later, splenocytes were assessed for IFN- production after a 6-h incubation in the presence of BFA (10 µg/ml) and the indicated peptides, at concentrations 10-4 M (), 10-5 M (), and 10-6 M ().

We sought to delineate the reactivities of anti-MVE₁₇₈₅ and anti-MVE₁₉₇₁ Tc cells further, with an emphasis on the possible recognition of mouse-derived determinants. We constructed consensus motifs with the aim of defining other peptides recognized by these effectors. Sequences of mouse proteins were searched for the presence of octamer peptides with sequence similarity to that of MVE₁₇₈₅ or MVE₁₉₇₁. In addition, naturally H-2K^k-bound peptides that had been eluted from two cell lines by others (49) were tested (Table IVA). For MVE₁₇₈₅-like peptides, His at position 3 was considered important, with only conservative substitutions allowed at the anchor residues and at positions 5 and 7. This yielded a "1785-motif": x(ED)Hx(GA)x(ED)(VI), where "x" may represent any amino acid. For MVE₁₉₇₁-like peptides, we considered that G3 and V7 were most important (conservative substitutions permitted), because recognition of YF₁₉₇₁ and failure to recognize NP_50–57 indicated that positions 4 to 6 are neither sufficient nor required for recognition by at least some of the clones expanding in response to MVE₁₉₇₁. This resulted in the "1971-motif" x(ED)(GA)xxx(VAI)(VI). A database search revealed 11 distinct murine sequences conforming to the 1785-motif, and 24 for the 1971-motif. The 5 and 12 sequences, respectively, that most closely resembled the MVE peptides were selected and tested on targets for recognition by anti-MVE₁₇₈₅ and anti-MVE₁₉₇₁ Tc cells (Table IV, B and C). Three of the 1971 motif-containing peptides (indicated in Table IVC) are probably of very low affinity for K^k because they did not generate significant up-regulation of K^k using the MHC class I stabilization assay. Nevertheless, they were included in the analysis because the data presented in Table III indicated that such peptides may still sensitize target cells for lysis. All other peptides tested in cytotoxicity assays induced intermediate or high up-regulation of K^k in the stabilization assay (data not shown). None of the peptides was recognized on T2.K^k target cells, suggesting an exquisite fine specificity of the Tc cells generated in MVE infection with regard to self-peptides. Furthermore, to test the possibility that the two K^k anchors are sufficient for recognition by anti-MVE₁₇₈₅ Tc cells, we also tested a heptamer and an octamer consisting of Gly residues except at the position 2 (Glu) and the C terminus (Ile). As expected, the octamer peptide was able to bind to K^k, whereas the heptamer was not (data not shown). Neither of the two peptides was recognized by MVE₁₇₈₅- or MVE₁₉₇₁-reactive Tc cells (Table IVD).

Target cells infected with viruses unrelated to flaviviruses are not recognized by anti-MVE Tc cells

Finally, we asked whether target cells infected by flavi-unrelated viruses are recognized by anti-MVE effectors. For analysis we chose influenza virus A and B, Sendai virus, SFV, and adenovirus-5. The data shown in Table V show no significant cross-reactivity on any targets infected with the unrelated viruses. The conditions for infection used have been shown in many previous experiments to be optimal to sensitize these targets for lysis by the relevant virus-immune Tc cells (35, 50, 51). This is in addition to VV (25, 33) and influenza virus A/WSN (52, 53), where lack of cross-reactivity has been shown previously.

	Discussion

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We investigated peptide cross-reactivity using secondary Tc cells against MVE, WNV, or KUN. Except for secondary KUN-immune Tc cells, which did not recognize YF₁₉₇₁-pulsed targets, these Tc cells cross-reacted on all targets pulsed with the various peptides, and the degree of lysis correlated well with the degree of peptide homology. The YF-derived peptides differed most from the MVE peptides with YF₁₇₈₅ sharing only three residues and YF₁₉₇₁ sharing four, but significant cross-reactivity was seen. When peptides were used to restimulate MVE-primed Tc cells, YF₁₇₈₅ and DEN ₁₉₇₁ were poor stimulators, correlating with their low affinity for K^k. All other peptides stimulated strong and generally cross-reactive Tc cell responses against targets pulsed with one of the panel of corresponding (1971 or 1785) peptides. Thus, cells restimulated with 1971 peptides lysed targets pulsed with 1971 peptides, with the degree of cross-reactivity generally corresponding to the degree of peptide sequence homology. Similar results were seen with 1785 peptides. Furthermore, YF₁₇₈₅ and DEN₁₉₇₁ gave lysis when used to pulse target cells, in contrast to their poor stimulating ability. This confirms that triggering effector cytolytic function in activated Tc cells is less demanding than stimulating clonal expansion of Tc cells over 5 days in vitro as reported previously (50, 54).

However, there were some striking examples of unexpected cross-reactivity between peptides. For example, target cells pulsed with DEN₁₉₇₁ were lysed well by Tc cells stimulated by MVE₁₇₈₅, KUN₁₇₈₅, and JE₁₇₈₅ although only the C-terminal anchor residue is shared between the stimulating and target peptides. Furthermore, this cross-reactivity could be detected despite both a very low affinity for K^k of DEN₁₉₇₁ and a short peptide-K^k complex half-life, which probably results in a comparatively low determinant density on target cells. Because the shared anchor residue is probably buried in the K^k peptide binding groove (45) this cross-reactivity may depend upon similar conformational change in the K^k helix residues caused by octamer peptides with quite distinct amino acid sequences. A similar explanation may account for cross-reactivity between YF₁₉₇₁ and DEN₁₇₈₅ where only the two anchor residues are shared. The opposite phenomenon was also seen, with no lysis of DEN₁₉₇₁-pulsed targets by Tc cells stimulated by JE₁₉₇₁, even though five amino acids were shared in the peptide sequences.

The general restimulation of lytic activity against 1785 peptides by WNV₁₉₇₁, KUN₁₉₇₁, and YF₁₉₇₁ also suggests a property shared by these determinants that is not obvious in the primary sequences. For two reasons, this cross-stimulation is unlikely to be due to a bystander "carryover" of activated anti-MVE₁₇₈₅ Tc cells in the in vitro culture, perhaps mediated by cytokines: 1) JE₁₉₇₁ restimulated most 1971 peptide-reactive Tc cells, thus it should also provide nonspecific activation signals, but it fails to generate lytic activity against 1785-pulsed targets; and 2) similar results were obtained when the in vitro culture was initiated 5 wk after priming (data not shown), suggesting that survival of activated T cells is unlikely to be the case.

There were some puzzling nonreciprocal relationships. For example, Tc cells stimulated by WNV₁₉₇₁, KUN₁₉₇₁, or YF₁₉₇₁ lysed target cells pulsed with JE₁₉₇₁, but Tc cells stimulated with JE₁₉₇₁ gave only weak lysis on target cells pulsed with WNV₁₉₇₁, KUN₁₉₇₁, or YF₁₉₇₁. We tested JE₁₉₇₁ for antagonist activity when mixed with other peptides during stimulation but without success. At present, we have no explanation for these nonreciprocal results.

We were interested to determine whether peptide cross-reactivity seen in cytolytic assays performed with secondary Tc cells expanded over 5 days in vitro was also reflected at the level of IFN- production in T cells primed by MVE infection and boosted in vivo with a recombinant VV encoding MVE₁₇₈₅. With this protocol, which restimulates only MVE₁₇₈₅- but not MVE₁₉₇₁-reactive memory Tc cells, reactivity against DEN₁₉₇₁ was also observed. This substantiates the cross-reactivities seen in the cytotoxic response.

Given the extraordinary cross-reactivity between peptides sharing only the C-terminal anchor residue, and because of prominent anti-self activity seen in ex vivo and secondary anti-flavivirus Tc cell populations (30, 42, 52, 53), we investigated cross-reactivity on a panel of self-peptides known to be naturally present in K^k (49) or a panel of putative self-peptides with motifs related to MVE₁₇₈₅ or MVE₁₉₇₁ (Table IV). No cross-reactivity was seen with 10 known naturally presented self K^k-binding peptides, even though four of them shared three residues, including the anchors, with either MVE₁₉₇₁ or MVE₁₇₈₅, and one shared four residues with MVE₁₉₇₁. Similarly, no cross-reactivity was seen with five examples of MVE₁₇₈₅-like putative self-peptides, even though they shared two or three amino acids with MVE₁₇₈₅ plus another two or three conservative substitutions, usually in the buried anchor residues at position 2 and at the C terminus. Another panel of 12 MVE₁₉₇₁-like putative self-peptides also gave no cross-reactivity, despite sharing up to four residues with MVE₁₉₇₁ and with additional conservative substitutions in one to three additional positions. In addition, no cross-reactivity occurred with an octameric peptide possessing the K^k anchor residues and glycine in all other positions. (The observed lack of binding of a similar but heptamer peptide strongly indicates that binding of octamer peptides to K^k results in a stretched configuration of the peptide within the groove.) These results indicate that either thymic negative selection or peripheral tolerance induction maintained Tc cell tolerance during MVE infection with respect to a substantial sample of self-peptides with potential cross-reactivity to the dominant viral peptides. As previously discussed (55), Ohno raised the issue of self-nonself discrimination with respect to minimal nonamer and octamer peptides binding to MHC class I molecules (56). The analysis of Ohno was based on the assumption that amino acid sequence similarity was central to cross-reactivity between self and viral peptides. Our results show that this assumption is invalid for cross-reactivity between viral peptides. We also found no cross-reactivity between Tc cells stimulated by MVE₁₇₈₅ or MVE₁₉₇₁ and target cells infected with influenza viruses A and B, human adenovirus type 5, Sendai virus, SFV, or ectromelia virus, all of which are known to stimulate K^k-restricted Tc cell responses (Table V, and Refs. 25, 53). Finally, the immunodominant influenza virus nucleoprotein determinant, NP_50–57, did not cross-react with the MVE peptides in either cytotoxicity or IFN- assays, despite sharing four amino acids with MVE₁₉₇₁ (plus a conservative substitution in the anchor residue at position 2) and two residues with MVE₁₇₈₅ (plus a conservative substitution at position 2).

The lack of recognition of self-peptides with sequence similarity to MVE determinants as well as determinants presented by cells infected with viruses other than flaviviruses suggests that despite the high peptide cross-reactivity among flaviviruses Tc cell recognition is highly peptide-discriminatory, but primary sequence homology is not the crucial factor. Such evidence of structural similarity in the absence of sequence homology in proteins with similar functions has been found to be not uncommon (57). These results emphasize the fact that viral peptides may cross-react with no sequence similarity in amino acid residues accessible to the TCR, strongly suggesting that the basis for cross-reactivity may include common conformational changes induced in the MHC class I helices following binding of the viral Tc cell determinants and their homologs from antigenically related viruses. Furthermore, self-tolerance can be maintained despite self-peptides sharing up to four amino acids with viral peptides that dominate anti-viral Tc cell responses.

It may be significant that all the cross-reactive peptides are derived from one viral protein, NS3. Perhaps the relevant stretches of amino acids in NS3, despite their different sequences, have structural similarity imposed by NS3 structure/function, thus giving rise to conformational similarity in MHC class I when they are acting as peptide ligands.

	Footnotes

 
¹ Current address: World Health Organization Center for Vaccinology and Neonatal Immunology, Pathologie, Centre Médical Universitaire, 1 rue Michel Servet, 1211 Genève 4, Switzerland.

² Address correspondence and reprint requests to Dr. Arno Müllbacher, Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia.

³ Abbreviations used in this paper: YF, Yellow Fever; MVE, Murray Valley Encephalitis; Tc, cytotoxic T; DEN, Dengue; JE, Japanese Encephalitis; WNV, West Nile virus; NS, nonstructural; KUN, kunjin; VV, vaccinia virus; SFV, Semliki Forest virus; 1785 peptides, peptides with sequences corresponding to MVE_1785–1792; 1971 peptides, peptides with sequences corresponding to MVE_1971–1979; BFA, brefeldin A; FI, fluorescence index.

Received for publication July 6, 2000. Accepted for publication January 4, 2001.

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