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H2-M3 Presents a Nonformylated Viral Epitope to CTLs Generated In Vitro¹

^* Howard Hughes Medical Institute, Departments of Microbiology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75235

	Abstract

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Most CTL responses to epitopes from influenza virus are restricted by MHC class Ia molecules. However, a synthetic peptide corresponding to residues 173 to 190 of influenza A/JAP/305/57 hemagglutinin (HA) can induce, in vitro, a CTL response to peptide presented by a mouse class Ib molecule encoded by a gene telomeric to H2-Q. Here, we identify the molecule as H2-M3 and show that the last five residues of HA173–190, MLIIW, is the minimal epitope for CTL recognition. Cells that express M3^wt, from C57BL/6 or BALB/c mice, are sensitized by both MLIIW and the longer peptide HA173–190, whereas cells that express M3^f, from A.CA or B10.M mice, are sensitized only by MLIIW; a single amino acid change at residue 31 (VM) of M3 accounts for this difference. Although M3-restricted CTLs preferably recognize N-formylated epitopes, i.e., those of mitochondrial or prokaryotic origin, our findings show that M3-restricted primary CTL responses can be generated in vitro against nonformylated epitopes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
MHC (Mhc) class I molecules mediate the elimination of virally infected cells by presenting viral epitopes to CTLs. In mice, the class I molecules involved in these responses typically are expressed from the H2-K, D, or L (class Ia) loci, which represent only a fraction of the >50 class I genes in the mouse Mhc. The remaining genes, encoded in the H2-Q, T, and M regions, are nonclassical class I, or class Ib, genes, and their roles in antiviral responses are uncertain.

In 1991, Milligan et al. reported evidence for class Ib presentation of an epitope from influenza virus (1). By stimulating naive mouse spleen cells in vitro, they demonstrated that a peptide corresponding to residues 173 to 190 of influenza (A/JAP/305/57) hemagglutinin (HA)³ induces a cytotoxic response by CD8⁺ T cells with ß receptors. These CTLs are restricted by a molecule encoded telomeric to the H2-Q region of the mouse Mhc. The molecule associates with ß₂-microglobulin (ß₂m) and has limited polymorphism, as lymphoblast target cells of H2 haplotypes b, d, k, s, and a, but not f, are sensitized to lysis by HA173–190. Interestingly, antipeptide CTLs were unable to lyse virus-infected cells.

Over 40 class I genes are mapped to the H2-T and M regions. Only four products from these regions are known to be recognized by T cells, and none appears to be a good candidate for presenting the HA peptide. Least likely is T10/T22, a ligand for T cells that does not require peptide for stable surface expression (2, 3, 4). A second candidate, TL (encoded by duplicate genes T3 and T18), is capable of binding peptides and being recognized by ß T cells when expressed as a transgene (5, 6), but normal expression is limited to the gut epithelium and the thymus (7, 8); moreover, both C57BL/6 and CBA/J, the strains used for characterizing the HA173–190 response, carry the b allele, but their CTLs are capable of recognizing peptide-sensitized lymphoblasts encoding either a-, b-, or c-type TL molecules (1). The other two candidates, Qa1 (encoded by T23) and M3, are ubiquitously expressed and recognized by ß CTLs in peptide-specific responses. But Qa1 seems unlikely for the same reason as TL: anti-HA CTLs lyse both Qa1^a-positive and Qa1^b-positive lymphoblasts equally well, whereas CTLs to Qa1 typically distinguish allelic forms (9, 10). M3 is unlikely because it preferentially binds peptides containing N-terminal formylmethionine (fMet), and all M3-restricted CTLs known are directed to fMet peptides (11, 12, 13, 14, 15); HA173–190 begins with a nonformylated valine and the only methionine is located 12 residues inward and 5 residues from the C terminus. Thus, with hopes of identifying a new class Ib molecule, we characterized the anti-HA173–190 response.

	Materials and Methods

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Mice

C57BL/6J (B6), C57BL/10J (B10), BALB/c, and A.CA mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B10.BR-mt^BOM, B10.CAS3/Kfl, B10.CAS3(R1)/Kfl (B10.R1), B10.CAS4(R2)/Kfl (B10.R2-l), B6.CAS3(R4-N₂F_n)/Kfl (B6.R4-l), B6.CAS3(R10)/Kfl (B6.R10-l), B10.SH1(R27)/Kfl (B10.R27), and B10.CAS4(R34)/Kfl (B10.R34) were bred and maintained in our mouse colony. (For review of these strains, see Ref. 15.)

Peptides

HA173–190 (VAKGSYNNTSGEQMLIIW), HA186–190 (MLIIW), HA186–189 (MLII), HA183–190 (GEQMLIIW), and formylated mitochondrial peptides (15) were synthesized on a Rainin Symphony peptide synthesizer, using standard F-moc chemistry, as described previously (16). Lyophilized peptides were dissolved in DMSO for 1 to 2 mM stock solutions.

Peptides were analyzed by reverse-phase HPLC and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Each eluted as a major peak by HPLC and contained a single major peak corresponding to the predicted m.w. by mass spectrometry.

Cell lines

The NZB/Icr-derived plasmacytoma cell line, Pc11198 (H2^d2) (17), was maintained in RP10 (RPMI 1640 supplemented to a final concentration of 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 50 µM 2-ME).

Fibroblast cell lines were maintained in RP10 and harvested with EDTA-saline (140 mM NaCl, 5 mM KCl, 12.5 mM Na₂HPO₄, 5.8 mM NaH₂PO₄, 0.2% glucose, and 0.9 mM Na₂EDTA; pH 7.2). CM3 and M31 are derivatives of the B10.CAS2 cell line that have been transfected with cDNAs encoding M3^wt (16) or M3 with a change at residue 31(ValMet) (18).

Cytotoxic T lymphocytes

HA173–190 peptide-reactive CTL lines were generated in vitro as described by Milligan et al. (1), or with some modifications according to Alsheikhly (19). Briefly, 7 to 10 x 10⁷ cells, prepared from fresh spleens of 6- to 12-wk old C57BL/6J mice, were incubated at 37°C in the presence of 5 µM peptide (from stock solutions) in 10 ml RP10, in upright 25-cm² tissue culture flasks (430168; Corning, Corning, NY). For lines generated according to Alsheikhly, splenocytes were incubated with 5 or 25 µM peptide in 4 ml of RP10 for 4 h, then diluted to 20 ml with RP10. After 7 to 10 days, 1 to 2 x 10⁶ cells from primary cultures were restimulated with 6 to 8 x 10⁶ -irradiated (1500 rads) syngeneic splenocytes, 1 to 5 µM HA173–190, and mouse IL-2 in 12-well tissue culture plates (3 ml RP10 per well). Restimulations were continued weekly thereafter to generate the long-term lines used in cytotoxicity assays. IL-2 for restimulations was partly purified from the supernatant of EL4.IL2 cells (American Type Culture Collection, Manassas, VA; no. TIB 181), assayed, and used as described in (20). The alloreactive anti-M3^wt CTLs used as controls for some experiments are described elsewhere (16, 21).

Cytotoxicity assay

CTL reactivities were measured in a standard 3.5-h ⁵¹Cr-release assay (20). Briefly, threefold serial dilutions of effector cells in RP10 were added in triplicate to a 96-well round-bottom microtiter plate (100 µl/well). Target cells were labeled with ⁵¹Cr for 1 h, washed, and resuspended to 10⁵ cells/ml. Stock solutions of peptide in DMSO were diluted, at least 100 times, into the resuspended target cells, and 100 µl of target cells were added per well to the microtiter plate. For lymphoblasts, 4 x 10⁶ spleen cells/ml were incubated in the presence of 2.5 µg/ml Con A for 48 h at 37°C, and cells (10⁶/ml) were incubated with peptide before labeling. Spontaneous and maximal release controls were prepared by adding 100 µl of targets to 100 µl of either RP10 or 1% Triton X-100 in water, respectively. Plates were centrifuged at 200 x g for 10 s, and incubated for 3.5 h at 37°C. One hundred microliters of supernatant was harvested from each well, and the radioactivity was measured in a gamma counter. Percent specific lysis represents the mean of duplicate or triplicate samples and was calculated as % specific lysis = 100 x ((experimental release - spontaneous release)/(maximal release)) (22). Errors were less than 5% of maximal release, and spontaneous release varied from 5 to 35% of maximal release depending on the target cells being used.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
To characterize the response to peptide HA173–190 of influenza A/JAP/57/305, we generated B6 (H2^b) CTLs in vitro by peptide stimulation according to Milligan et al. (1). Consistent with their results, our anti-HA173–190 CTLs (anti-HA173 CTLs), which could be induced within 1 wk, were peptide specific and not class Ia restricted, as H2^b-, H2^d-, and H2^k-derived target cells were lysed equally well. Nevertheless, to avoid class Ia (H2^b)-restricted killing, we used the NZB/Icr-derived (H2^d2) plasmacytoma cell line, Pc11198, as a target in many of the CTL assays. This line also provided a control against Qa1-restricted killing as it has the a allele of Qa1, whereas B6, used to induce and stimulate the anti-HA173 CTLs, is Qa1^b.

Recognition of HA186–190 by CTLs

Most class I molecules bind peptides 8 to 10 amino acids long, so HA173–190, an 18-mer peptide, is probably not the minimal epitope for the anti-HA173 CTL response. Already, Milligan et al. demonstrated that a series of overlapping 12-mer peptides, all containing HA186–190, effectively sensitized target cells to lysis (1). These results indicated that residues 173–185 were not required; however, the pentamer spanning 186–190 was not tested alone. Thus, to define the minimal epitope required for anti-HA173 CTL recognition, we synthesized both the pentamer, HA186–190 (MLIIW), and the tetramer, HA186–189 (MLII), and tested their abilities to sensitize Pc11198 cells. In Figure 1, MLIIW sensitized target cells as well as HA173–190, losing potency in the 100 nM range, but the tetramer, which lacks the C-terminal tryptophan, was not recognized at all, even at 10 µM. Because HA186–190 is the last five residues of HA173–190 and has similar potency, if not greater, we concluded that MLIIW represents the minimal epitope for the anti-HA173 response and used it for target cell sensitization in subsequent experiments.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. HA186–190 is the minimal epitope for recognition by anti-HA173 CTL lines. 51Cr-labeled Pc11198 cells (H2d2) were mixed with the indicated concentration of either HA173–190 (VAKGSYNNTSGEQMLIIW) , HA186–190 (MLIIW) •, or HA186–189 (MLII) and used as targets in a 3.5-h assay. B6 anti-HA173 CTLs were used at an E:T ratio of 5:1.

Lysis by anti-HA173 CTLs is restricted by a ß₂m-associated MHC class I molecule encoded in the Mhc distal to the H2-Q region (1). To more precisely define the restriction element, we tested a panel of Con A lymphoblasts derived from congenic and Mhc recombinant mice (Table I). Experiment 1 showed that, in the presence of HA186–190, anti-HA173 CTLs recognized H2^b (B6) and H2^k2(B10.BR-mt^BOM) lymphoblasts but not those carrying H2^cas (B10.CAS3) genes. This allowed us to use lymphoblasts from castaneus (cas) recombinants to determine the region encoding the restriction element. Consistent with the previous results, K, D, and Q regions were not involved, as B10.R1 lymphoblasts, which are cas proximal to the T region, were lysed. However, the CTL response was not restricted by an element from the T region, as B10.R2-l lymphoblasts, which are cas in the T region, were lysed and B6.R4-l lymphoblasts, which are k in the T region, were not. Therefore, the response must be directed to an element expressed from the M region, as was most clearly shown in experiment 3, with B10.R2-l lymphoblasts being lysed and B6.R10-l not lysed by anti-HA173 CTLs. In all experiments, lysis required the HA186–190 peptide; a formylated ND1 peptide, which binds to H2-M3^wt, was not recognized. Lymphoblasts sensitized with formylated ND1 were recognized by M3-restricted, ND1-specific clones and verified restriction to the M region. Thus, these findings confirmed our prediction that the anti-HA173 response was not directed to a T region molecule, such as T10/T22, TL, or Qa1.

Transcripts of three class I genes from the M region, M2, M3, and M10, have been identified by Northern hybridization or reverse transcription-PCR; of these, M3 is the only gene for which a functional class I molecule has been described (15). Although M3 binds nonformylated epitopes poorly (16), it was an easily tested candidate for presentation of the HA186–190 epitope. In Table I, we observed that anti-HA173 CTLs did not recognize lymphoblasts that express the cas alleles in the M region. Thus, to determine whether M3 presented HA186–190, we used CM3, a B10.CAS2 fibroblast cell line transfected with an M3^wt cDNA; B10.CAS2 cells express M3^cas, a phenotypic null allele of M3, and are not lysed by M3^wt-restricted CTLs (16). To our surprise, anti-HA173 CTLs lysed CM3 cells sensitized with peptide (Fig. 2). Lysis of the fibroblasts was peptide specific, as CM3 cells alone were not lysed, and required M3^wt expression, as B10.CAS2 cells were not recognized. These findings strongly suggested that the nonformylated epitope binds to M3 for presentation to anti-HA173 CTLs.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. H2-M3wt expression by target cells is required for peptide-sensitized lysis by anti-HA173 CTL lines. H2-M3wt-transfected CM3 (circles) and untransfected B10.CAS2 (H2-M3cas) (squares) fibroblast cell lines were mixed with 4 µM HA186–190 peptide (filled symbols) or DMSO (open symbols) and used as targets in a 51Cr release assay. M3wtexpression was verified with an M3cas anti-M3wt CTL clone (data not shown).

To verify presentation by M3, we tested the ability of HA186–190 to compete with various formylated peptides representing the amino termini of mitochondrially encoded proteins. If M3^wt presents the HA186–190 epitope, addition of formylated peptides known to bind M3 with high affinity should inhibit the ability of HA186–190 to sensitize target cells (11, 23). Pc11198 cells were mixed with equimolar concentrations (1 µM) of HA186–190 and the individual formylated peptides, and lysis by anti-HA173 CTLs was assayed (Fig. 3). CTLs lysed targets sensitized with HA186–190 in the presence of formylated peptides ND5, ATPase6, ATPase8, and COIII, but not ND1, COI, or ND4. These results indicate that formylated ND1, COI, and ND4 competed away HA186–190 binding to M3, whereas formylated ND5, ATPase6, ATPase8, and COIII did not. Similar experiments have shown that ND1, COI, and ND4 have the highest affinity for M3, and the other peptides bind with low to negligible affinity (15, 21). Thus, peptides with high affinity for M3 outcompete HA186–190, whereas lower affinity peptides compete less well. Together, the results in Figures 2 and 3 indicate that the B6 anti-HA173 CTL response is restricted by M3^wt.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Peptide competition assay using B6 anti-HA173 CTL lines. Light bars represent the specific lysis of 51Cr-labeled Pc11198 targets mixed with 1 µM HA186–190 and 1 µM of the formylated peptide indicated on the left. Black bars represent the lysis of Pc11198 targets sensitized with the formylated peptides alone; these values were near zero, excluding cross-reactivity, and are therefore hidden by the baseline for some peptides, e.g., f-ND1. E:T ratio was 10:1.

The presence of a formyl group is not required for T cell recognition but is important for anchoring peptides in M3 for high affinity binding (16, 24). Some M3-restricted CTLs are capable of recognizing nonformylated peptides, when 100- to 10,000-fold more peptide is used for sensitization (14, 16), and this was consistent with the anti-HA173 response as well. As shown in Figure 4, anti-HA173 CTLs recognize the formylated HA186–190 as well as the nonformylated form with the same level of maximal lysis, but, as predicted, the formylated peptide was more potent: approximately 100-fold less formyl-peptide was needed for sensitization. The formyl group on MLIIW did not appear to alter the recognition of peptide bound to M3, but it did increase the affinity of the peptide for M3 (as measured by the concentration needed for half-maximal lysis).

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Formylated HA186–190 sensitizes target cells for lysis at lower concentrations than unformylated HA186–190. Anti-HA173 CTLs were incubated with 51Cr-labeled Pc11198 target cells mixed with either formylated HA186–190 (•) or unformylated HA186–190 () at the concentrations indicated. E:T ratio was 14:1.

M3^f (of H2^f mice) differs from M3^wt (of most other laboratory strains of mice) at three residues, 31(ValMet), 219(LysArg), and 236(SerAla), and is not recognized by some CTLs specific for mitochondrial ND1 or Listerial Fr38 (21, 25), although alloreactive M3^cas anti-M3^wt CTLs capably recognize M3^f (21). Similarly, Milligan et al. (1) showed that anti-HA173–190 CTLs do not lyse H2^flymphoblast targets sensitized with HA173–190 and even used this difference for mapping the gene encoding the restriction element. However, we noticed that our antipeptide CTLs lysed both A.CA and B10.M (H2^f) lymphoblasts sensitized with the short peptide, HA186–190. To resolve this conflict, lymphoblasts from B10, BALB/c, B10.M, and a recombinant strain, B10.R27 (Table II), were sensitized with 10 µM concentrations of either HA186–190, HA173–190, or HA183–190 peptides and then tested for recognition by anti-HA173 CTLs. (a 10 µM peptide concentration corresponds to the concentration (10 µg/ml) used by Milligan et al. (1)) All three peptides sensitized M3^wt, as expected, but only the short peptide sensitized M3^f (Fig. 5). The longer peptides, including HA183–190, which has only three residues before the MLIIW epitope, did not sensitize M3^f.

View this table: [in this window] [in a new window]   Table II. Haplotypes of lymphoblast targets used in Figure 5

View larger version (19K): [in this window] [in a new window]   FIGURE 5. M3f presents HA186–190 but not HA173–190 or HA183–190. Con A lymphoblasts from B10, BALB/c, B10.M, and B10.R27 mice (Mhc genotypes given in Table II) were preincubated with no peptide (x) or 10 µM of either HA186–190 (), HA173–190 (•), or HA183–190 () for 4 h, then 51Cr-labeled and tested for recognition by anti-HA173 CTLs. Peptide sequences are as follows: HA186–190 (MLIIW), HA183–190 (GEQMLIIW), HA173–190 (VAKGSYNNTSGEQMLIIW).

To determine how well HA186–190 sensitizes M3^f, we titrated the pentamer on A.CA (H2^f) and BALB/c (H2^d) blasts. Figure 6 shows that 10 µM was the minimal concentration capable of sensitizing A.CA, whereas BALB/c could be equally sensitized with 100-fold less peptide. Together, Figures 5 and 6 indicate that anti-HA173 CTLs recognize M3^f, but M3^f presents the HA epitope poorly; HA186–190 sensitizes only at high concentrations and the longer peptides do not sensitize at all.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Ten micromolar is the minimal concentration of HA186–190 capable of sensitizing M3f lymphoblasts. BALB/c () and A.CA () lymphoblasts were preincubated with the indicated concentration of HA186–190 for 4 h, then labeled and assayed as in Figure 5 using an E:T ratio of 10:1. B10.M (H2f) lymphoblast targets gave the same results as A.CA (data not shown).

In M3^wt, residue 31 is in the 1 domain and points away from the peptide binding groove, making no direct contact with peptide (24). Residues 219 and 236 are in the 3 domain, even farther from the groove; the Ser236 is more significant of the two, forming a hydrogen bond with ß₂m. Of the three changes in M3^f, Met³¹ is likely to have the greatest effect on peptide binding. To test this hypothesis, we used a B10.CAS2 cell line, M31, which has been transfected with an M3 cDNA encoding Met³¹ instead of a Val³¹. CM3 (M3^wt), M31 (M3^31M), and B10.CAS2 (M3^cas) cells were incubated with either HA173–190 or HA186–190 and tested for recognition by anti-HA173 CTLs (Fig. 7). Both peptides sensitized CM3 cells to lysis, confirming presentation of the HA peptides by M3^wt. HA186–190 capably sensitized M31 cells to lysis, indicating that the short peptide could be presented by the mutated M3 as well. However, M31 cells incubated with HA173–190 were not recognized at all, even at a peptide concentration of 10 µM, indicating that M3^31M did not bind the long peptide. Just like cells expressing M3^f, M31 cells can present the pentamer peptide but not HA173–190 (compare Figs. 5 and 6). These results indicate that the Met³¹ of M3^f is largely responsible for its inability to bind the long peptide.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Met31 causes poor binding of HA peptides to M3f. Untransfected M3cas (B10.CAS2) fibroblast cells or cells that had been transfected with M3wt (CM3) or M3 with a Met31 (M31) were incubated overnight with 10 µM HA186–190 (), HA173–190 (•), or no peptide (x) and used as targets in a 3.5-h 51Cr-release assay with anti-HA173 CTLs. All CM3 and M31 targets were lysed with similar efficiency by alloreactive anti-M3wt CTLs in parallel experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recognition of nonformylated epitopes

M3-restricted CTLs directed to fMet peptides can recognize analogous nonformylated peptides, but the formylated ones are 100- to 10,000-fold more potent than the nonformylated ones (14, 16). Equally, anti-HA173 CTLs, which are induced against a nonformylated peptide, recognize the formylated peptide as well; again, the formylated analogue is 100-fold more potent than the nonformylated peptide used for inducing the response (Fig. 4). In all cases, the formyl group does not appear to alter T cell recognition, as peptide-specific CTLs lyse cells displaying either form of the peptides at the same maximal level, but it does affect the potency of the peptide for sensitizing target cells to lysis. These findings support that the key role of the formyl group in M3^wt-restricted responses is for binding peptides to the groove.

Binding by long peptides

We are uncertain how, and if, the long peptides HA173–190 and HA183–190 bind to M3. Anti-HA173 CTLs were induced with HA173–190, but the pentameric epitope can induce the response as well. Figures 5 and 7 suggest that the longer peptides are not degraded by serum proteases to the HA186–190 epitope before binding M3: HA173–190 and HA183–190 were incubated with cells overnight, which is ample time for degradation to occur (21). If they were degraded, cells expressing M3^f should have been sensitized by the MLIIW proteolytic product. Furthermore, HA173–190 sensitizes cells to lysis in serum-free conditions (data not shown). These results suggest that peptides with N-terminal extensions might bind M3^wt. This ability would be limited to nonformylated epitopes, because the N terminus of formylated epitopes is blocked by the formyl group. However, all peptides were synthesized using F-moc chemistry, which builds peptides from the C terminus, so the first five residues constructed for the longer peptides were still MLIIW: the minimal epitope. As peptide synthesis involves cycles of protecting and deprotecting the growing peptide substrate, failure sequences can occur that prevent extension of the growing peptide. These failures could indeed be responsible for the sensitization of target cells that we see in our experiments. Both HA173–190 and HA183–190 were fractionated by reverse-phase HPLC (data not shown). HA173–190 resolved poorly, because full-length product was detected in every fraction that sensitized target cells to lysis by anti-HA CTLs. By contrast, the most active fractions of HA183–190 did contain truncated peptides, although all fractions were capable of sensitizing cells to lysis as well. Coelution of MLIIW with HA173–190 or HA183–190 showed that the pentamer is hard to separate from the longer peptides, so purification of the long peptides would be difficult. If failures are responsible for sensitization of target cells, then they are 1) rare compared with full-length product, as they were not detected by mass spectrometry of the stock peptides; 2) uncapped, whereas failure sequences are typically N--acetylated (capped) with acetic anhydride, and acetylated methionine does not bind M3 (11); and 3) reproducible, as we have used three different syntheses of HA173–190 in our experiments to induce the same CTL response as Milligan et al. (1).

Presentation by M3^f

Like other class Ib molecules, M3 is not very polymorphic. Five alleles have been described that together encode proteins differing at 10 residues total; the spretus allele alone accounts for five of these changes (15). Furthermore, all molecules but M3^cas, which has two amino acid changes, 31(ValMet) and 95(LeuGln), can be recognized, more or less well, by CTLs specific for M3^wt. In particular, M3^f can be recognized like M3^wt by allospecific lines and clones but is recognized less well by some peptide-specific CTLs, e.g., anti-Listeria (25), anti-ND1 (21), and anti-HA (Fig. 5).

Poor recognition of M3^f by anti-HA CTLs could be due to low surface expression or a structural change that affects peptide binding and interaction with TCRs. We believe that M3^f and M3^wt are expressed similarly based on the equal recognition by some allospecific CTL clones, but we lack the M3-specific Abs to measure surface expression more directly. Our results with transfected cell lines indicate that poor recognition of M3^f is largely due to a structural change caused by the Met³¹ (Fig. 7). The structure of M3^wt shows that Val³¹ points away from the groove and makes a van der Waals contact with the Tyr²⁰⁹ in the 3 domain (24). The bulkier methionine substitution must alter the groove of M3 such that peptides bind poorly and recognition by peptide-specific CTLs is abrogated (Fig. 6). In hindsight, earlier studies in our laboratory using M31 cells support this: allospecific CTLs recognized M3^wt and M3^31M similarly, whereas M3^31M was recognized less well by peptide-specific CTLs (18).

Competition with mitochondrial fMet peptides

CTL recognition of virus-infected cells requires endogenous synthesis of viral proteins, cytosolic proteolysis to short peptides, and TAP-transport of peptides into the endoplasmic reticulum (ER), where they are loaded onto class I molecules and subsequently conveyed to the cell surface. In their original report, Milligan et al. demonstrated that anti-HA173–190 CTLs failed to lyse flu-infected cells or cells infected with an HA173–190 minigene and suggested that the undefined class Ib molecule may acquire peptides through a unique pathway not used for processing viral Ags (1). However, M3 acquires peptides through the classical pathway: anti-ND1 CTLs are TAP dependent, because neither RMA-S (TAP-2 negative) cells nor TAP-1° lymphoblasts are lysed by M3-restricted T cells (15). These results indicate that ND1 must be transported into the ER to bind M3 for surface expression.

Influenza HA is a transmembrane protein that is cotranslationally translocated into the ER. Although antigenic processing of transmembrane proteins is poorly understood, CTL responses to the HA of influenza A/JAP/305/57 have been well characterized. One strongly immunodominant region, HA202-221, which is close to HA186–190, contains two overlapping H2-K^d-restricted epitopes, HA202-212 and HA211-221 (26). Presentation of these epitopes is proteasome dependent and requires translocation into the ER (27, 28). Thus, HA epitopes are loaded onto class I molecules in the ER, where M3 acquires peptides as well. So why did Milligan et al.’s anti-HA173–190 CTLs not lyse cells expressing the epitope? It is possible that HA186–190 is proteolytically cleaved and not available for binding M3, but our most compelling explanation is provided in Figure 3. Formylated ND1, COI, and ND4 have very high affinities for M3: at equimolar concentration, they prevent HA186–190 from binding and, because they are formylated, are at least 100-fold more potent than the nonformylated HA186–190. Because most naturally presented peptides have a high affinity and slow off-rate for class I molecules (29, 30), the nonformylated epitope may be unable to compete against the high affinity mitochondrial peptides for binding to M3, and it is therefore not presented on the cell surface.

Historically, the generation of M3-restricted CTLs has required immunization of mice with either spleen cells or bacteria to prime the response, but here we show that stimulation in vitro with nonformylated peptide epitopes can induce M3-restricted responses as well. The response is not unique to HA173–190, as a nonformylated Bla-z (MFVLNKFF) peptide (the formylated form is known to bind well to M3 (23)) can also induce specific, M3-restricted CTLs. Lenz and Bevan have shown recently that the two formylated epitopes from Listeria monocytogenes, f-LemA and f-MIVIL, similarly can induce primary responses in vitro (31). However, in contrast to their finding that antipeptide CTLs are generated only from mice raised in conventional, not specific pathogen-free (SPF) conditions (or from SPF mice immunized with Listeria), our preliminary data indicate, at least for C57BL/6J, that spleen cells from SPF mice respond to HA173–190 just like cells from conventional mice do (data not shown). It is not clear whether there is a real discrepancy between these results, as we are comparing responses of different strains of mice (C57BL/6J vs BALB/c) against separate epitopes (HA186–190 vs f-LemA and f-MIVIL).

We still doubt whether M3 can naturally present nonformylated peptides: at least 10 nM peptide is required for half-maximal response to HA186–190 (Fig. 4), whereas naturally presented peptides are potent in the picomolar range. But the ease with which all these responses can be generated ex vivo suggests that a portion of the T cell repertoire in mice may be devoted to recognizing similar peptides presented by M3.

	Acknowledgments

 
Special thanks to Vik Dabhi for providing mitochondrial peptides, M3-restricted CTL clones, and words of wisdom about keeping CTLs happy. We thank Annie Eubanks, Margaretan Stolfo, and Henry Taylor for their excellent upkeep of and assistance with our mouse facility and Steve Madden, Lynn Mayfield, and Clive Slaughter of the Howard Hughes Biopolymers Facility at the University of Texas Southwestern Medical Center for peptide syntheses and helpful discussion.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI37818 and the Medical Scientist Training Program (D.E.B.).

² Address correspondence and reprint requests to Dr. Kirsten Fischer Lindahl, Howard Hughes Medical Institute, 5323 Harry Hines Boulevard, Dallas, TX 75235-9050. E-mail address:

³ Abbreviations used in this paper: HA, hemagglutinin; ß₂m, ß₂-microglobulin; fMet, formylmethionine; anti-HA173 CTL, cytotoxic T lymphocyte lines induced by stimulation with HA173–190; cas, castaneus; ER, endoplasmic reticulum; TAP, transporters associated with antigen processing; SPF, specific pathogen-free.

Received for publication September 8, 1997. Accepted for publication February 27, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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