A. Müllbacher, Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT 2601, Australia
Cytotoxic T (Tc)-cell responses against influenza virus infection in BALB/c (H-2d) mice are dominated by Tc clones reactive to the viral nucleoprotein (NP). Here, we report investigations using recombinant vaccinia viruses (VV) encoding major histocompatibility complex (MHC) class I H-2Kd molecules differing by a single amino acid from glutamine (wild-type, Kdw) to histidine (mutant, Kdm) at position 114 located in the floor of the peptide-binding groove. Influenza-infected target cells expressing Kdw were strongly lysed by Kd-restricted Tc cells against A/WSN influenza virus or the immunodominant peptide of viral NP (NPP147–155), whereas infected Kdm-expressing targets gave little or no lysis, respectively, thus showing the immunodominance of NPP147–155. Kdm-expressing target cells saturated with synthetic NPP147–155 (10−5m) were lysed similarly to Kdw-expressing targets by NPP147–155-specific Tc cells. Thus the defect in influenza-infected Kdm-expressing targets was quantitative; insufficient Kdm–peptide complexes were expressed. Tc-cell responses against four other viruses or alloantigens showed no effect of Kdm. When peptide transport-defective cells were infected with VV-Kdw or VV-Kdm and co-infected with a recombinant VV encoding an endoplasmic reticulum-targeted viral peptide, two influenza haemaglutinin peptides caused higher expression of Kdw than NPP147–155 indicating their higher affinity for Kdw. These results are inconsistent with the hypothesis that immunodominance in the anti-influenza response reflects high affinity of the immunodominant peptide, but are consistent with skewing of the Tc-cell receptor repertoire.
The cytotoxic T-cell response against viral infections and minor histocompatibility antigens, including H-Y, is often characterized by a phenomenon known as immunodominance in which the majority of effector T cells are reactive to a particular epitope [1–4]. The epitope to which the antigen receptors of CD8+ cytotoxic T (Tc) cells bind is an area on the surface of major histocompatibility complex (MHC) class I heavy chain molecule containing an embedded viral peptide of 8–10 amino acids [5, 6]. The majority of the surface of the epitope is provided by two alpha helices of the MHC molecule, which form the boundaries of a groove in which the peptide binds. The floor of the groove is formed by a beta sheet. Polymorphism in the MHC class I amino acid sequence is concentrated in the alpha helices and beta sheet, thus affecting pockets which accept side chains of the amino acids of viral peptides, and endowing individual MHC class I molecules with a characteristic peptide-binding motif [7–9]. In this paper, we have studied the effect on Tc-cell responses of a spontaneous mutation (Kdm) causing a change from wild-type (Kdw) in the amino acid (glutamine to histidine) at position 114 in the floor of the groove of the H-2Kd molecule  with respect to the immunodominant peptide derived from influenza virus nucleoprotein (NP). The results suggest that in this case, immunodominance is a reflection of a skewed Tc-cell receptor repertoire, rather than a high affinity of NP-derived peptide(s) for Kd.
MATERIALS AND METHODS
BALB/c (KdDd), C57BL/6 (KbDb), C3H.H-2° (KdDk) and B10.HTG (KdDb) were bred under pathogen-free conditions at the JCSMR breeding facilities. Only females were used at ≥ 12 weeks of age.
Viruses and synthetic peptides
The vaccinia viruses (VV) WR strain (VV-WR), the thymidine kinase-deficient virus (VV-Tk−), the VV-recombinants encoding the mutant mouse MHC class I heavy chain Kd (VV-Kdm) , the VV-recombinants encoding the wild-type mouse MHC class I heavy chain Kd (VV-Kdw) , the VV encoding the Kd-binding influenza NP peptide determinant, NPP147–155 , as a minigene (VV-NP147–155), the VV-encoding NPP147–155 as a minigene plus the ER targeting signal sequence (VV-ES/NPP)  and the VV containing the full NP gene of A/influenza virus A/Pr8 (VV-NP)  were grown on CV-1 cell monolayers and titrated as described previously .
The A strain influenza virus A/WSN  and the flavivirus Kunjin (KUN)  were prepared and titrated as described previously.
The Kd-binding synthetic peptide TYQRTRALV(NPP) derived from the sequence of amino acids 147–155 of the NP of A/influenza virus  was synthesized at the Biomolecular Resource Facility (ANU, Canberra, Australia).
The mouse cell lines L929 (H-2k), MC57 (H-2b), HTG (KdDb), the monkey cell line CV-1 and the human cell line 143-B were grown in Eagle's minimal essential medium (EMEM) supplemented with 10% fetal calf serum (FCS). The cells were labelled with 51Cr for 1 h and infected with VV at a multiplicity of infection (MOI) of 10–20 plaque-forming units (PFU) per cell. Target cells were infected with influenza virus A/WSN, VV  or KUN , or treated with NPP as described previously . Mock infection comprised similar treatment with virus-free material.
Animals were immunized with 107 PFU of VV-WR, 104 haemagglutinating units (HAU) of A/WSN or 5 × 106 PFU KUN intraperitoneally in 0.2 ml.
MC57 (H-2b) cells were infected at a MOI of 20 with VV-Kdw or VV-Kdm as described for target cells, or were left uninfected. At 3 h after infection the cells were washed, resuspended at 107 cells/ml, and labelled to saturation at 4 °C for 45 min with monoclonal antibody (MoAb) HB-159 (ATCC) specific for Kd, followed by fluorescein isothiocyanate-conjugated sheep anti-mouse immunoglobulin (Silenus, Hawthorn, Australia) staining. Cells were examined using a FACScan flow cytometer (Becton Dickinson).
For the generation of alloreactive Tc cells, 8 × 107 responder splenocytes were co-cultured with 4 × 107 irradiated (2000 rad) allogeneic stimulator cells for 5 days in 40 ml EMEM, 10% FCS plus 10−5m 2-mercaptoethanol. The generation of secondary influenza-immune Tc cells, secondary VV-immune Tc cells, secondary KUN-immune Tc cells  and secondary anti-NPP Tc cells  has been described previously.
51Cr release cytotoxicity assay
The methods used for cell line targets have been described in detail elsewhere [14, 16, 17]. The duration of the assays was 6 h. Percentage specific lysis was calculated by the formula:
Data given are the means of triplicate determinations. SEM values were always < 5%.
Class I MHC stabilization assays on T2 cells
T2 cells were infected at a MOI of 10 with VVs encoding NPP147–155, HA189–197, HA529–537, the malaria peptide CSP252–260 or ICAM-1 behind the Adenovirus 2 E19 leader sequence [18–20] co-infected with either VV-Kdw or VV-Kdm and incubated overnight at 37 °C in serum-free RPMI-1640 supplemented with cytosine arabinoside at 40 mg/ml to minimize VV-associated cytotoxicity. Cells were then incubated with a saturating concentration of the Kd-specific MoAb HB159 conjugated to fluorescein (Pharmingen, San Diego, CA) for 30 min at 0 °C, and viable cells were analysed by cytofluography in a FACScan.
Affect of the Kdm mutation on the Tc-cell response to influenza virus infection
In the Tc-cell response of BALB/c mice to influenza, ≈ 75% of the total responding Tc cells are reactive against the viral NP . Secondary Tc-cell responses were generated against either influenza virus A/WSN or the immunodominant peptide (NPP147–155) of the viral NP  and tested against target cells expressing either Kdw or Kdm as a result of infection with recombinant VV. Control secondary anti-VV Tc cells restricted by Kdw lysed targets expressing Kdw or Kdm to a comparable extent (Fig. 1B, D). This indicated similar levels of expression of Kdw and Kdm and no effect of the mutation on the anti-VV response. In contrast, lysis of influenza-infected target cells expressing Kdm by either anti-A/WSN (Fig. 1A) or anti-NPP Tc cells (Fig. 1C) was virtually eliminated in comparison with similar targets expressing Kdw. These results show that the majority of Kd-restricted Tc cells responding to influenza infection in BALB/c mice recognize NPP147–155 and strongly suggest that NPP binding to MHC class I is adversely affected by the mutation at amino acid 114 (see Table 1).
Table 1. . Cell-surface expression* of Kdw or Kdm on T2 cells infected with VV-Kdw or VV-Kdm and co-infected with recombinant VV expressing ER-targeted Kd-binding peptides or ICAM-1 *Data are mean channel fluorescence; background value of 16 subtracted from each.
Kdm and Kdw were also compared with respect to Tc-cell responses against Sendai, West Nile virus  and Kunjin (Fig. 2). As with VV, these responses were not significantly affected by Kdm.
Cell-surface expression of wild-type and mutant Kd in T2 cells using ER-targeted peptide minigenes encoded by recombinant VV
The assembly of Kdw and Kdm molecules with biosynthesized ER-targeted peptides was examined using T2 cells. These human cells lack TAP and express low levels of native class I HLA molecules at the cell surface due to the virtual absence of transport to the lumen of the endoplasmic reticulum (ER) of peptides that bind class I molecules . Co-infection of T2 cells with VV-Kd and rVVs expressing ER-targeted (containing signal sequences), Kd-binding peptides can enhance the cell-surface expression of Kd, providing a simple means of testing the binding of peptides to the different class I molecules in the ER .
T2 cells were infected simultaneously overnight with VVs expressing either Kdw or Kdm and a second VV expressing one of four ER-targeted, Kd-binding peptides or a control VV expressing ICAM-1. Kd expression on the surface of viable cells was determined by binding of a fluorescein-conjugated antibody (HB159) that binds only Kd molecules. As expected, all four of the ER-targeted peptides enhanced Kdw expression (Table 1) with NPP147–155 being less efficient than the others, implying a lower affinity for Kdw. Three of these peptides also enhanced expression of Kdm. Indeed the presence of HA189–197 resulted in greater expression of Kdm than Kdw, however, HA529–537 and CSP252–260 showed less enhancement with Kdm than with Kdw. By contrast, NPP147–155 was unable to rescue cell surface Kdm over levels observed in cells co-infected with the control VV expressing ICAM-1. This demonstrates that Kdm has a lower affinity for NPP147–155 than Kdw when the peptide is delivered to the ER.
Wild-type, but not mutant, VV-encoded Kd can present NPP derived from translation of the full-length NP gene
L929 (H-2k) target cells were infected with VV-Kdw or VV-Kdm and co-infected with VV-NP, and their susceptibility to lysis by VV- and influenza virus NP-immune BALB/c (H-2d) Tc cells was tested. Kd-restricted VV-immune Tc cells lysed VV-Kdw- and VV-Kdm-infected target cells to a similar extent (Fig. 3). This indicates similar levels of expression of Kdw and Kdm loaded with VV peptides on the target cells. In contrast, NP-immune Tc cells did not lyse targets infected with VV-Kdm and co-infected with VV-NP, as shown previously . However, target cells infected with VV-Kdw-and co-infected with VV-NP were lysed efficiently. This indicates that VV-NP is a source of NP peptides (NPP) but that the low affinity of Kdm for NPP147–155 results in insufficient cell surface expression of Kdm-NPP147–155 complexes to trigger Tc-cell function. These data explain why influenza-infected Kdm-expressing target cells were not lysed in the experiment shown in 1Fig. 1A.
Mutant Kdm can bind and present exogenous NPP
From the experiments shown above it is clear that Kdm is unable to present NPP generated endogenously from full-length NP genes in either influenza virus or VV-NP. Therefore, we investigated its ability to present exogenously supplied synthetic NPP. L929 target cells were infected with the control VV-Tk− or VV-Kdm and treated with 10−5m synthetic NPP147–155 for 1 h and assayed for susceptibility to lysis by BALB/c (H-2d) influenza- or VV-immune Tc cells (Fig. 4). Target cells infected with VV-Kdm and treated with NPP147–155 were lysed efficiently by H-2d-restricted influenza virus-immune Tc cells. The value of 10−5m is on the saturation concentration plateau of NPP147–155 for interaction with Kdm and Kdw; at lower concentrations around 10−7m, Kdm-expressing target cells are lysed much less than Kdw -expressing targets .
The result in Fig. 4 shows that despite its low affinity for Kdm, NPP147–155, at high concentrations, can form immunogenic complexes with Kdm that are bound by Tc cell receptors originally selected by Kdw-NPP147–155 (during priming and restimulation). However, during influenza infection of target cells the supply of NPP147–155 is quantitatively limiting in the case of Kdm.
Cell-surface expression of mutant and wild-type Kd using vaccinia virus expression on allogeneic targets
MC57 tumour cells were infected with either VV-Kdw or VV-Kdm and cell surface expression was determined by FACS analysis after staining with a Kd-specific MoAb (Fig. 5). Infection with either of these viruses caused a similar increase of fluorescence intensity over that of mock-infected cells, which suggests that neither cell surface expression nor binding of the specific antibody HB159 is affected by the mutation at position 114.
Alloreactive Tc cells do not distinguish between mutant and wild-type Kd
To investigate whether Kdm can be distinguished from Kdw by alloreactive Tc cells, we used Kdm and Kdw in the induction as well as target-cell-recognition phases of alloreactive Tc cells. In one type of experiment, as responder splenocytes in vitro we used B10.HTG (KdDb), and syngeneic stimulator splenocytes infected with VV-Kdm or VVKdw. In addition, we used C57Bl/6 (H-2b) responders and stimulated them with syngeneic splenocytes infected with VV-Kdm, VVKdw, or with irradiated splenocytes from B10.HTG. Primary in vivo anti-VV responses were also generated in either B10.HTG or C57BL/6 mice. Lysis was evaluated on HTG and MC57 target cells, infected with either VV-Kdm or VV−Kdw, or mock-infected (Table 2). In the case of B10.HTG responders there was no evidence of a Kdm-specific response. With HTG targets, infection with VV-Kdm led to slightly higher lysis than infection with VV-Kdw but this was true after stimulation with all three types of vaccinia-infected stimulator cell. In the case of C57BL/6 responders there was no evidence of a Kdm-specific response except that MC57 targets infected with VV-Kdw were lysed slightly more than MC57 targets infected with VV-Kdm. We investigated whether this differential lysis was due to individual alloreactive Tc cell clones which lysed targets expressing the Kdw molecule but not targets expressing Kdm. Split clone limiting dilution experiments were performed. C57BL/6 splenocytes at a responder concentration of 3 × 103–12 × 103 per well were stimulated with 2 × 105 B10.HTG stimulator cells per well for 6 days. Individual wells were split and tested for lysis of MC57 target cells infected with either VV-Kdm or VV-Kdw. In no instance out of 150 clones screened did we find clones that lysed VV-Kdw-infected targets and did not lyse VV-Kdm-infected targets (data not shown).
Table 2. . Induction and recognition of alloreactive anti-Kd Tc cells using VV-encoded mutant and wild-type Kd % specific lysis of target cells* *Target cells were labelled with 51Cr and infected with recombinant VV at MOI of 20 PFU/cell or mock-infected as described previously . Assay duration was 6 h. SEM were never > 5%. †Splenocytes of B10.HTG or C57BL/6 were infected with 10 PFU/cell of VV-Kdm or VV-Kdw and cocultured with uninfected syngeneic splenocytes at a ratio of 1 : 5 for 5 days. Primary in vivo‡VV-immune and in vitro§alloreactive Tc cells were generated as described previously .
Since alloreactive Tc cells recognize MHC class I molecules loaded with endogenous cell-derived peptides , these results emphasize that the Kdm mutation has no detectable effect on such complexes. Tc-cell clones distinguishing between Kdm and Kdw seem to be nonexistent or at low frequency (< 1/150).
The Kdm mutation presumably arose spontaneously in the mouse colony from which the original cDNA encoding Kd was obtained [11, 25]. We have shown here that the Kd-specific MoAb HB159, which binds to the α3 domain , does not differentiate between the wild-type and mutant molecules. Furthermore, alloreactive Tc cells were unable to distinguish between mutant and wild-type molecules at the induction and effector levels, using both bulk culture and split clone limiting dilution approaches in vitro over 6 days of culture. It would be interesting to see whether the mutation could be detected in skin graft experiments. Rare T-cell clones distinguishing between mutant and wild-type Kd could possibly produce delayed graft rejection.
Antiviral Tc-cell responses against Sendai virus, West Nile virus , VV and Kunjin were also minimally affected by the Kdm mutation. Thus, in the case of alloreactive and most antiviral Tc cells, there is little or no evidence of qualitative or quantitative differences between epitopes with various peptides and Kdw or Kdm.
With respect to the Tc-cell response against influenza virus infection, previous reports have clearly shown the immunodominance of the viral NP. Wysocka and Bennink  showed that 75% of Tc-cell clones were NP-reactive, with other viral proteins sharing the remaining clones.
Furthermore, in routine secondary responses in vitro, HA-specific Tc cells are often undetectable and the response is dominated by NP-specific Tc cells. The HA-specific response requires repeated restimulation . The present experiments involving comparison of Kdm and Kdw as restriction elements during the Tc-cell response to influenza virus infection reinforced the case for the immunodominance of the viral NP. When Tc cells specific for the immunodominant NP peptide (NPP147–155) were tested on target cells infected with influenza virus, expression of Kdw via VV-Kdw gave substantial lysis, but this was completely abrogated when VV-Kdm provided the restriction element. When anti-influenza Tc cells were used against the same targets, there was again little lysis of Kdm-expressing targets, indicating that the majority of effector Tc cells in the bulk culture were NPP147–155-specific. The lack of lysis of influenza-infected, Kdm-expressing target cells is not due to a qualitative difference between Kdw- and Kdm–NPP147–155 complexes, since we have shown here that saturation levels of exogenous, synthetic NPP147–155 (10−5m) gave equal lysis of Kdw- and Kdm-expressing targets. The defect in Kdm-expressing, influenza-infected target cells is quantitative; insufficient Kdm–NPP147–155 complexes are expressed on the cell surface to trigger Tc cells as discussed in detail elsewhere .
The weak response to HA is not due to antigen-processing problems. Previous results indicated that complexes of Kdm and HA-derived peptides were expressed on target cells and recognized by Tc cells, even when Kdm and HA were expressed via separate infections with recombinant VV vectors . Additional information pertinent to this issue is provided by the present experiments. When interactions between Kdm or Kdw and various influenza peptides were examined in more detail by co-infection with VV vectors and detection of MHC–peptide complexes by Kd-specific antibody, it was shown that the immunodominant NP peptide (NPP147–155) had considerably reduced affinity for Kdm in comparison with Kdw. In contrast, HA peptides (HA189–197 and HA529–537) were affected much less by the mutation in Kdm; in fact the affinity of HA189–197 was increased. Both of the HA peptides had a higher affinity for Kdw than NPP147–155, a finding of particular interest in the context of immunodominance.
One possible explanation for immunodominance in Tc-cell responses is that a particular (immunodominant) viral peptide has much higher affinity for a particular MHC class I molecule than others and therefore dominates numerically in the complexes expressed on infected cell surfaces . In turn, assuming an unbiased Tc-cell precursor repertoire, these complexes would activate the majority of Tc-cell precursors during viral infection. However, this explanation does not account for the dominance of NPP147–155 in Kd-restricted responses against influenza virus infection, since NPP147–155 has a lower affinity for Kdw than two HA peptides, HA189–197 and HA529–537 as discussed above. An alternative explanation of immunodominance of NPP147–155 in Kd-restricted anti-influenza Tc-cell responses is that the Tc-cell repertoire is numerically biased towards NPP147–155-reactive clones, with a minority of precursors reactive to HA-derived peptide–MHC complexes. Self-tolerance may impose this bias via cross-reactivity between self MHC–peptide complexes and self MHC–viral peptide complexes. Thus, clonal deletion and/or anergy imposed by self-tolerance may skew the antiviral precursor Tc-cell pool as first reported over 15 years ago .
We would like to thank Dr Frank Momburg (Heidelberg Germany) for helpful discussions.