Department of Immunology, Imperial College School of Medicine (St Marys Campus), Norfolk Place, London W2 1PG, UK1
Author for correspondence: Keith Gould. Fax +44 20 7402 0653. e-mail k.gould{at}ic.ac.uk
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Abstract |
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Introduction |
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Influenza virus epitopes that are recognized by CD8+ T lymphocytes in H-2b and H-2d mice have been well-characterized and their relative immunodominance has been investigated thoroughly (Vitiello et al., 1996 ; Belz et al., 2000
; Chen et al., 2000
). However, much less is known about the CD8+ T cell response to influenza virus in H-2k mice. Previously, we have investigated the specificity of the CTL response to influenza virus A/PR/8/34 in CBA/Ca (H-2k) mice and defined four different epitopes presented by the Kk molecule: two epitopes in the haemagglutinin (HA), one epitope in the nucleoprotein (NP) and one epitope in the non-structural protein NS1 (Gould et al., 1991
; Cossins et al., 1993
). Although it has long been thought that epitopes in the NP are immunodominant, the dominance hierarchy of the Kk-restricted epitopes has not been determined, and there is also an additional, uncharacterized Dk-restricted response to influenza virus in H-2k mice. This response was described as long ago as 1979 (Blanden et al., 1979
), but as yet no influenza virus peptide epitopes presented by the Dk major histocompatibility complex (MHC) class I molecule have been reported. Other studies have confirmed a prominent Dk-restricted response to influenza virus infection (Stringfellow et al., 1983
) and to the PB1 polymerase protein in H-2k mice (Bastin et al., 1987
; Bennink et al., 1987
; Bennink & Yewdell, 1988
; Reay et al., 1989
) and demonstrated, using T cell hybridomas, that there is a Dk-restricted response to PB1 (Daly et al., 1995
).
In this study, we have identified a Dk-restricted epitope derived from PB1 and shown, in polyclonal cell lines, that a large majority of CD8+ T cells recognize this peptide, implying that this is the major epitope within the influenza virus PB1 protein. Enzyme-linked immunospot (ELISpot) experiments demonstrated that this new epitope is at least as immunodominant during primary influenza virus infection of H-2k mice as any of the previously defined Kk-restricted epitopes.
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Methods |
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Recombinant VV.
PB1-VAC, a recombinant WR strain expressing the PB1 polymerase of influenza virus A/PR/8/34 has been described previously (Smith et al., 1987 ). Four new recombinant VV expressing overlapping fragments of PB1 were constructed. PB1 fragments were inserted into the thymidine kinase (tk) gene of VV using the shuttle vector pKG18 (Gould et al., 1991
) by standard procedures (Mackett et al., 1984
). PB1 DNA fragments were generated by PCR using Pfu polymerase (Stratagene), PB1-specific oligonucleotides containing restriction enzyme recognition sites and the plasmid that was used to make PB1-VAC as template DNA. The new recombinant viruses were designed to express influenza virus A/PR/8/34 PB1 gene fragments comprising amino acid residues 1150, 1300, 1450 and 1600. All plasmid constructs were confirmed by DNA sequencing using a Perkin Elmer ABI 373 DNA sequencer. Recombinant viruses were also assessed for the presence of the VV tk gene by PCR using viral genomic DNA and oligonucleotide primers specific for the tk gene (5' AATTAGACGAGTTAGACG 3' and 5' ATCTCGGTTTCCTCACCC 3').
Synthetic peptides.
An overlapping set of 71 different peptides, each nine amino acid (9-mer) residues in length, was synthesized by Chiron Mimotopes. Individual peptides were supplied by Research Genetics. Peptides were dissolved in RPMI 1640 medium for use in assays.
Cytotoxic assay.
A standard 5 h 51Cr-release assay was used with modifications for adherent target cell lines, as described previously (Townsend et al., 1984 ; Gould et al., 1991
). The target cell lines L929 (KkDk), C3H.OH (KdDk) and NA (KkDdLd) (Bennink & Yewdell, 1988
) were used. Virus infections of target cells, use of synthetic peptides and calculation of percentage of specific lysis were all as described previously (Gould et al., 1991
). The average values of all experimental points, which were in duplicate with quadruplicate controls, are shown.
Intracellular interferon (IFN)-
staining.
Effector T cell lines were incubated for 5 h at 37 °C either without peptide or with 10 µg/ml of peptide ARLGKGYMF in DMEM containing 10% foetal bovine serum (FBS) and 2 µg/ml of brefeldin A (Sigma). Cells were then washed and stained with anti-CD8-cychrome (Pharmingen) for 30 min on ice. After incubation, cells were washed in PBS containing 3% FBS and 0·1% NaN3 and fixed with 4% formaldehyde in PBS for 20 min. After washing, cells were permeablized with 0·5% saponin (Sigma) in PBS for 10 min, centrifuged and resuspended in 50 µl of 0·5% saponin in PBS. Cells were then stained with anti-IFN--PE (Pharmingen) for 20 min. After incubation, cells were washed in PBS containing 3% FBS and 0·1% NaN3 and analysed on an EPICS XL flow cytometer using Expo 2 software (Beckman Coulter).
ELISpot assay.
Female 8- to 10-week-old CBA/Ca mice were infected with 0·05 HAU influenza virus A/PR/8/34 in 50 µl PBS by intranasal inoculation or with 300 HAU in 500 µl PBS by intraperitoneal injection. After 8 days (Belz et al., 2000 ), the numbers of IFN-
-producing cells in spleen cell populations from individual mice were determined by ELISpot analysis (Power et al., 1999
). Nitrocellulose-bottomed 96-well plates (Millipore) were coated for 2 h at 37 °C followed by overnight incubation at 4 °C with rat anti-mouse IFN-
antibody (clone R4-6A2; Pharmingen). Dilutions of responder spleen cells in complete medium were cultured without or with 10 µM peptide epitope for 48 h. Plates were then washed and incubated with biotinylated IFN-
antibody (clone XMG1.2; Pharmingen) followed by streptavidin conjugated to alkaline phosphatase (Boehringer Mannheim). Spots were visualized using BCIP/NBT alkaline phosphatase substrate (Promega) and counted using an automated ELISpot plate counter (Autoimmun Diagnostika). Test wells were assayed in triplicate and the frequency of peptide-specific T cells present was calculated by subtracting the mean number of spots obtained in the presence of no peptide from the mean number of spots obtained in the presence of peptide.
IFN- ELISpot assay was also used to confirm the MHC restriction of the PB1 peptide epitope by using restimulated PB1-specific effector CTL (as responder cells), peptide epitope and transfected P815 feeder cells (H-2d haplotype) expressing either Dk or Kk MHC class I molecules. P815-Dk cells were obtained from Diane Scott, Imperial College School of Medicine, London, UK. Stable transfectant P815-Kk cells were generated by electroporation of P815 cells with an expression plasmid containing a full-length Kk cDNA under the control of the SV40 early promoter. MHC class I expression in both cell lines was verified by flow cytometry using specific antibodies.
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Results |
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To address whether peptide 24 was the dominant epitope within the entire PB1 protein, a polyclonal PB1-specific T cell line which had only been restimulated with influenza virus was incubated in the presence or absence of peptide 24 and the cells were stained for both CD8 and intracellular IFN- (Fig. 5
). Approximately 90% of the CD8+ cells in the polyclonal line recognized peptide 24, amino acid sequence ARLGKGYMF, and responded by producing IFN-
. This clearly showed that peptide 24 is by far the most immunodominant epitope within the PB1 polymerase protein. We confirmed that peptide 24 is presented by the Dk MHC class I molecule in an IFN-
ELISpot assay (Table 2
). P815 cells expressing Dk efficiently stimulated PB1-specific effector CTL to produce IFN-
in the presence of peptide 24, whereas P815 cells expressing Kk did not.
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Discussion |
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The recognition of many different peptides by PB1-specific T cells (Fig. 3) was surprising. Following previous experience of mapping Kk-restricted epitopes using sets of overlapping peptides, the expected result was for a few closely grouped peptides encompassing a single epitope to be recognized. However, this was clearly not the case and although a single peptide was recognized much more efficiently than all the others, suggesting the existence of a single epitope, multiple unrelated peptide sequences were recognized by the T cells. Although it was originally assumed that T cell receptors must be highly specific, more recent evidence has suggested that they may be cross-reactive (Brock et al., 1996
; Basu et al., 2000
) and indeed it has been argued that this is an essential feature of the T cell receptor (Mason, 1998
). Our results support the idea that T cell receptors may be cross-reactive for different peptides and further work to characterize this cross-reactivity using PB1-specific T cell clones is in progress.
It is intriguing that in the only other Dk-restricted T cell response studied in detail, that against polyomavirus middle T protein, similar results were observed and T cells specific for an immunodominant epitope were shown to recognize a second peptide from within the same protein (Wilson et al., 1999 ). This could, to some extent, reflect particular properties of the Dk class I molecule and the T cell receptors capable of recognizing it. However, even in the influenza virus model, there are examples of cross-reactivity not involving Dk. In H-2d mice, two PB2 polymerase peptides were found to be recognized by NP-specific CD8+ T cells (Anderson et al., 1992
) and we have observed that the NS1-derived peptide FDRLETLI causes T cells specific for the Kk NP epitope SDYEGRLI to proliferate, although not they do not sensitize target cells for lysis (K. G. Gould, unpublished data). All of these observations suggest that although there may be relatively few immunodominant epitopes in the response to a virus infection, there may be other peptide sequences within the virus that are capable of stimulating the T cells that recognize these immunodominant epitopes and therefore contribute to the overall response.
The importance of the Dk-restricted response against influenza virus was first suggested when 6 of 11 T cell hybridomas derived from influenza virus-infected H-2k mice were found to be Dk-restricted and specific for PB1 (Daly et al., 1995 ). We have confirmed the significance of this response after primary influenza virus infection using an IFN-
ELISpot assay. Our results showed that the PB1 epitope is at least as prominent as any of the other influenza virus epitopes in H-2k mice defined to date. Influenza virus NP has long been thought of as the immunodominant antigen for CD8+ T cells in mice, but while this may still be the case in H-2d mice (Chen et al., 2000
), the results presented here, and recently reported results obtained with H-2b mice (Belz et al., 2000
), have shown that this is not always the case, particularly in primary infection. In H-2b mice, it was thought that the Db-restricted NP 366374 epitope is immunodominant, but identification of a new Db-restricted epitope in the PA polymerase protein led to the demonstration that the response to this epitope is equally prominent during primary infection (Belz et al., 2000
). Interestingly, the NP 366374 epitope was still dominant in secondary responses and the PA-specific T cells did not lyse influenza virus-infected target cells efficiently in a chromium-release assay (Belz et al., 2000
). Our PB1-specific CTL were efficient at lysing influenza virus-infected target cells, but it would be worthwhile to test the prominence of PB1-specific CTL after secondary infection to see if the response was less apparent. It may also be the case that the uncharacterized Dd-restricted response to the influenza virus polymerase protein PB2 (Bennink & Yewdell, 1988
) is very prominent during infection of H-2d mice, but testing this hypothesis awaits the identification of the epitope in question.
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Acknowledgments |
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References |
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Bastin, J. M., Townsend, A. R. M. & McMichael, A. J. (1987). Specific recognition of influenza virus polymerase protein (PB1) by a murine cytotoxic T-cell clone. Virology 160, 278-280.[Medline]
Basu, D., Horvath, S., Matsumoto, I., Fremont, D. H. & Allen, P. M. (2000). Molecular basis for recognition of an arthritic peptide and a foreign epitope on distinct MHC molecules by a single TCR. Journal of Immunology 164, 5788-5796.
Belz, G. T., Xie, W., Altman, J. D. & Doherty, P. C. (2000). A previously unrecognized H-2Db-restricted peptide prominent in the primary influenza A virus-specific CD8+ T-cell response is much less apparent following secondary challenge. Journal of Virology 74, 3486-3493.
Bennink, J. R. & Yewdell, J. W. (1988). Murine cytotoxic T lymphocyte recognition of individual influenza virus proteins. High frequency of nonresponder MHC class I alleles. Journal of Experimental Medicine 168, 1935-1939.[Abstract]
Bennink, J. R., Yewdell, J. W., Smith, G. L. & Moss, B. (1987). Anti-influenza virus cytotoxic T lymphocytes recognize the three viral polymerases and a nonstructural protein: responsiveness to individual viral antigens is major histocompatibility complex controlled. Journal of Virology 61, 1098-1102.[Medline]
Blanden, R. V., Mullbacher, A. & Ashman, R. B. (1979). Different D end-dependent antigenic determinants are recognized by H-2-restricted cytotoxic T cells specific for influenza and Bebaru virus. Journal of Experimental Medicine 150, 166-173.[Abstract]
Brock, R., Wiesmuller, K.-H., Jung, G. & Walden, P. (1996). Molecular basis for the recognition of two structurally different major histocompatibility complex/peptide complexes by a single T-cell receptor. Proceedings of the National Academy of Sciences USA 93, 13108-13113.
Brown, E. L., Wooters, J. L., Ferenz, C. R., OBrien, C. M., Hewick, R. M. & Herrmann, S. H. (1994). Characterization of peptide binding to the murine MHC class I H-2Kk molecule. Sequencing of the bound peptides and direct binding of synthetic peptides to isolated class I molecules. Journal of Immunology 153, 3079-3092.
Chen, W., Anton, L. C., Bennink, J. R. & Yewdell, J. W. (2000). Dissecting the multifactorial causes of immunodominance in class I-restricted T cell responses to viruses. Immunity 12, 83-93.[Medline]
Cossins, J., Gould, K. G., Smith, M., Driscoll, P. & Brownlee, G. G. (1993). Precise prediction of a Kk-restricted cytotoxic T cell epitope within the NS1 protein of influenza virus using an MHC allele-specific motif. Virology 193, 289-295.[Medline]
Daly, K., Nguyen, P., Woodland, D. L. & Blackman, M. A. (1995). Immunodominance of major histocompatibility complex class I-restricted influenza virus epitopes can be influenced by the T-cell receptor repertoire. Journal of Virology 69, 7416-7422.[Abstract]
de Bergeyck, V., De Plaen, E., Chomez, P., Boon, T. & Van Pel, A. (1994). An intracisternal A-particle sequence codes for an antigen recognized by syngeneic cytolytic T lymphocytes on a mouse spontaneous leukemia. European Journal of Immunology 24, 2203-2212.[Medline]
Flynn, K. J., Belz, G. T., Altman, J. D., Ahmed, R., Woodland, D. L. & Doherty, P. C. (1998). Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 8, 683-691.[Medline]
Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G. & Garcia-Sastre, A. (1999). Rescue of influenza A virus from recombinant DNA. Journal of Virology 73, 9679-9682.
Gould, K. G., Scotney, H. & Brownlee, G. G. (1991). Characterization of two distinct major histocompatibility complex class I Kk-restricted T-cell epitopes within the influenza A/PR/8/34 virus hemagglutinin. Journal of Virology 65, 5401-5409.[Medline]
Hackett, C. J. & Askonas, B. A. (1981). H-2 expression by lymphoid cells of different mouse strains: quantitative interaction of H-2 with monoclonal antibodies and their Fab fragments. Immunology 42, 207-215.[Medline]
Lukacher, A. E. & Wilson, C. S. (1998). Resistance to polyoma virus-induced tumors correlates with CTL recognition of an immunodominant H-2Dk-restricted epitope in the middle T protein. Journal of Immunology 160, 1724-1734.
Mackett, M., Smith, G. L. & Moss, B. (1984). General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. Journal of Virology 49, 857-864.[Medline]
Mason, D. (1998). A very high level of crossreactivity is an essential feature of the T-cell receptor. Immunology Today 19, 395-404.[Medline]
Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., Hobom, G. & Kawaoka, Y. (1999). Generation of influenza A viruses entirely from cloned cDNAs. Proceedings of the National Academy of Sciences, USA 96, 9345-9350.
Norda, M., Falk, K., Rotzschke, O., Stevanovic, S., Jung, G. & Rammensee, H.-G. (1993). Comparison of the H-2Kk- and H-2Kkml-restricted peptide motifs. Journal of Immunotherapy 14, 144-149.[Medline]
ONeill, H. C. & McKenzie, I. F. C. (1980). Quantitative variation in H-2-antigen expression. I. Estimation of H-2K and H-2D expression in different strains of mice. Immunogenetics 11, 225-239.[Medline]
Parker, C. E. & Gould, K. G. (1996). Influenza A virus a model for viral antigen presentation to cytotoxic T lymphocytes. Seminars in Virology 7, 61-73.
Power, C. A., Grand, C. L., Ismail, N., Peters, N. C., Yurkowski, D. P. & Bretscher, P. A. (1999). A valid ELISPOT assay for enumeration of ex vivo, antigen-specific, IFN-producing T cells. Journal of Immunological Methods 227, 99-107.[Medline]
Reay, P. A., Jones, I. M., Gotch, F. M., McMichael, A. J. & Brownlee, G. G. (1989). Recognition of the PB1, neuraminidase, and matrix proteins of influenza virus A/NT/60/68 by cytotoxic T lymphocytes. Virology 170, 477-485.[Medline]
Smith, G. L., Levin, J. Z., Palese, P. & Moss, B. (1987). Synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses. Virology 160, 336-345.[Medline]
Stringfellow, M., Wraith, D. C. & Askonas, B. A. (1983). Cytotoxic T-cell recognition of influenza-infected target cells varies in different H-2k mouse strains. Immunogenetics 18, 177-181.[Medline]
Townsend, A. R. M., McMichael, A. J., Carter, N. P., Huddleston, J. A. & Brownlee, G. G. (1984). Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells. Cell 39, 13-25.[Medline]
Vitiello, A., Yuan, L., Chesnut, R. W., Sidney, J., Southwood, S., Farness, P., Jackson, M. R., Peterson, P. A. & Sette, A. (1996). Immunodominance analysis of CTL responses to influenza PR8 virus reveals two new dominant and subdominant Kb-restricted epitopes. Journal of Immunology 157, 5555-5562.[Abstract]
Wilson, C. S., Moser, J. M., Altman, J. D., Jensen, P. E. & Lukacher, A. E. (1999). Cross-recognition of two middle T protein epitopes by immunodominant polyoma virus-specific CTL. Journal of Immunology 162, 3933-3941.
Winter, G. & Fields, S. (1982). Nucleotide sequence of human influenza A/PR/8/34 segment 2. Nucleic Acids Research 10, 2135-2143.[Abstract]
Yewdell, J. W. & Bennink, J. R. (1999). Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annual Review of Immunology 17, 51-88.[Medline]
Zinkernagel, R. M., Althage, A., Cooper, S., Kreeb, G., Klein, P. A., Sefton, B., Flaherty, L., Stimpfling, J., Shreffler, D. & Klein, J. (1978). Ir-genes in H-2 regulate generation of anti-viral cytotoxic T cells. Mapping to K or D and dominance of unresponsiveness. Journal of Experimental Medicine 148, 592-606.[Abstract]
Received 16 January 2001;
accepted 20 March 2001.