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Differential effect of cytotoxic T lymphocyte variant epitopes on generation and cytotoxicity in chronic hepatitis C virus infection
Hepatology Research 24 (2002) 91 – 94
www.elsevier.com/locate/ihepcom
Differential effect of cytotoxic T lymphocyte variant epitopes on generation and cytotoxicity in chronic hepatitis C virus infection Kazumasa Hiroishi a,*, Jun-ichi Eguchi a, Shigeaki Ishii a, Hiroaki Okamoto b, Keiji Mitamura a, Michio Imawari c,* a
Second Department of Internal Medicine, Showa Uni6ersity School of Medicine, 1 -5 -8 Hatanodai, Shinagawa-ku, Tokyo 142 -8666, Japan b Immunology Di6ision, Jichi Medical School, Tochigi, Japan c Di6ision of Gastroenterology, Omiya Medical Center, Jichi Medical School, 1 -847 Amanuma-cho, Saitama 330 -8503, Japan Received 7 March 2001; received in revised form 14 February 2002; accepted 12 March 2002
Abstract Human leukocyte antigen (HLA) B44-restricted, hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) recognize HCV nucleoprotein amino acid residues 88 – 96 as an epitope. We previously reported the existence of variant peptide sequences at the epitope locus in three of 27 patients with HCV infection and HLA B44. Here we studied the effects of the variant peptide sequences on generation and cytotoxicity of CTLs. Two of the three variant peptides generated CTLs poorly although they activated well the cytotoxicity of CTLs. Such a differential activation of proliferation and cytotoxicity may contribute to the emergence of HCV with variant epitopes for CTLs. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis C virus; Cytotoxic T lymphocyte; Variant epitope; HLA B44
1. Introduction Cytotoxic T lymphocytes (CTLs) are thought to be one of the major host defense arms against viral infection [1] and are also implicated in the immunopathogenesis of viral infection [2]. We previ-
* Corresponding authors. Tel.: + 81-3-3784-8535; fax: + 81-3-3784-7553 or tel.: + 81-48-647-2111; fax: + 81-48-6485166 E-mail addresses: [email protected] (K. Hiroishi), [email protected] (M. Imawari).
ously demonstrated the existence of hepatitis C virus (HCV)-specific CTLs that recognize HCV nucleoprotein residues 88–96 in association with human leukocyte antigen (HLA) B44 in the peripheral blood of some patients with HCV infection [3,4]. We found that a detectable CTL response to the HCV nucleoprotein was associated with a low titer of serum HCV RNA [5], suggesting that HLA B44-restricted CTLs might inhibit the outgrowth of HCV. In the same study, we found that some of the patients were infected with HCV that had variant peptide sequences at the HCV nucleoprotein residues 88–96.
1386-6346/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 0 2 ) 0 0 0 7 8 - 5
K. Hiroishi et al. / Hepatology Research 24 (2002) 91–94
92
In the present study, we studied the effects of the variant HCV peptides on the cytotoxicity and generation of HCV-specific CTLs.
2. Materials and methods
wild-type peptide of HCV of genotypes 1a and 2b. 9-mer peptides, NETCGWAGW designated as N9-90T91C, DEGLGWAGW designated as N988D91L and NEGLGWTGW designated as N991L94T, are the variant peptides of HCV. All peptides were purchased from Mimotopes Pty (Victoria, Australia).
2.1. Subjects 2.4. CTL Assay Three HLA B44-positive patients infected with HCV that had variant peptide sequences at HCV nucleoprotein amino acid residues 88– 96 were studied. They were among 27 HLA B44-positive patients that were studied previously [5]. Characteristics of the three patients are shown in Table 1. Informed consent was obtained from all subjects, and the study was approved by the Ethical Review Committees of Jichi Medical School.
2.2. Serum HCV RNA, HCV genotype, an amino acid sequence of HCV nucleoprotein residues 88 – 96 Serum HCV RNA, the HCV genotype and the sequence of part of nucleoprotein gene of HCV were determined and the titer was expressed as described previously [5].
2.3. Synthetic peptides A 9-mer peptide corresponding to HCV nucleoprotein residues 88– 96, NEGLGWAGW designated as N9-91L, is the wild-type peptide of HCV of genotype 1b as well as 2a [6]. A 9-mer peptide, NEGCGWAGW designated as N9-91C, is the
Peripheral blood lymphocytes (PBLs) were isolated from heparinized peripheral blood and stimulated twice with the individual 9-mer peptides as described previously [5]. The cytotoxic activity of peptide-stimulated effector cells was assessed against Epstein–Barr virus-transformed HLA B44-positive B cell lines (BCLs) sensitized with the individual HCV peptides with a 4-h europium (Eu)-release assay [4].
3. Results Patient 1 was infected with HCV of genotype 2a, but the amino acid at position 91 of the nucleoprotein was cysteine and was that of isolates of HCV of genotypes 1a and 2b [6]. When we stimulated the PBLs of patient 1 with the 9-mer N9-91C peptide and the variant peptide N9-90T91C, N9-91C-stimulated effector cells recognized both targets sensitized with the N9-91C peptide and those sensitized with the N9-90T91C; the CTLs recognized the latter more efficiently than the former (Fig. 1a). The variant peptide N9-90T91C also generated CTLs although the
Table 1 The characteristics of patients studied and the amino acid sequences of the nucleoprotein residues 88–96 of infected HCV Subjects
Age
Sex
Diagnosis (grade)
ALT
HCV RNA
Genotype
Amino acid Residues 88–96*
Patient 1 Patient 2 Patient 3
58 59 63
Male Male Male
LC, HCC (mild) CH (mild) CH (mild)
69 115 26
102 103 ]104
2a 1b 1b
NETCGWAGW DEGLGWAGW NEGLGWTGW
Abbreviations: LC, liver cirrhosis; HCC, hepatocellular carcinoma; CH, chronic hepatitis; ALT, alanine aminotransferase (normalB 30 U/l). * The common sequences between the HCV nucleoprotein amino acid residues 88–96 are NEGLGWAGW for genotypes 1b and 2a, and NEGCGWAGW for genotype 2b. The residues 88–96 is the minimal and optimal epitope for HLA B44-restricted CTL. Amino acids are identified by one-letter codes.
K. Hiroishi et al. / Hepatology Research 24 (2002) 91–94
93
Fig. 1. Cytotoxicity of effector cells stimulated by HCV wild-type or variant peptides. PBLs of patient 1 were stimulated with (a) the N9-91C (NEGCGWAGW; a wild-type peptide of residues 88 – 96 of genotypes 1a and 2b HCV) or (d) the variant N9-90T91C peptide (NETCGWAGW). Since autologous PBLs of patient 2 were not available, PBLs of another patient infected with prototype HCV of genotype 1b were stimulated with (b) the wild-type peptide N9-91L (NEGLGWAGW) or (e) the variant peptide N9-88D91L (DEGLGWAGW). PBLs of patient 3 were stimulated with (c) the wild-type peptide N9-91L (NEGLGWAGW) or (f) the variant peptide N9-91L94T (NEGLGWTGW). The concentration of the peptides used for stimulation of PBLs was 10 mM. The cytotoxic activity of the effector cells was assessed against HLA B44-positive BCLs sensitized with the indicated concentrations of the wild-type peptide or variant peptides at an effector to target cell ratio of 40. Results are reported as mean specific lysis (%)9S.D.
N9-90T91C peptide-stimulated CTLs lysed target cells a little less efficiently than the N9-91C peptide-stimulated CTLs (Fig. 1d). PBLs of patient 2 had had a detectable CTL response to the wild-type peptide (data not shown). However, since PBLs of patient 2 were not available thereafter, PBLs of another patient infected with HCV with the common sequence of residues 88– 96 of genotype 1b was used to study whether the N9-88D91L peptide affected the cytotoxicity and generation of CTLs. Peptide N9-91L-stimulated CTLs recognized both targets sensitized with the N9-91L peptide and those sensitized with the variant peptide N9-88D91L although the lysis of the variant peptide-sensitized targets by the CTLs was a little less efficient than that of the wild-type peptidesensitized targets (Fig. 1b). However, the N9-
88D91L peptide was not able to generate CTLs efficiently (Fig. 1e). When the autologous PBLs of patient 3 were stimulated with the N9-91L peptide or the N991L94T peptide, peptide N9-91L-stimulated CTLs recognized equally both targets sensitized with the N9-91L peptide and those sensitized with the peptide N9-91L94T, but the N9-91L94T peptide was more than 1000-fold less efficient to generate CTLs compared with the peptide N991L (Fig. 1c, f).
4. Discussion Chang et al. [7] have reported that variant viral peptide sequences at HCV-specific CTL epitope loci were observed in 8– 69% of HCV sequences
94
K. Hiroishi et al. / Hepatology Research 24 (2002) 91–94
depending on the loci and that some of them were potential CTL escape variants. We found the variant peptide sequences at the immunodominant HLA B44-restricted HCV-specific CTL epitope locus in three of 27 patients with chronic HCV infection and HLA B44 [5]. Two of the variant peptides activated the cytotoxicity of CTLs, but did not generate CTLs effectively. Such a differential activation of proliferation and cytotoxicity has been demonstrated in human T-cell lymphotropic virus type I Tax-specific CTLs by an altered peptide ligand [8]. In addition, it has been reported that natural altered peptide ligands of HIV have the capacity to inhibit the range of CTLs directed against an epitope [9], and the impaired reaction to variants of HIV could contribute to the loss of immune control of the infection [10]. We have also reported that co-existence of the variant peptide of patient 3 and the wild-type peptide selectively induced CTLs recognizing the wild-type peptide but not those recognizing the variant peptide [11]. Thus the shift in viral sequence from wild-type to the variants may lead to the loss of immune control of the variant HCV and contribute to the emergence of HCV variants in some patients by affecting the generation of CTLs even if CTLs directed to a wild-type peptide sequence and cross-reactively recognizing the variant peptides were primed. Further studies will be needed to confirm our hypothesis and to clarify the role of mutations at CTL epitope loci in HCV persistence.
Acknowledgements We thank Drs Makoto Mayumi, Takashi Kaneko, Takashi Moriyama (Jichi Medical School), Tatsuya Aikawa (Aikawa Hospital), and Kazuki Ando (Gifu University) for helpful discussion.
References [1] Yap KL, Ada GL, McKenzie IFC. Transfer of specific cytotoxic T lymphocytes protects mice inoculated with influenza virus. Nature 1978;273:238 – 9. [2] Zinkernagel RM, Haenseler E, Leist T, Cerny A, Hengartner H, Althage A. T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay? J Exp Med 1986;164:1075 – 92. [3] Kita H, Hiroishi K, Moriyama T, et al. A minimal and optimal cytotoxic T-cell epitope within hepatitis C virus nucleoprotein. J Gen Virol 1995;76:3189 – 93. [4] Kita H, Moriyama T, Kaneko T, et al. HLA B44-restricted cytotoxic T lymphocytes recognizing an epitope on hepatitis C virus nucleocapsid protein. Hepatology 1993;18:1039 – 44. [5] Hiroishi K, Kita H, Kojima M, et al. Cytotoxic T lymphocyte response and viral load in hepatitis C virus infection. Hepatology 1997;25:705 – 12. [6] Weiner AJ, Christopherson C, Hall JE, et al. Sequence variation in hepatitis C viral isolates. J Hepatol 1991;13(Suppl. 4):S6 – S14. [7] Chang K-M, Rehermann B, McHutchison JG, et al. Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus. J Clin Invest 1997;100:2376 – 85. [8] Ho¨ lsberg P, Weber W, Dangond F, Batra V, Sette A, Hafler D. Differential activation of proliferation and cytotoxicity in human T-cell lmphotropic virus type I Taxspecific CD8 T cells by an altered peptide ligand. Proc Natl Acad Sci USA 1995;92:4036 – 40. [9] Klenerman P, Meier U-C, Phillips R, McMichael A. The effects of natural altered peptide ligands on the whole blood cytotoxic T lymphocyte response to human immunodeficiency virus. Eur J Immunol 1995;25:1927 – 31. [10] McAdam S, Klenerman P, Tussey L, et al. Immunogenic HIV variant peptides that bind to HLA-B8 can fail to stimulate cytotoxic T lymphocyte responses. J Immunol 1995;155:2729 – 36. [11] Kaneko T, Moriyama T, Hiroishi K, et al. Impaired induction of cytotoxic T lymphocytes by antagonism of a weak agonist borne by a variant hepatitis C virus epitope. Eur J Immunol 1997;27:1782 – 7.
www.elsevier.com/locate/ihepcom
Differential effect of cytotoxic T lymphocyte variant epitopes on generation and cytotoxicity in chronic hepatitis C virus infection Kazumasa Hiroishi a,*, Jun-ichi Eguchi a, Shigeaki Ishii a, Hiroaki Okamoto b, Keiji Mitamura a, Michio Imawari c,* a
Second Department of Internal Medicine, Showa Uni6ersity School of Medicine, 1 -5 -8 Hatanodai, Shinagawa-ku, Tokyo 142 -8666, Japan b Immunology Di6ision, Jichi Medical School, Tochigi, Japan c Di6ision of Gastroenterology, Omiya Medical Center, Jichi Medical School, 1 -847 Amanuma-cho, Saitama 330 -8503, Japan Received 7 March 2001; received in revised form 14 February 2002; accepted 12 March 2002
Abstract Human leukocyte antigen (HLA) B44-restricted, hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) recognize HCV nucleoprotein amino acid residues 88 – 96 as an epitope. We previously reported the existence of variant peptide sequences at the epitope locus in three of 27 patients with HCV infection and HLA B44. Here we studied the effects of the variant peptide sequences on generation and cytotoxicity of CTLs. Two of the three variant peptides generated CTLs poorly although they activated well the cytotoxicity of CTLs. Such a differential activation of proliferation and cytotoxicity may contribute to the emergence of HCV with variant epitopes for CTLs. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis C virus; Cytotoxic T lymphocyte; Variant epitope; HLA B44
1. Introduction Cytotoxic T lymphocytes (CTLs) are thought to be one of the major host defense arms against viral infection [1] and are also implicated in the immunopathogenesis of viral infection [2]. We previ-
* Corresponding authors. Tel.: + 81-3-3784-8535; fax: + 81-3-3784-7553 or tel.: + 81-48-647-2111; fax: + 81-48-6485166 E-mail addresses: [email protected] (K. Hiroishi), [email protected] (M. Imawari).
ously demonstrated the existence of hepatitis C virus (HCV)-specific CTLs that recognize HCV nucleoprotein residues 88–96 in association with human leukocyte antigen (HLA) B44 in the peripheral blood of some patients with HCV infection [3,4]. We found that a detectable CTL response to the HCV nucleoprotein was associated with a low titer of serum HCV RNA [5], suggesting that HLA B44-restricted CTLs might inhibit the outgrowth of HCV. In the same study, we found that some of the patients were infected with HCV that had variant peptide sequences at the HCV nucleoprotein residues 88–96.
1386-6346/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 0 2 ) 0 0 0 7 8 - 5
K. Hiroishi et al. / Hepatology Research 24 (2002) 91–94
92
In the present study, we studied the effects of the variant HCV peptides on the cytotoxicity and generation of HCV-specific CTLs.
2. Materials and methods
wild-type peptide of HCV of genotypes 1a and 2b. 9-mer peptides, NETCGWAGW designated as N9-90T91C, DEGLGWAGW designated as N988D91L and NEGLGWTGW designated as N991L94T, are the variant peptides of HCV. All peptides were purchased from Mimotopes Pty (Victoria, Australia).
2.1. Subjects 2.4. CTL Assay Three HLA B44-positive patients infected with HCV that had variant peptide sequences at HCV nucleoprotein amino acid residues 88– 96 were studied. They were among 27 HLA B44-positive patients that were studied previously [5]. Characteristics of the three patients are shown in Table 1. Informed consent was obtained from all subjects, and the study was approved by the Ethical Review Committees of Jichi Medical School.
2.2. Serum HCV RNA, HCV genotype, an amino acid sequence of HCV nucleoprotein residues 88 – 96 Serum HCV RNA, the HCV genotype and the sequence of part of nucleoprotein gene of HCV were determined and the titer was expressed as described previously [5].
2.3. Synthetic peptides A 9-mer peptide corresponding to HCV nucleoprotein residues 88– 96, NEGLGWAGW designated as N9-91L, is the wild-type peptide of HCV of genotype 1b as well as 2a [6]. A 9-mer peptide, NEGCGWAGW designated as N9-91C, is the
Peripheral blood lymphocytes (PBLs) were isolated from heparinized peripheral blood and stimulated twice with the individual 9-mer peptides as described previously [5]. The cytotoxic activity of peptide-stimulated effector cells was assessed against Epstein–Barr virus-transformed HLA B44-positive B cell lines (BCLs) sensitized with the individual HCV peptides with a 4-h europium (Eu)-release assay [4].
3. Results Patient 1 was infected with HCV of genotype 2a, but the amino acid at position 91 of the nucleoprotein was cysteine and was that of isolates of HCV of genotypes 1a and 2b [6]. When we stimulated the PBLs of patient 1 with the 9-mer N9-91C peptide and the variant peptide N9-90T91C, N9-91C-stimulated effector cells recognized both targets sensitized with the N9-91C peptide and those sensitized with the N9-90T91C; the CTLs recognized the latter more efficiently than the former (Fig. 1a). The variant peptide N9-90T91C also generated CTLs although the
Table 1 The characteristics of patients studied and the amino acid sequences of the nucleoprotein residues 88–96 of infected HCV Subjects
Age
Sex
Diagnosis (grade)
ALT
HCV RNA
Genotype
Amino acid Residues 88–96*
Patient 1 Patient 2 Patient 3
58 59 63
Male Male Male
LC, HCC (mild) CH (mild) CH (mild)
69 115 26
102 103 ]104
2a 1b 1b
NETCGWAGW DEGLGWAGW NEGLGWTGW
Abbreviations: LC, liver cirrhosis; HCC, hepatocellular carcinoma; CH, chronic hepatitis; ALT, alanine aminotransferase (normalB 30 U/l). * The common sequences between the HCV nucleoprotein amino acid residues 88–96 are NEGLGWAGW for genotypes 1b and 2a, and NEGCGWAGW for genotype 2b. The residues 88–96 is the minimal and optimal epitope for HLA B44-restricted CTL. Amino acids are identified by one-letter codes.
K. Hiroishi et al. / Hepatology Research 24 (2002) 91–94
93
Fig. 1. Cytotoxicity of effector cells stimulated by HCV wild-type or variant peptides. PBLs of patient 1 were stimulated with (a) the N9-91C (NEGCGWAGW; a wild-type peptide of residues 88 – 96 of genotypes 1a and 2b HCV) or (d) the variant N9-90T91C peptide (NETCGWAGW). Since autologous PBLs of patient 2 were not available, PBLs of another patient infected with prototype HCV of genotype 1b were stimulated with (b) the wild-type peptide N9-91L (NEGLGWAGW) or (e) the variant peptide N9-88D91L (DEGLGWAGW). PBLs of patient 3 were stimulated with (c) the wild-type peptide N9-91L (NEGLGWAGW) or (f) the variant peptide N9-91L94T (NEGLGWTGW). The concentration of the peptides used for stimulation of PBLs was 10 mM. The cytotoxic activity of the effector cells was assessed against HLA B44-positive BCLs sensitized with the indicated concentrations of the wild-type peptide or variant peptides at an effector to target cell ratio of 40. Results are reported as mean specific lysis (%)9S.D.
N9-90T91C peptide-stimulated CTLs lysed target cells a little less efficiently than the N9-91C peptide-stimulated CTLs (Fig. 1d). PBLs of patient 2 had had a detectable CTL response to the wild-type peptide (data not shown). However, since PBLs of patient 2 were not available thereafter, PBLs of another patient infected with HCV with the common sequence of residues 88– 96 of genotype 1b was used to study whether the N9-88D91L peptide affected the cytotoxicity and generation of CTLs. Peptide N9-91L-stimulated CTLs recognized both targets sensitized with the N9-91L peptide and those sensitized with the variant peptide N9-88D91L although the lysis of the variant peptide-sensitized targets by the CTLs was a little less efficient than that of the wild-type peptidesensitized targets (Fig. 1b). However, the N9-
88D91L peptide was not able to generate CTLs efficiently (Fig. 1e). When the autologous PBLs of patient 3 were stimulated with the N9-91L peptide or the N991L94T peptide, peptide N9-91L-stimulated CTLs recognized equally both targets sensitized with the N9-91L peptide and those sensitized with the peptide N9-91L94T, but the N9-91L94T peptide was more than 1000-fold less efficient to generate CTLs compared with the peptide N991L (Fig. 1c, f).
4. Discussion Chang et al. [7] have reported that variant viral peptide sequences at HCV-specific CTL epitope loci were observed in 8– 69% of HCV sequences
94
K. Hiroishi et al. / Hepatology Research 24 (2002) 91–94
depending on the loci and that some of them were potential CTL escape variants. We found the variant peptide sequences at the immunodominant HLA B44-restricted HCV-specific CTL epitope locus in three of 27 patients with chronic HCV infection and HLA B44 [5]. Two of the variant peptides activated the cytotoxicity of CTLs, but did not generate CTLs effectively. Such a differential activation of proliferation and cytotoxicity has been demonstrated in human T-cell lymphotropic virus type I Tax-specific CTLs by an altered peptide ligand [8]. In addition, it has been reported that natural altered peptide ligands of HIV have the capacity to inhibit the range of CTLs directed against an epitope [9], and the impaired reaction to variants of HIV could contribute to the loss of immune control of the infection [10]. We have also reported that co-existence of the variant peptide of patient 3 and the wild-type peptide selectively induced CTLs recognizing the wild-type peptide but not those recognizing the variant peptide [11]. Thus the shift in viral sequence from wild-type to the variants may lead to the loss of immune control of the variant HCV and contribute to the emergence of HCV variants in some patients by affecting the generation of CTLs even if CTLs directed to a wild-type peptide sequence and cross-reactively recognizing the variant peptides were primed. Further studies will be needed to confirm our hypothesis and to clarify the role of mutations at CTL epitope loci in HCV persistence.
Acknowledgements We thank Drs Makoto Mayumi, Takashi Kaneko, Takashi Moriyama (Jichi Medical School), Tatsuya Aikawa (Aikawa Hospital), and Kazuki Ando (Gifu University) for helpful discussion.
References [1] Yap KL, Ada GL, McKenzie IFC. Transfer of specific cytotoxic T lymphocytes protects mice inoculated with influenza virus. Nature 1978;273:238 – 9. [2] Zinkernagel RM, Haenseler E, Leist T, Cerny A, Hengartner H, Althage A. T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay? J Exp Med 1986;164:1075 – 92. [3] Kita H, Hiroishi K, Moriyama T, et al. A minimal and optimal cytotoxic T-cell epitope within hepatitis C virus nucleoprotein. J Gen Virol 1995;76:3189 – 93. [4] Kita H, Moriyama T, Kaneko T, et al. HLA B44-restricted cytotoxic T lymphocytes recognizing an epitope on hepatitis C virus nucleocapsid protein. Hepatology 1993;18:1039 – 44. [5] Hiroishi K, Kita H, Kojima M, et al. Cytotoxic T lymphocyte response and viral load in hepatitis C virus infection. Hepatology 1997;25:705 – 12. [6] Weiner AJ, Christopherson C, Hall JE, et al. Sequence variation in hepatitis C viral isolates. J Hepatol 1991;13(Suppl. 4):S6 – S14. [7] Chang K-M, Rehermann B, McHutchison JG, et al. Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus. J Clin Invest 1997;100:2376 – 85. [8] Ho¨ lsberg P, Weber W, Dangond F, Batra V, Sette A, Hafler D. Differential activation of proliferation and cytotoxicity in human T-cell lmphotropic virus type I Taxspecific CD8 T cells by an altered peptide ligand. Proc Natl Acad Sci USA 1995;92:4036 – 40. [9] Klenerman P, Meier U-C, Phillips R, McMichael A. The effects of natural altered peptide ligands on the whole blood cytotoxic T lymphocyte response to human immunodeficiency virus. Eur J Immunol 1995;25:1927 – 31. [10] McAdam S, Klenerman P, Tussey L, et al. Immunogenic HIV variant peptides that bind to HLA-B8 can fail to stimulate cytotoxic T lymphocyte responses. J Immunol 1995;155:2729 – 36. [11] Kaneko T, Moriyama T, Hiroishi K, et al. Impaired induction of cytotoxic T lymphocytes by antagonism of a weak agonist borne by a variant hepatitis C virus epitope. Eur J Immunol 1997;27:1782 – 7.