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. 2010 Jul;84(13):6527-35.
doi: 10.1128/JVI.00519-10. Epub 2010 Apr 21.

Cytotoxic T lymphocytes established by seasonal human influenza cross-react against 2009 pandemic H1N1 influenza virus

Wenwei Tu  1 , Huawei Mao, Jian Zheng, Yinping Liu, Susan S Chiu, Gang Qin, Ping-Lung Chan, Kwok-Tai Lam, Jing Guan, Lijuan Zhang, Yi Guan, Kwok-Yung Yuen, J S Malik Peiris, Yu-Lung Lau
Affiliations

Cytotoxic T lymphocytes established by seasonal human influenza cross-react against 2009 pandemic H1N1 influenza virus

Wenwei Tu et al. J Virol. 2010 Jul.

Abstract

While few children and young adults have cross-protective antibodies to the pandemic H1N1 2009 (pdmH1N1) virus, the illness remains mild. The biological reasons for these epidemiological observations are unclear. In this study, we demonstrate that the bulk memory cytotoxic T lymphocytes (CTLs) established by seasonal influenza viruses from healthy individuals who have not been exposed to pdmH1N1 can directly lyse pdmH1N1-infected target cells and produce gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Using influenza A virus matrix protein 1 (M1(58-66)) epitope-specific CTLs isolated from healthy HLA-A2(+) individuals, we further found that M1(58-66) epitope-specific CTLs efficiently killed both M1(58-66) peptide-pulsed and pdmH1N1-infected target cells ex vivo. These M1(58-66)-specific CTLs showed an effector memory phenotype and expressed CXCR3 and CCR5 chemokine receptors. Of 94 influenza A virus CD8 T-cell epitopes obtained from the Immune Epitope Database (IEDB), 17 epitopes are conserved in pdmH1N1, and more than half of these conserved epitopes are derived from M1 protein. In addition, 65% (11/17) of these epitopes were 100% conserved in seasonal influenza vaccine H1N1 strains during the last 20 years. Importantly, seasonal influenza vaccination could expand the functional M1(58-66) epitope-specific CTLs in 20% (4/20) of HLA-A2(+) individuals. Our results indicated that memory CTLs established by seasonal influenza A viruses or vaccines had cross-reactivity against pdmH1N1. These might explain, at least in part, the unexpected mild pdmH1N1 illness in the community and also might provide some valuable insights for the future design of broadly protective vaccines to prevent influenza, especially pandemic influenza.

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Figures

FIG. 1.
FIG. 1.
Bulk memory CTL cross-response against pdmH1N1 in healthy individuals. (A) PBMCs from healthy individuals were stimulated with both H1N1 and H3N2 viruses from 2008-2009 seasonal influenza vaccine strains to expand virus-specific bulk CTLs, of which IFN-γ-secreting CD8 T cells (effector [E]) were purified and then cocultured with autologous MDMs (target [T]) infected or not with H1N1, H3N2, and pdmH1N1 viruses at an E/T ratio of 10:1. Data for 12 different individuals are shown. (B) Human influenza virus-specific bulk CTLs were cocultured with pdmH1N1 virus-infected autologous MDMs at the indicated E/T ratios. After 4 h, cell death of MDMs was analyzed by flow cytometry. Data for 12 different individuals are shown. (C) Effector cells were cocultured with MDMs at an E/T ratio of 10:1 in the presence of anti-human CD107a antibody. After 4 h, CD107a expression on CD8 T cells was examined by flow cytometry. Data for 12 different individuals are shown.
FIG. 2.
FIG. 2.
Cytokine expressions of bulk memory CTLs against pdmH1N1. PBMCs from healthy individuals were stimulated with both H1N1 and H3N2 viruses from 2008-2009 seasonal influenza vaccine strains to expand virus-specific bulk CTLs, of which IFN-γ-secreting CD8 T cells (effector [E]) were purified and then cocultured with autologous MDMs (target [T]) infected with or without H1N1, H3N2, and pdmH1N1 viruses at an E/T ratio of 10:1 for 4 h in the presence of brefeldin A. The cells were then fixed, permeabilized, and examined for IFN-γ and TNF-α expression in CD8 T cells. Data for eight different individuals are shown.
FIG. 3.
FIG. 3.
Ex vivo expansion of M158-66 epitope-specific memory CTLs. (A) PBMCs from healthy HLA-A2-seropositive individuals were stimulated with influenza M158-66 peptide to expand the epitope-specific CTLs. HLA-A2/M158-66 peptide tetramer+ CD8 T cells were analyzed by flow cytometry based on the gating of CD3+ CD8+ T cells. (B) Expression of perforin and granzyme B in HLA-A2/M158-66 peptide tetramer+ CD3+ CD8+ T cells. The results shown are representative of four different individuals.
FIG. 4.
FIG. 4.
Cross-responses of M158-66 epitope-specific memory CTLs against pdmH1N1. (A) PBMCs from healthy HLA-A2-seropositive individuals were stimulated with influenza M158-66 peptide to expand the epitope-specific CTLs. T2 cells were loaded with influenza virus M1 (M1-T2) or negative control HPV peptide (HPV-T2) and then cocultured with M158-66 epitope-specific CTLs at the indicated E/T ratios. After 4 h, specific lysis was determined by flow cytometry. (B) M158-66 epitope-specific CTLs were cocultured with peptide-pulsed T2 cells in the presence of anti-CD107a antibody for 4 h. The surface expression of CD107a on CD8 T cells was examined. (C) Autologous B cells infected with or without H1N1, H3N2, or pdmH1N1 virus were cocultured with M158-66 epitope-specific CTLs for 6 h. B-cell death was analyzed by flow cytometry. (D) B cells were loaded with M158-66 (M1-B) or negative control HPV peptide (HPV-B) and then cocultured with M158-66 epitope-specific CTLs in the presence of anti-CD107a antibody. After 4 h, the surface expression of CD107a on CD8 T cells was examined. The results shown are representative of four independent experiments.
FIG. 5.
FIG. 5.
Effector memory phenotype and trafficking receptor expression in M158-66 epitope-specific memory CTLs. M158-66 epitope-specific CTLs were examined for the expression of memory phenotype CD45RO, CD62L, and CCR7 and of the trafficking receptor CXCR3 and CCR5 by flow cytometry. The results shown are representative of four independent experiments.
FIG. 6.
FIG. 6.
In vivo expansion of M158-66 epitope-specific CTLs by seasonal influenza vaccine. PBMCs from healthy HLA-A2-seropositive individuals were isolated before and 1 month after seasonal 2008-2009 influenza vaccination. HLA-A2/M158-66 tetramer staining was performed and analyzed by flow cytometry. The numbers shown here indicate the frequency of HLA-A2/M158-66-tetramer+ cells within the population of CD3+ CD8+ T cells. Among 20 HLA-A2-positive individuals, only 4 individuals had an increase of HLA-A2/M158-66 tetramer-positive CTLs 1 month after vaccination. Results from those four donors are shown.
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