Human cytotoxic T lymphocytes directed to seasonal influenza A viruses cross-react with the newly emerging H7N9 virus

Abstract

In February 2013, zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported, a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population, there is interest in identifying other correlates of protection, such as cross-reactive CD8(+) T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8(+) T cells are known to recognize conserved internal proteins of influenza A viruses predominantly, but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here, we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8(+) T cells, obtained from HLA-typed study subjects, with the novel H7N9 virus. The cross-reactivity of CD8(+) T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that, apart from recognition of individual H7N9 variant epitopes, CD8(+) T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8(+) T cells may afford some protection against infection with the new virus.

FIG 1

Epitope-specific IFN-γ production by seasonal influenza virus-specific CD8⁺ T cells after stimulation with peptide-loaded BLCLs. Polyclonal CD8⁺ T cells were isolated from PBMCs in vitro stimulated with sH3N2, sH1N1, or pH1N1, as indicated. No pH1N1 in vitro stimulation was performed for subject 6, since those PBMCs were isolated in 2008, prior to the 2009 pandemic outbreak. The polyclonal CD8⁺ T cells were subsequently stimulated with peptide-loaded and untreated HLA class I-matched BLCLs. Stimulation with homologous peptides is indicated by black bars, stimulation with H7N9 variant peptides is indicated by gray bars, and control cells without peptide are indicated by white bars. The number of IFN-γ-producing cells per 10,000 polyclonal CD8⁺ T cells was determined by ELISpot assay. The results represent the averages of triplicate wells. Peptides were selected based on the variation in the H7N9 sequence and their compatibility with the HLA haplotypes of our study subjects. The error bars indicate standard deviations of results from the triplicate wells.

FIG 2

Virus-specific IFN-γ production by polyclonal CD8⁺ T cells after stimulation with BLCLs infected with homologous or H7N9 virus. (A to Q) Seasonal influenza virus-specific polyclonal CD8⁺ T cells were isolated from PBMCs stimulated with sH3N2 (A, D, G, J, M, and P), sH1N1 (B, E, H, K, N, and Q), or pH1N1 (C, F, I, L, and O). PBMCs of subject 6 were not stimulated in vitro with pH1N1, since they were isolated prior to the pH1N1 outbreak. The CD8⁺ T cells were subsequently cocultured with BLCLs infected with homologous virus (sH3N2, sH1N1, or pH1N1) (black bars) or the heterologous novel H7N9 virus (gray bars). The number of IFN-γ-producing cells per 5,000 polyclonal CD8⁺ T cells was determined by ELISpot assay. Uninfected BLCLs were used as negative controls (white bars). Experiments were performed in triplicate. The error bars indicate standard deviations for the triplicates. (R, S, and T) The symbols represent the averages of triplicate experiments for each individual subject, and the horizontal bars represent the average responses of all study subjects combined.

FIG 3

Lytic activity of virus-specific polyclonal CD8⁺ T cells against BLCLs infected with the homologous or H7N9 virus. Seasonal influenza virus-specific polyclonal CD8⁺ T cells from study subjects 1, 3, and 5 were isolated after stimulation with sH3N2 (A, D, and G), sH1N1 (B, E, and H), or pH1N1 (C, F, and I) virus, as indicated. Lytic activity against CFSE-labled BLCLs infected with the homologous virus (sH3N2, sH1N1, or pH1N1) (solid squares) or the heterologous novel H7N9 virus (open squares) was assessed as lytic background activity against uninfected cells (open circles). Experiments were performed in triplicate. The error bars indicate standard deviations for the triplicates.