We have studied the kinetics and specificity of the cytotoxic T lymphocyte (CTL) response to influenza A/PR/8 (H1N1) virus pulmonary infection in the mouse detected using spleen cells from infected mice which were stimulated in bulk and limiting dilution cultures. A hybrid protein designated D-peptide, which contains the terminal 157 amino acids of the HA2 subunit of A/PR/8 virus, was used to stimulate influenza virus subtype-specific secondary CTL in vitro. Infection induced two specificities of precursor CTL, cross-reactive and subtype-specific. The kinetics of the subtype-specific CTL response detected by the D-peptide were similar to the cross-reactive CTL response detected by stimulation with live virus. The majority of the precursor CTL (CTL-p) are able to lyse virus-infected target cells in a cross-reactive fashion. The number of memory subtype-specific and cross-reactive CTL increased by approximately 2.5 logs10 during the first 3 weeks after infection.

We analysed the production of interferons (IFN)-alpha and -gamma during the generation of human influenza-virus specific cytotoxic T lymphocyte (CTL) responses using monoclonal antibodies in a specific radioimmunoassay. The results showed that the peripheral blood mononuclear cells (PBM) of all donors tested produced IFN-gamma and had influenza A virus-specific CTL activity after stimulation. The amount of IFN-gamma produced and the level of CTL activity were significantly correlated. The PBM of some donors also produced IFN-alpha. The level of IFN-gamma produced was low during the first few days and increased subsequently, but IFN-alpha, when it was detected, was produced on day 1. The kinetics of the increase in IFN-gamma correlated with the increase in CTL activity. We also observed an increased percentage of cells bearing interleukin-2 receptors, which may have been a response to the production of IFN-gamma. The T cells active in lysing influenza A virus-infected target cells and in producing IFN-gamma were determined after separating effector cells with monoclonal antibodies. The CTL effector cells were mainly in the T8+ subset, but IFN-gamma-producing cells were found in both T4+ and T8+ subsets. These results suggest that influenza virus-specific T8+ CTL produce IFN-gamma in response to virus, and that T4+ cells which are not CTL effectors also produce IFN-gamma after restimulation with influenza A virus-infected cells.

We have tested the ability of the c13 protein, which is a hybrid protein of the first 81 amino acids of the viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutination produced in E. coli, to render target cells susceptible to the lytic activity of influenza virus-specific cytotoxic T lymphocytes (CTL). The results showed that P815 cells coated with c13 protein were lysed by PR8 virus-induced secondary CTL derived from BALB/c mice. Cold-target inhibition tests clearly demonstrated that c13 protein-coated P815 cells were recognized by an H1 subtype-specific CTL population. Furthermore, PR8 virus-induced CTL derived from C3H mice did not lyse c13 protein-coated P815 cells, suggesting that c13 protein was recognized by CTL in conjunction with H-2d products. These findings suggest that this protein interacts with the cellular plasma membrane and makes target cells recognizable by H-2-restricted, influenza virus subtype-specific CTL.

We have tested the abilities of various polypeptides of A/PR/8/34 (H1N1) virus, constructed by recombinant DNA techniques, to induce influenza virus-specific secondary cytotoxic T lymphocyte (CTL) responses. A hybrid protein (c13 protein), consisting of the first 81 amino acids of viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutinin (HA), induced H-2-restricted, influenza virus subtype-specific secondary CTL in vitro, although other peptides did not. Using a recombinant virus, the viral determinant responsible for recognition was mapped to the HA2 portion of c13 protein. Immunization of mice with c13 protein induced the generation of memory CTL in vivo. The CTL precursor frequencies of A/PR/8/34 virus- and c13 protein-immune mice were estimated as one in 8,047 and 50,312, respectively. These results indicate that c13 protein primed recipient mice, even though the level of precursor frequency was below that observed in virus-immune mice.