Using a sensitive ex vivo assay, we have directly characterized unrestimulated low frequency antigen-specific CD8+
memory T cells freshly isolated from peripheral blood. In individuals in the memory state with respect to influenza virus infection, we have demonstrated that influenza-specific memory CD8+
T cells circulate in a state wherein they can display effector function within 6 h of antigen contact. The fact that IFN-γ release within 6 h was triggered by exposure to cognate peptide alone and in the absence of exogenous cytokines, suggests that these CD8+
T cells are capable of immediate effector function in their natural state in vivo. This novel population of CD8+
T cells thus does not conform to the conventional view of memory T cells which require restimulation to divide and differentiate to become effectors (1
). The identification of influenza-specific CD8+
effector T cells in our donors is remarkable given that the donors have all been exposed to influenza in the past but none had acute influenza at the time of venesection and most have not experienced clinical influenza for several years. This does not exclude the possibility of a more recent subclinical infection, but even this would have been at least 5 mo before venesection, since influenza virus stopped circulating in this region by early April 1996 (as per records of viral isolates, Virology Department, Public Health Laboratory, John Radcliffe Hospital, Oxford, UK) and most subjects were studied from September through mid-November. Thus, long after an acute viral infection in humans with a nonpersistent virus, we have identified memory CD8+
T cells capable of rapid effector activity which are, in a functional sense, in a relatively activated state.
During acute (19
) and some persistent (20
) viral infections, the frequency of circulating antigen-specific CD8+
effectors is markedly raised and lytic activity can be demonstrated in freshly isolated PBMCs (19
). However, after recovery, such activated effector cells are not detectable in humans. Murine studies using peripheral (mucosal, cutaneous, and solid organ) rather than intravenous routes of challenge with cytopathic and noncytopathic viruses have demonstrated that antiviral protective immunity in vivo depends upon circulating activated CTLs, capable of rapid effector function as measured ex vivo in CTL assays with freshly isolated PBMCs and in vivo by cytokine-mediated foot pad swelling (4
). These cells are similar to the expanded populations of CD8+
effector T cells found during acute viral infections; their continued presence in a relatively activated state at a low frequency after recovery is thought to reflect ongoing low-level stimulation by persisting antigen (21
). We provide evidence for an analogous population of CD8+
T cells long after recovery from an acute viral infection in humans.
Influenza virus is cytopathic and causes disease by replicating (with a life cycle of 3–6 h) and causing tissue damage at its site of entry, the nasopharyngeal mucosa. In this situation, cellular antiviral protective immunity in vivo would require circulating, influenza-specific CD8+
T cells capable of rapid effector function; this is the phenotype displayed by the influenza-specific CD8+
T cells characterized here, and we propose that these cells subserve protective immunological memory to influenza virus infection. The almost immediate release of IFN-γ by these lymphocytes would have rapid antiviral effects (12
) acting in a paracrine fashion on both infected and uninfected cells at mucosal surfaces. Confirmation of this hypothesis will require the demonstration of a protective association of the presence of specific CD8+
effectors during the next influenza pandemic with an antigenically shifted virus that circumvents humoral immunity.
Since the freshly isolated influenza-specific CD8+
T cells we have characterized are present at such low frequencies, it is not possible to test whether they can directly lyse target cells since this is only measurable when they constitute 0.1–1% of the population. However, the frequency of virus-specific CD8+
T cells reaches this value in HIV-infected patients and here there is a close correlation between the frequency of effector IFN-γ SFCs and percentage-specific lysis in fresh ex vivo CTL assays across a range of epitopes (Lalvani, A., G. Ogg, and A.J. McMichael, unpublished observations). Further indirect evidence that the cells defined here may be cytotoxic is provided by the experiment illustrated in Fig. B
which shows that all the freshly isolated IFN-γ–secreting CD8+
T cells specific for the HLA-A2.01–restricted peptide M1 58–66 bear Vβ17+
TCRs. It is known that CTL lines and clones specific for the M1 58– 66 influenza epitope are dominated by TCRs incorporating the Vβ17 gene segment and the magnitude of peptide-specific lysis correlates with the proportion of CD8+
T cells bearing Vβ17+
). Therefore, it seems likely that a proportion of the M1 58–66-specific IFN-γ SFCs are CTLs, and Fig. B
also confirms that Vβ17 restriction of the M1 58–66 response is not merely a bias introduced by in vitro restimulation since it also defines the fresh ex vivo response to this epitope.
The frequency of antigen-specific T cells enumerated by ex vivo ELISPOT is generally severalfold higher than that calculated from LDA, the traditional method for quantitating CD8+
T cells. This suggests that the LDA detects only a subset of the specific CD8+
T cells quantitated by the ELISPOT. This may be because the LDA measures only those CTL precursors with a capacity to proliferate on antigenic stimulation in vitro (20
); a proportion of the effectors detected by the ELISPOT assay probably lack this proliferative potential. It would appear that these novel CD8+
memory T cells capable of rapid effector function have thus been previously overlooked by standard techniques based on in vitro stimulation and proliferation.