The nature of the CD8+ T cells that underlie antiviral protective immunological memory in vivo is unclear. We have characterized peptide-specific CD8+ T lymphocytes directly ex vivo from peripheral blood in humans with past exposure to influenza virus, using single cell interferon γ (IFN-γ) release as a measure of effector function. In individuals in the memory state with respect to influenza virus infection, unrestimulated antigen-specific CD8+ T cells displayed IFN-γ release within 6 h of antigen contact, identifying a population of memory CD8+ T cells that exhibit effector function without needing to divide and differentiate over several days. We have quantified circulating CD8+ effector T cells specific for six different MHC class I–restricted influenza virus epitopes. Enumeration of these CD8+ T cells gives frequencies of peptide-specific T cells that correlate with, but are in general severalfold higher than, CTL precursor frequencies derived from limiting dilution analysis, indicating that this novel population of memory CD8+ T cells has hitherto been undetected by standard means. The phenotype of these cells, which persist at a low frequency long after recovery from an acute viral infection, suggests that they play a role in protective immunological memory.

Heterologous prime-boost immunization strategies can evoke powerful T cell immune responses and may be of value in developing an improved tuberculosis vaccine. We show that recombinant modified vaccinia virus Ankara, expressing Mycobacterium tuberculosis Ag 85A (M.85A), strongly boosts bacille Calmette-Guérin (BCG)-induced Ag 85A specific CD4+ and CD8+ T cell responses in mice. A comparison of intranasal (i.n.) and parenteral immunization of BCG showed that while both routes elicited comparable T cell responses in the spleen, only i.n. delivery elicited specific T cell responses in the lung lymph nodes, and these responses were further boosted by i.n. delivery of M.85A. Following aerosol challenge with M. tuberculosis, i.n. boosting of BCG with either BCG or M.85A afforded unprecedented levels of protection in both the lungs (2.5 log) and spleens (1.5 log) compared with naive controls. Protection in the lung correlated with the induction of Ag 85A-specific, IFN-γ-secreting T cells in lung lymph nodes. These findings support further evaluation of mucosally targeted prime-boost vaccination approaches for tuberculosis.

Protective immunity to Mycobacterium tuberculosis is poorly understood, but mounting evidence, at least in animal models, implicates major histocompatibility complex class I-restricted CD8 ؉ T cells as an essential component. By using a highly sensitive assay for single cell interferon ␥ release, we screened an array of M. tuberculosis antigenderived peptides congruent with HLA class I allele-specific motifs. We identified CD8؉ T cells specific for epitopes in the early secretory antigenic target 6 during active tuberculosis, after clinical recovery and in healthy contacts. Unrestimulated cells exhibited peptide-specific interferon ␥ secretion, whereas lines or clones recognized endogenously processed antigen and showed cytolytic activity. These results provide direct evidence for the involvement of CD8 ؉ cytotoxic T lymphocytes in host defense against M. tuberculosis in humans and support current attempts to generate protective cytotoxic T lymphocyte responses against M. tuberculosis by vaccination.