Background: After recognition of cognate peptide, various effector functions are elicited in CD8⁺ T-cells, including cytokine production and cytotoxicity. It has been proposed that HIV-specific CD8⁺ T-cells are impaired in their cytotoxic capacity; however, HIV-specific CD8⁺ T-cells are clearly cytotoxic directly ex vivo, despite the observed phenotype of low perforin expression. To address this discrepancy, we examined CD8⁺ T-cell granule exocytosis by measuring the deposition of granular membrane proteins on the T-cell surface simultaneously with measurement of intracellular IFNγ production to assess the functional capacity of HIV^- and CMV-specific CD8⁺ T-cells.

Methods: HIV^- and CMV peptides at various concentrations were used to stimulate PBMC from 11 HIV⁺ and HIV^- patients (pts). The cells were later stained with fluorescent-labeled antibodies to T-cell surface markers, cytotoxic granule membrane proteins, various cytokines, and/or specific MHC-class I tetrameric complexes, then analyzed by flow cytometry. In some pts, responding T-cells were sorted and the clonality of degranulating and non-degranulating T-cells was compared.

Results: Our results indicate that HIV^- and CMV-specific CD8⁺ T-cells degranulate in response cognate antigen directly ex vivo. As expected, degranulation was associated with loss of perforin and granzyme B. IFNγ expression by CD8⁺ T-cells was concordant with degranulation in the same cells in all pts tested, although a significant proportion of responding T-cells degranulate without producing IFNγ. Degranulation was observed at peptide concentrations below which IFNγ production was induced (≤ 10^-10M); this phenomenon occurred without substantial T-cell receptor down-regulation. Sequence analysis of the T-cell receptors in responding and non-responding antigen-specific T-cell populations, as measured by degranulation, showed that the same T-cell clones were present in both populations, suggesting that response threshold heterogeneity exists within individual CD8⁺ T-cell clonotypes.

Conclusions: Assessment of degranulation provides a novel and important measure of CD8⁺ T-cell effector function. Our results indicate that HIV-specific CD8⁺ T-cells are not defective in their ability to degranulate and release cytotoxic granule contents upon recognition of cognate peptide. We also demonstrate separation of effector function within individual CD8⁺ T-cell clonotypes directly ex vivo.