None of the Mamu-A*02-Nef159-167YY9-specific clones from elite controller r95061 exhibited effective suppression (Table ). However, all of these ineffective clones produced IFN-γ and tumor necrosis factor alpha (TNF-α) (Fig. ). Furthermore, both Nef195-203MW9 clones with effective (98% reduction in Gag-p27-positive cell frequency) and ineffective (24.6% reduction) suppression of SIVmac239 replication had robust IFN-γ, TNF-α, and interleukin-2 (IL-2) responses to 10 μM cognate peptide stimulation (Fig. ). A less effective Env830-838FW9 clone had stronger IFN-γ, TNF-α, and IL-2 responses than an effective FW9 clone (Fig. ). In addition, two Vif66-73HW8 clones with highly effective suppression exhibited notable differences in IFN-γ, TNF-α, and IL-2 responses (Fig. ). Interestingly, an effective HW8 clone had no IL-2 response after stimulation with 10 μM cognate peptide. In t-test statistical analyses with log-transformed data from both suppressive and nonsuppressive groups of clones, there was no significant correlation between the TNF-α or IL-2 response and virus suppression efficacy (P = 0.94 and 0.44, respectively) (Fig. ). Interestingly, the IFN-γ response was significantly correlated with the ability of clones to suppress viral replication (P = 0.002) (Fig. ). It should be noted, however, that there were many suppressive clones with very low IFN-γ responses.
FIG. 3. IFN-γ, TNF-α, and IL-2 responses were not always associated with the ability to suppress SIVmac239 replication. Gag p27 staining was carried out with day 8 VSA target cells to determine the frequency of SIV-infected target cells. Clones (more ...)
Macaques with CD8+
T-lymphocyte responses to similar epitope sets after infection with molecularly cloned SIVmac239 have variable disease courses, suggesting that epitope specificity alone cannot account for effective control of viremia (3
). The ability of a clone to reduce SIV replication in vitro likely depends upon its cytolytic mechanism, as has been demonstrated previously (22
). Loss of T cells with an effective TCR repertoire in an epitope-specific CD8+
T-lymphocyte population may result in a poor clinical outcome for HIV-infected humans or SIV-infected macaques (14
). However, other findings suggested that CD8+
T cells from healthier individuals might be functional, independent of TCR expression (39
). In previous studies, only one or two CD8+
T-cell clones specific for a few HIV or SIV epitope-specific CD8+
T-cell lines were used to determine the ability to suppress virus replication (50
). The variation in antiviral efficacy among epitope-specific CD8+
T cells in HIV or SIV infection has not, until now, been comprehensively assessed. Using a recently developed in vitro functional assay to evaluate the antiviral efficacy of epitope-specific CD8+
T-cell clones, we identified clonal variation in the ability to suppress virus replication in five of the seven CD8+
T-cell specificities tested. Some epitope-specific CD8+
T cells derived from particular animals all had effective clones with relatively minor variation in clonal efficacy. Clones with effective suppression of SIV replication could diminish the frequency of SIV-infected cells by >99% in our 8-day coculture assay. Therefore, variations in antiviral efficacy among certain epitope-specific CD8+
T lymphocytes may result in different disease courses in MHC class I-matched animals.
The association between cytokine-secreting T-cell responses and HIV or SIV control remains controversial. HIV-1-specific IFN-γ-secreting T-cell responses were significantly and inversely correlated with viral load in previous studies (8
), whereas others showed no clear correlation (1
). In this study with SIV epitope-specific clones, all of the ineffective clones in the VSA secreted IFN-γ and TNF-α and/or were positive for tetramer staining. Furthermore, some effective suppressor clones had no IL-2 response after cognate peptide stimulation. In addition, functional avidities of suppressive and nonsuppressive clones were not associated with viral suppression efficacy (data not shown). Therefore, current assays using cytokine secretion may not actually measure CD8+
The specificity and magnitude of HIV- or SIV-specific CD8+ T lymphocytes can be identified by using MHC class I tetramers. The function of these antigen-specific T lymphocytes is currently assessed by enzyme-linked immunospot and intracellular cytokine staining assays that measure the ability of these cells to secrete a range of cytokines. Unfortunately, tetramer-positive epitope-specific CD8+ T cells varied in the ability to suppress SIV replication. These results suggest that currently available cytokine-based assays, including enzyme-linked immunospot and intracellular cytokine staining assays, may not be reliable tools to evaluate protective CD8+ T-lymphocyte responses.