Studies using transformed human cell lines suggest that most SIV strains use CCR5 as co-receptor. Our analysis of primary rhesus macaque CD4(+) T-cell clones revealed marked differences in susceptibility to SIV(mac)239 infection. We investigated whether different levels of CCR5 expression account for clonal differences in SIV(mac)239 susceptibility. Macaque CD4(+) T-cells showed significant CCR5 downregulation 1-2days following CD3 mAb stimulation, which gradually recovered at resting state, 7-10days after activation. Exposure of clones to SIV(mac)239 during their CCR5(low) or CCR5(high) expression states revealed differences in SIV susceptibility independent of surface CCR5 levels. Furthermore, a CCR5 antagonist similarly reduced SIV(mac)239 infection of clones during their CCR5(low) or CCR5(high) expression states. Our data suggest a model where i) very low levels of CCR5 are sufficient for efficient SIV infection, ii) CCR5 levels above this threshold do not enhance infection, and iii) low level infection can occur in the absence of CCR5.

CD8(+) cytotoxic T lymphocyte (CTL) responses play an important role in controlling the replication of primate lentiviruses. Induction of these responses is a key objective for most current AIDS vaccine approaches. Despite a variety of approaches for measuring properties and activities of CTL, the functions responsible for controlling viral replication in vivo have not been clearly identified. Assays measuring CTL-mediated suppression of viral replication in vitro are beginning to be used as possible correlates of in vivo virus suppressive activity, but the utility and interpretive value of these assays are typically limited by properties of the cells that have been used. We investigated the capacity of SIV-specific CTL clones (effectors), immortalized by transduction with human telomerase reverse transcriptase (hTERT), to suppress SIV replication in autologous hTERT immortalized CD4(+) T-cell clones (targets). Immortalized and non-immortalized SIV-specific effector cells showed IFN-gamma production and degranulation in response to viral antigen specific stimulation and significantly inhibited SIV(mac)239 replication (2 to 4 log decrease in viral RNA or cell-associated proviral DNA) (p<0.0005). Our in vitro assays of inhibition of viral replication, using T-cell clones as effectors and targets, provide a well-defined approach for evaluating possible mechanisms of CTL-mediated control of viral production which may involve direct killing of infected target cells and/or release of proinflammatory cytokines such as IFN-gamma and TNF-alpha. The use of hTERT immortalized effector and target cells for such assays preserves relevant functional properties while providing a convenient, reproducible means of conducting studies over time.

CD8(+) T lymphocytes (CTL) play a role in controlling HIV/SIV infection. CTL antiviral activity is dependent on recognition of antigenic peptides associated with MHC class I molecules on infected target cells, and CTL activation can be impaired by Nef-mediated down-regulation of MHC class I molecules. We tested the ability of a series of rhesus macaque CD8(+) T-cell clones specific for the SIV Gag CM9 peptide to suppress SIV infection of autologous CD4(+) T cells. We used a set of SIV(mac)239 viruses with either wild-type Nef or Nef mutations that impair MHC class I down-regulation. All CTL clones efficiently suppressed virus replication in cells infected with mutant viruses with altered Nef function, phenotypically MHC class I(high) or MHC class I(intermediate). However, the ability of the clones to suppress virus replication was variably reduced in the presence of wild-type Nef (MHC class I(low)) despite the observations that all CTL clones showed similar IFN-gamma responses to titrated amounts of cognate peptide as well as to SIV-infected cells. In addition, the CTL clones showed variable CD107a (CTL degranulation marker) responses that did not correlate with their capacity to suppress virus replication. Thus, the clonal differences are not attributable to TCR avidity or typical effector responses, and point to a potential as yet unknown mechanism for CTL-mediated suppression of viral replication. These data emphasize that current assays for evaluating CTL responses in infected or vaccinated individuals do not fully capture the complex requirements for effective CTL-mediated control of virus replication.

CD8(+) cytotoxic T lymphocytes (CTL) play an important role in controlling virus replication in HIV- and SIV-infected humans and monkeys, respectively. Three well-studied SIV CTL determinants are the two Mamu A()01-restricted epitopes Gag CM9 and Tat SL8, and the Mamu B()17-restricted epitope Nef IW9. Point mutations leading to amino acid replacements in these epitopes have been reported to mediate SIV escape from CTL control. We found that synthetic peptides containing mutations in SIV Gag CM9 and Tat SL8 were no longer recognized by the respective CTL. On the other hand, the described I-to-T replacement at the N-terminal amino acid residue of the SIV Nef IW9 epitope only moderately affected CTL recognition of the variant peptide, TW9. In an attempt to dissect the mechanism of escape of the Nef TW9 mutation, we investigated the effect of this mutation on CTL recognition of CD4(+)T cells infected with an engineered SIV(mac)239 that contained the TW9 mutation in Nef. Although, the wild type and mutant virus both infected and efficiently replicated in rhesus macaque CD4(+)T cells, the TW9 mutant virus failed to induce IFN-gamma expression in an SIV Nef IW9-specific CTL clone. Thus, unlike escape from Gag CM9- or Tat SL8-specfic CTL control presumably by loss of epitope binding, these results point to a defect at the level of processing and/or presentation of the variant TW9 epitope with resultant loss of triggering of the cognate TCR on CTL generated against the wild type peptide. Our data highlight the value of functional assays using virus-infected target cells as opposed to peptide-pulsed APC when assessing relevant escape mutations in CTL epitopes.