A NOVEL METHOD FOR DETECTION AND EX-VIVO EXPANSION OF HIV TYPE 1-SPECIFIC CYTOLYTIC T-LYMPHOCYTES

Studies have shown that cytolytic T lymphocyte (CTL) responses may be critical to the clearance of the early viremia in acute HIV-1 infectio n. It is likely that these cells play an important role in prolonging the asymptomatic phase of the infection. Although HIV-1-specific CTL a ctivity can be detected in direct assays of freshly isolated periphera l blood lymphocytes (PBL) from some infected individuals, this method fails to detect CTL that are present at low frequency and resting, mem ory CTL. For these reasons, direct CTL assays on PBL from seropositive individuals may underestimate the level of CTL immunity. As part of o ngoing investigations of CTL activity in HIV-1-infected individuals, w e developed a novel strategy for the detection and ex vivo expansion o f HIV-1-specific CTL. This technique involves selective stimulation of PBL from seropositive individuals with autologous Epstein-Barr virus (EBV)-transformed, B-lymphoblastoid cell lines (B-LCL) infected with v accinia vectors expressing various HIV-1 genes. Prior to their use for in vitro stimulation, B-LCL are treated with psoralen and UV light to inactivate vaccinia virus. After 1 week of stimulation, CTL activity in stimulated cultures is measured in a standard Cr-51 release assay. This ex vivo expansion method can selectively increase the bulk cultur e CTL activity against env, gag and nef, even in some seropositive ind ividuals with low CD4 counts and little evidence of HIV-1-specific CTL in assays of freshly isolated PBL. These expanded CTL are predominant ly of the CD8(+) phenotype. This method provides an efficient and sens itive method for the detection and ex vivo expansion of HIV-1-specific CTL from infected individuals at all stages of disease and may prove useful in following changes in CTL responses with disease progression and therapy. In addition, this method may allow for the ex vivo expans ion of HIV-1-specific CTL.