A SIMPLE METHOD TO SELECTIVELY EXPAND HIV-1 SPECIFIC CYTOTOXIC T-LYMPHOCYTES IN-VITRO

Cytotoxic T lymphocytes (CTL) may play a critical role in controlling the progression of HIV-1 disease. Conventional assays for demonstratio n of CTL against HIV-1 have used either fresh PBMC or T cell lines and clones generated by non-specific stimulation. These methods are limit ed in their sensitivity since without specific secondary stimulation i n vitro, epitopes recognized at low frequency may not be detected. Mor eover, derivation of CTL clones is labor intensive and not practical f or studying a large number of patients. We have developed a simple met hod to enrich HIV-1 specific CTL in vitro. Autologous antigen presenti ng cells (APC), either adherent macrophages or EBV transformed B-lymph oblastoid cells, are infected with recombinant vaccinia virus encoding individual HIV-1 proteins and after overnight culture the vaccinia vi rus is inactivated by uv irradiation in the presence of psoralin. The infected APC are then cultured with patient's T cells and CTL activity determined 10-14 days later. We have used this method to stimulate pa tients' T cells obtained directly from PBMC and also after mitogenic s timulation. In both systems, the HIV-1 specific response could be enha nced up to five to ten fold. This enhancement is comparable to CTL sel ection by exposure to HIV-1 immunodominant peptide incubated APC. In s ome patients, viral-specific CTL could be detected after HIV-vaccinia selection even though the mitogen stimulated cultures had no demonstra ble antiviral CTL activity. Selective expansion of CTL directed agains t multiple HIV-1 proteins (env, gag and RT) could be obtained from PBM C as well as from mitogen-stimulated lines from individual patients. A s these lines are predominantly CD8+ T cells by flow cytometric analys is and are free of vaccinia virus as ascertained by the lack of cytopa thic effect in culture, in vitro vaccinia selection might also be usef ul to generate CTL lines for adoptive immunotherapy.