A Simple Method to Selectively Expand HIV-1 Specific Cytotoxic T Lymphocytes in Vitro

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Abstract
Cytotoxic T lymphocytes (CTL) may play a critical role in controlling the progression of HIV-1 disease. Conventional assays for demonstration of CTL against HIV-1 have used either fresh PBMC or T cell lines and clones generated by non-specific stimulation. These methods are limited in their sensitivity since without specific secondary stimulation in vitro, epitopes recognized at low frequency may not be detected. Moreover, derivation of CTL clones is labor intensive and not practical for studying a large number of patients. We have developed a simple method to enrich HIV-1 specific CTL in vitro. Autologous antigen presenting cells (APC), either adherent macrophages or EBV transformed B-lymphoblastoid cells, are infected with recombinant vaccinia virus encoding individual HIV-1 proteins and after overnight culture the vaccinia virus 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 patients' T cells obtained directly from PBMC and also after mitogenic stimulation. In both systems, the HIV-1 specific response could be enhanced up to five to ten fold. This enhancement is comparable to CTL selection by exposure to HIV-1 immunodominant peptide incubated APC. In some patients, viral-specific CTL could be detected after HIV-vaccinia selection even though the mitogen stimulated cultures had no demonstrable antiviral CTL activity. Selective expansion of CTL directed against multiple HIV-1 proteins (env, gag and RT) could be obtained from PBMC as well as from mitogen-stimulated lines from individual patients. As these lines are predominantly CD8+ T cells by flow cytometric analysis and are free of vaccinia virus as ascertained by the lack of cytopathic effect in culture, in vitro vaccinia selection might also be useful to generate CTL lines for adoptive immunotherapy.

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