The ex vivo expansion of antigen-specific cytotoxic T lymphocyte (CTL) lines is being developed for immunotherapy of viral infections and cancer and is critically dependent on the precise cell expansion and stimulation conditions. In this article, we investigate medium requirements for the development of HIV-specific CTL in cell lines generated from the peripheral blood of seven asymptomatic HIV-infected individuals. We find that HIVspecific CTL do not readily develop in the serum-free medium AIM V but do develop if the medium is supplemented with 1% plasma or serum. T cell lines with antigen-specific cytolytic activity express more cellsurface CD57 than do cell lines grown in the absence of serum or plasma. Three sources of serum (human autologous, human AB, or fetal calf) are comparable. Human plasma is somewhat less effective than serum from an identical source. 0 1996 John Wiley & Sons, Inc. Key words: HIV cytotoxic T lymphocytes (CTL) serum * To whom all correspondence should be addressed.
specific CTL memory cells and activated CTL in HIVinfected individuals before they develop opportunistic infection^.^^^'^^^^ Because of this, it is possible to generate, by polyclonal activation, T cell lines that have readily detectable antiviral CTL activity. In this article, we use polyclonal activation of peripheral blood mononuclear cells (PBMC) from infected patients to compare different media for their ability to support the development of viral-specific CTL.
Serum-free media have been developed and approved for the ex vivo expansion of T cells used for treatment. These media have several potential advantages for clinical use. There is little lot-to-lot variation, often a problem with undefined fetal calf or human sera. They are free of possible infectious agents, especially viruses, that may cause clinical disease, especially in immunosuppressed patients. Because they are not contaminated with multiple ill-defined proteins, they are less likely to induce allergic or antibody responses. Serum-free media also have been reported to reduce nonspecific or background proliferation."
In this article we will show that a widely used serumfree medium, AIM V, does not support the development of HIV-specific CTL. Cells grown in serum-free AIM V proliferate as well as cells grown in serum-containing media. However, they have undetectable HIV-specific CTL activity compared with readily apparent antiviral cytolytic activity when grown in serum-containing media. Another serum-free medium, X-VIVO 20, is also unable to support efficiently the ex vivo generation of HIV-specific CTL. The addition of as little as 1% serum can support antigen-specific CTL generation from the PBMC of HIV-infected donors. Either human or bovine serum can support the development of antiviral CTL activity. Human plasma is less effective than serum at supporting CTL generation.