The intent of this proposal is to develop a new assay for the functional assessment of HIV-specific cytotoxic T lymphocytes (CTL), and in the process compare the new assay with other methods for measuring these cells. This assay will be based on the recent description of a novel means of identifying and enumerating antigen-specific CTL. This new technique, tetramer binding analysis, uses soluble multimers of the major histocompatibility complex (MHC) and peptide to directly stain antigen-specific T cells. While excellent for quantitation, this technique does not address function of the identified CTL. The investigators propose to use analysis of conjugate formation between the virus-specific CTL and their specific target cells as a means to examine function in the CTL population. The experiments proposed may more clearly define the role of HIV-1-specific CTL in killing virus-infected cells. In addition, directly comparing different methods of measuring CTL may improve the ability to assess this critical arm of the immune response to viral infection. The proposal is intended to ultimately address the functional activity of real-time cellular immune responses. The application has four Specific Aims. (1) To use tetramer-specific flow cytometric techniques to examine the lytic relationship between an HIV-specific CTL clone and its target cell. (2) To determine the effect of MHC tetramer binding to T cell receptors on the function of the CTL. (3) To analyze conjugates between freshly isolated HIV-specific T cells and autologous infected target cells. (4) To compare the conjugate formation functional analysis of antigen-specific CTL with other methods of antigen-specific CTL measurement in both the HIV and murine LCMV systems.
