Tuberculosis (TB) is the leading cause of death in the world resulting from a single infectious disease. Efforts to develop a new generation of effective TB vaccines or immunotherapy ar severely restricted by the lack of understanding of the mechanisms involved in the immunopathogenesis of the disease in man. Although T lymphocyte (T cells)-mediated immunity is important in both the protective as well as the pathogenic host responses to mycobacterium tuberculosis (Mtb), it is not known if they are mediated by the same or different populations of T cells. The long term goal of this research is to develop new vaccine therapies for TB. The goal of the research described in this proposal is to identify the T cell subsets involved in the pathogenesis of pulmonary TB in humans. We have proposed a model of an immune cell """"""""circuit"""""""" consisting of macrophages and subsets of alpha beta and gamma delta T cells that is responsible for the initiation, homeostatic regulation and resolution of the inflammatory immune reaction to Mtb. Although this circuit would normally provide protection, its absence or dysfunction may be responsible for promoting disease reactivation. This model provides the conceptual framework for the further characterization of the T cell populations involved in the pathogenesis of TB. To test this model, we propose to identify the T cell subsets present, and the soluble factors (cytokines) they produce, in the lung at different times during the natural development of the disease. Peripheral blood (PB) and the bronchoalveolar lavage (BAL) lymphocyte populations will be isolated from normal, healthy (PPD) individuals and from different groups of patients at distinct stages of Mtb-infection. T cell subsets will initially be identified by flow cytometry and alpha beta and alpha beta T cell receptor (TCR) usage will be determined using a sensitive polymerase chain reaction (PCR)/Southern hybridization technique thereby defining the profile of TCR variable (V) region gene expression. TCR gene expression will be correlated with TCR protein expression by analyzing fractionated populations of cell surface TCR+ cells. DNA sequence analysis of the TCR-transcripts will serve to distinguish TCRs expressed by cells present in the site of infection as well as providing a means of estimating the diversity of the T cell repertoire and antigens they respond to. In situ hybridization will be used to identify and enumerate subsets of alpha beta and gamma delta T cells expressing TCRs encoded by different V-region genes in situ, in the site of infection. The profile of cytokines produced by T cell subsets at the site of Mtb- infection in the lung will be determined using a PCR assay to identify and quantitate cytokine gene expression in isolated T cell populations. Hybridization in situ will be used to determine the relative frequencies and population dynamics of T cells expressing different cytokine genes. The characterization of the T cell subset that are involved in the inflammatory immune response to Mtb is essential in order to understand how the hosts response is directed to generating protection or disease.
