Infection with Human T cell lymphotropic virus type I but not II (HTLV-I/II) can result in debilitating and life-threatening diseases such as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP)and adult T cell leukemia. Provirus from symptomatic vs. asymptomatic carriers is indistinguishable. Even though the factors leading to disease manifestation are unknown, the host's immune response seems to play a major role. Spontaneous lymphoproliferation is a common observation in infected individuals. HAM/TSP patients show unusually high precursor frequencies for virus-specific cytotoxic T cells (CTL), and almost all recognize the viral transactivator protein Tax. We showed that Tax-specific CTL arise from oligoclonal expansion, and are sensitized by femto-molar amounts of peptide. Crystallization of one of our virus-specific T cell receptors (TCR) revealed for the first time TCR recognition of an MHC/peptide complex on a molecular level. Despite high precursor frequencies, virus-specific CTL in patients seem to be less effective, as their viral load is typically 10 to 15-fold higher than that of asymptomatic carriers. We demonstrated CTL secretion of pro-inflammatory cytokines, chemokines and matrix metalloproteinases, all shown to contribute to inflammatory demyelinative processes such as occur in HAM/TSP. The cellular immune responses in asymptomatic carriers of HTLV-I are poorly defined, have not been studied at all for HTLV-II. Currently, HTLV-II infection is on the rise and predominates HTLV-I in IV drug users and HIV-positive individuals. The proposal aims to study anti-viral cellular immune responses in both cohorts. In contrast to previous analyses that depended on T-cell expansion in vitro, possibly leading to a skewing of the results, we will perform a direct examination of TCR repertoires. In particular, peripheral blood lymphocytes are obtained from asymptomatic viral carriers, and T cells are sorted based on cell surface markers that allow a distinction between naove, activated and memory phenotypes, and MHC class I and II-restricted T cells. RNA is extracted and the TCR repertoires are examined by PCR using TCR variable region alpha- and beta-specific primers. The PCR products are run on a special electrophoresis apparatus which allows the separation of DNA in a non-denaturing gel at pre-set temperatures. The PCR products are separated according to length and nucleotide composition (SSCP analysis). Every T cell clone will migrate to a unique spot in the gel. T-cell oligoclonal expansions are visible as prominent bands which will be excised and sequenced. The corresponding T-cell lines are cloned using traditional immunological methods (repeated in vitro stimulation with antigen) and further characterized for their cytokine, and chemokine profiles, and MMP expression.
