The P.I. has previously demonstrated in a mouse model that CD8+ cytotoxic T lymphocytes (CTL) are a major mechanism of immune control of T. cruzi. His laboratory has identified three T. cruzi proteins (TSA-1, pS12, and clone 4) which may serve as target molecules to stimulate the production of parasite-specific CTLs in infected mice. The current proposal seeks to determine if T. cruzi-infected humans generate CD8+ CTL responses and to determine if human patients recognize and respond to the same set of T. cruzi molecules as infected mice.
Three specific aims are given: (1) To determine if peripheral blood mononuclear cells of HLA-A2+ individuals with serological evidence of exposure to T. cruzi have cytotoxic T lymphocyte activity specific for T. cruzi. (2) To determine if these anti-parasite CTLs recognize as target molecules the same set of molecules which are recognized by CTLs generated in murine T. cruzi infections. (3) To determine if there is a wide variation among individual patients in the peptides recognized and in the strength of the CTL response to individual peptides. The foreign collaborators will identify Argentinean patients who are positive for T. cruzi infection and who type positive for the HLA-A2 MHC allele. Patients will be considered to be infected with T. cruzi if they test positive for at least two of three serological tests. Lymphocytes from these patients will be used to generate in vitro CTL cultures using a large variety of peptide antigens derived from T. cruzi molecules; the parasite molecules to be tested will include (but not be limited to) those which induce CTL responses in mice (TSA-1, pS12, and clone 4.) The resulting CTL effectors will then be assayed using a standard lytic assay for the ability to kill autologous target cells sensitized with these same parasite peptides. Controls will be included to ascertain whether the lytic response is peptide specific and MHC-restricted. Depletion of effector cell populations using anti-CD4 or anti-CD8 antibodies will allow determination of the phenotype (putatively CD8+) of the lytic effector cell. The overall goal of these studies is to determine if T. cruzi-infected individuals generate CTLs to any T. cruzi protein. Thus, each infected participant will be screened with 10 peptides so that a minimum of 20-25 peptides overall can be evaluated. All CTL studies will be conducted by the U.S. co-investigator at the foreign site. A major effort of the proposal is to identify T. cruzi peptides which can be presented by human class I MHC (HLA) molecules and thus stimulate human CTL for the in vitro CTL cultures described above. Several approaches will be used to identify candidate peptides. First, peptides that exhibit the HLA-A2 binding motif will be evaluated for their ability to bind HLA-A2.1. Binding of peptides to HLA-A2.1 will be measured using a quantitative competitive molecular binding assay and an MHC molecule membrane stabilization assay. Second, transgenic mice which express a chimeric human A2.1 MHC molecule will be infected with T. cruzi; CTLs isolated from the infected mice will be assayed in vitro for their cytolytic activity against HLA-A2.1+ target cells presenting various parasite peptides. Such studies will determine whether HLA-A2.1-binding peptides can elicit CTLs in vivo. Peptides identified with these approaches will be tested for their ability to generate in vitro CTL cultures, as indicated above.
