Identification of T cell epitopes of Trypanosoma cruzi antigen using HLA-B transgenic mice

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Analysis of the protective immunity against infection with Trypanosoma cruzi (TC) in the experimental animalmodel using a HLA-B35 transgenic mouse (HLA-B35-TG).CD8 positive cytotoxic T cells are known to be major effector cells to kill the TC infected host cells. Those T cells recognize the invaders antigen with their own HLA-class lmolecules. Using chronically infected HLA-B35-TG,we tried to identify the T cell epitopes that HLA molecule presents to the CD8 cytotoxic T cells. Using the information of the binding motif of HLA-B35, we selected and synthesized 61 different peptides (8mer to 10 mer) from TC cysteine protease. After the binding assay, 5 peptides were chosen for in vitro stimulation experiments. Two months after the infection with TC,HLA-B35-TG mice were sacrificed to get the splenocytes. One million cells in 1 ml of culture medium were incubated with 10muM of each peptide and 30Units of IL-2. Ten dyas later, re-stimulation was done by adding irradiated peptide pulsed autologous splenocytes and IL-2. Totally 2wks. later, the viable cells (T cells) were examined for their cytotoxic activity against peptide pulsed target cells (HLA-B35 Transfectant) by Chromium 51 release assay. None of the peptides stimulated the cytotoxic T cells. though we repeated the experiments twice. Main reasons why there were no effective stimulation were considered as follows.(1) HLA-B35 might not be ideal for the analysis of immune response against the cysteine protease antigen due to row affintiy.(2) Human CD8 might have been essential to complete the HLA-peptide-T cell interaction.2. Search for the major T cell antigens recognized by human T cells from the patients in the endemic area. We have produced T cell clones reactive to epimastigote antigen. Using a T cell clone, 55KDa protein were purified whose molecular weight is almost the same as the cysteine protease. The amino acid sequencing are now on going study.