| Summary: | Experiments have been performed to test the hypothesis that soluble HLA-DR-antigenic peptide complexes spontaneously released from antigen presenting cells might induce recruitment of antigen specific T cells as seen in early delayed type hypersensitivity reactions. Through the use of an enzyme-linked immunosorbent assay (ELISA) developed to measure soluble HLA-DR release, it was found that significant release did not occur from resting peripheral blood mononuclear cells (PBMC) until 5 days after the start of culture following cellular activation. In addition, release of soluble HLA-DR was positively correlated with the number of dead cells, suggesting that the release was not an active cellular process. Therefore it was concluded that these molecules would be likely to be responsible for the early recruitment of antigen specific T cells in delay type hypersensitivity reactions. Subsequently, related methods were used in an attempt to identify naturally processed, antigen-derived immunostimulatory peptides recovered from HLA-DR molecules. Large scale Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines were fed with the model antigen, purified protein derivative of Mycobacterium Tuberculosis (PPD). Solubilized HLA-DR-peptide complexes from detergent lysed cultures were affinity purified and the peptide dissociated by treatment with acid, and ultrafiltered peptides further purified by reverse phase high performance liquid chromatography (RP-HPLC). Assay of fractions for stimulation of PPD-specific T cell lines demonstrated the presence of antigenic peptide, although purification to homogeneity and structural identification were not achieved. The results demonstrated the potential feasibility of identifying T cell epitopes by this approach, although the difficulty in obtaining pure peptide in quantities sufficient for amino acid sequencing was recognised. The further problem encountered was the retention on the affinity column of biologically active peptide following standard elution procedures, resulting in contamination of subsequent samples with repeated column use.
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