Combining positional scanning peptide libraries, HLA-DR transfectants and bioinformatics to dissect the epitope spectrum of HLA class II cross-restricted CD4+ T cell clones

The use of positional scanning peptide libraries in combination with biometrical analysis is one of the few approaches, which allows the identification of stimulatory peptides for T cells of unknown specificity. Despite the successful application of this strategy in different studies, not every T cell is suited for analysis. For as yet unknown reasons some T cells do not recognize these highly complex libraries, and even more importantly the predictive capacity of the current approach shows high variability among individual T cell clones and their TCRs. A number of factors probably contribute to differences in T cell recognition and have to be taken into account in order to overcome these difficulties. Our results suggest that the ability of some T cells to recognize peptides in the context of more than one HLA class II molecule expressed by autologous APCs could diminish the predictive capacity of the approach. In contrast, the use of B cell lines expressing single HLA class II molecules as APCs instead of autologous peripheral blood mononuclear cells markedly improves the capacity to identify target peptides for this type of T cells.