PLASMACYTOID DENDRITIC CELL-BASED ASSAY AS AN IN VITRO ALTERNATIVE FOR CHEMICAL ALLERGENICITY SCREENING.

Human dendritic cells (DC) have been used as an alternative to existing animal models utilized throughout industry to monitor products for contact sensitization.  Such methods are necessary to comply with the ban on animal testing imposed by the Cosmetics Directive in the EU.  In this study, we investigated whether CD86 expression in plasmacytoid DC (pDC) can be used to identify contact allergens.  Human DC were generated from CD34+ progenitor cells and the pDC fraction (CD123+/CD11c-) was harvested using FACS sorting.  The pDC were exposed to an expanded list of chemical allergens (n=49) or irritants (n=42).  Concentrations of each chemical that resulted in >50% viability as determined by FACS analysis of propidium iodide stained cells were used.  Allergens were identified based on stimulation index (SI) calculated by the fold increase in CD86 expression levels.  A material that had an SI ≥1.5 in at least 50% of the pDC donors (n=2-5 donors) was considered an allergen.  For 75 of the 91 materials tested, historical mouse local lymph node assay (LLNA) and human clinical data were available. Using the in vitro pDC assay, CD86 expression increased ≥1.5 fold for 41 of 43 allergens but not for 26 of 32 non-allergens.  Based on these results, a prediction model was developed to classify chemicals as allergens or non-allergens.  The in vitro assay performance has; sensitivity=95%, specificity=81%, and accuracy=89%; these results were slightly better than those obtained using the LLNA assay: sensitivity=85%, specificity=84%, and accuracy=85%.  Transferability of the test method was evaluated using 7 test articles in 3 laboratories. The results showed that all samples were correctly identified in the 3 labs.  In conclusion, the CD86 expression level in pDC appears to be a sensitive and specific predictor of allergenicity.  The pDC assay is advantageous because high throughput screening of chemicals is possible, donor-to-donor variation can be monitored, the cells are of human origin, and the assay is cost effective.