Performance of a Novel In Vitro Assay for Skin Sensitization Based on Activation of T Lymphocytes

Abstract

There are currently no skin sensitization assays based on T cell activation. We built a novel in vitro test to assess T cell activation and assessed its performance to discriminate skin sensitizers from non-sensitizers using 52 reference chemicals. Jurkat Clone E6-1 human T lymphocytes were exposed to a series of concentrations of test substances for 24 hours and CD69 expression was measured as a marker of early T cell activation by flow cytometry. Most sensitizers tested induced an increased relative fluorescence intensity (RFI) of CD69 on the T cells, which was linearly correlated with the tested concentrations, indicating a statistically significant causal link between sensitizer concentration and increase of CD69 expression. CD69 RFI ≥ 1.5 was determined as the positive criterion for skin sensitizer classification when cell viability ≥ 50%. The sensitivity (79.4%), specificity (88.9%) and accuracy (82.7%) of our test for the 52 tested reference chemicals showed a good predictivity for skin sensitizers. Results were reproduced in at least two repeats, and the concurrent positive control, 2 µg/mL 2,4-dinitrochlorobenzene, was found positive in all 25 independent runs conducted, indicating in-house reproducibility. The EC1.5 value, i.e., the concentration at which a test chemical induces a CD69 RFI of 1.5, may be used to categorize skin sensitization potency of a chemical. This work may contribute to the development of an in vitro assay for skin sensitization based on the activation of T cells.