Optimization of an In Vitro Lymphocyte Blastogenesis Assay for Predictive Assessment of Immunologic Responsiveness to Contact Sensitizers

Abstract

Current methods for the predictive and diagnostic assessment of contact sensitization rely on the visual scoring of skin reactions. Predictive animal tests, generally using guinea pigs, require a relatively large number of animals to produce a sufficient database for interpreting skin reaction scores. In vitro assays have the potential of being more quantitative than skin testing and, if so, would require fewer animals. However, although in vitro assays are commonly used to study the cellular immune response to strong contact sensitizers, there has been little effort to validate them for predictive assessment purposes. We have optimized an in vitro lymphocyte blastogenesis assay for detecting the response of mouse lymphocytes to strong contact sensitizers with the eventual objective of applying this assay to moderate and weak sensitizers as well. Lymph node lymphocytes from mice sensitized to the strong contact allergens, dinitrochlorobenzene (DNCB), dinitrofluorobenzene (DNFB), or trinitrochlorobenzene (TNCB), responded [greater than or equal to 12,000 counts per minute (CPM) above background] when cultured with water soluble chemical analogues, di- or trinitrobenzene sulfonic acid (DNBS or TNBS). However, the strong sensitizer, oxazolone (OXAZ), has no water soluble analogue and lymphocytes from mice sensitized to OXAZ responded poorly in vitro (less than 2000 CPM) to an ethanol-solubilized OXAZ preparation in spite of very strong in vivo sensitization (ear swelling assay). To increase the assay sensitivity, for OXAZ, we modified the antigen presentation conditions by using 1) solubilized antigen-modified adherent spleen cells, 2) dendritic cells from the draining lymph nodes of antigen painted mice, and 3) antigen-modified Langerhans cell-enriched cultured epidermal cells (EC). These approaches increased OXAZ-directed responses to greater than 7000, greater than 20,000, and greater than 100,000 CPM, respectively, under culture conditions optimized for cell density, responder: stimulator cell ratio, culture duration, and responder cell type. Our results represent a first attempt to directly modify cultured epidermal cells with OXAZ and use these cells to stimulate OXAZ-directed blastogenesis in microtiter plate cultures. This optimized assay is now under evaluation for predictive assessment of contact sensitizers relevant to occupational and consumer exposures.