Assessment of cumulative allergen-activated lymph node cell proliferation using flow cytometry

The murine local lymph node assay (LLNA) is a method for the prospective identification of chemical contact allergens. The current validated protocol assesses lymphocyte proliferation induced in the draining lymph node as a function of in situ incorporation of radiolabeled thymidine. We have explored the potential utility of an alternative nonradioisotopic marker of cell division, the cytoplasmic dye carboxyfluoresein succinimidyl ester (CFSE). Using this method, the cell phenotype and the number of divisions each cell has undergone can be tracked using flow cytometry. BALB/c strain mice were exposed topically to various concentrations of the contact allergens 2,4-dinitrochlorobenzene (DNCB), oxazolone (ox) or hexyl cinnamic aldehyde (HCA), or to the nonsensitizing skin irritant methyl salicylate (MS). Five days later, lymph node cells (LNC) were labeled with CFSE, cultured for 96 h, then incubated with fluorescent labeled anti-CD4 (T helper) and -CD8 (T cytotoxic) cell antibodies, and proliferating CD4+ and CD8+ cells analyzed by flow cytometry. In LNC populations derived from vehicle-treated animals, less than 1% of either cell population had undergone one cell division or more. Topical exposure to MS (2.5 to 20%) did not increase the frequencies of proliferating cells. Exposure to all three allergens, however, resulted in a marked increase in the percentages of both CD4+ and CD8+ cells undergoing division, with up to 5% and 3% of these cells, respectively, proliferating in response to DNCB and oxazolone, and with lower levels of proliferation stimulated by HCA. These preliminary data suggest that this method may be applied to provide the basis of a nonradioisotopic end point for the LLNA, particularly for the identification of potent contact allergens.