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

Area of Science:

• Immunotoxicology
• Dermatology
• Flow Cytometry

Background:

• The murine local lymph node assay (LLNA) is a standard method for identifying chemical contact allergens.
• The current LLNA protocol relies on radiolabeled thymidine incorporation, posing potential health and disposal concerns.
• There is a need for non-radioisotopic alternatives to improve the safety and efficiency of allergy testing.

Purpose of the Study:

• To evaluate the utility of carboxyfluorescein succinimidyl ester (CFSE) as a non-radioisotopic marker for cell division in the LLNA.
• To assess the ability of CFSE labeling and flow cytometry to detect allergen-induced lymphocyte proliferation in mice.
• To determine if CFSE can serve as a viable alternative endpoint for the LLNA.

Main Methods:

• BALB/c mice were topically exposed to contact allergens (DNCB, oxazolone, HCA) or a non-sensitizing irritant (MS).
• Lymph node cells (LNC) were harvested, labeled with CFSE, and cultured.
• Proliferating CD4+ and CD8+ T cells were analyzed by flow cytometry after staining with fluorescent antibodies.

Main Results:

• CFSE labeling allowed tracking of cell division and phenotype via flow cytometry.
• Exposure to contact allergens DNCB, oxazolone, and HCA significantly increased the percentage of proliferating CD4+ and CD8+ T cells.
• The non-sensitizing irritant MS did not induce significant lymphocyte proliferation.
• Potent allergens like DNCB and oxazolone showed marked proliferation responses, up to 5% and 3% respectively.

Conclusions:

• CFSE labeling and flow cytometry provide a reliable, non-radioisotopic method for assessing lymphocyte proliferation in the LLNA.
• This approach can effectively distinguish between contact allergens and non-sensitizing irritants.
• The CFSE-based method shows promise as a non-radioisotopic endpoint for identifying potent contact allergens in the LLNA.