Binding of the Active Vitamin A Metabolite Retinoic Acid to the Major Cows Milk Allergen Bos d 5 Down-Regulates T-Cell Responses
Sunday, March 6, 2016
South Exhibit Hall H (Convention Center)
Stefanie Wagner, MSc, BSc, Karin Hufnagl, PhD, Luis F F Pacios, PhD, Prof., Franziska Roth-Walter, PhD, Ass.Prof., Rodolfo Bianchini, PhD, Gerlinde Hofstetter, MSc, BSc, Erika Jensen-Jarolim, MD

Recent research of our group has shown that the major cows milk allergen Bos d 5, a member of the lipocalin family, acts immunomodulatory depending on its load with siderophore-iron complexes. The aim of this study was to investigate whether Bos d 5 could influence Th1/Th2 immune reponses when complexed with the active Vitamin A metabolite retinoic acid (RA). 


Binding of RA to Bos d 5 was determined by autofluorescence quenching and ANS displacement assay. Activated PBMCs from 12 healthy donors were incubated with the milk allergen being “emptied” (apo-Bos d 5) or being loaded with RA (holo-Bos d 5). T-cell subsets (CD3+, CD4+, CD8+) were analyzed by FACS, cytokines (IFN-γ, IL-10, IL-13) measured by ELISA.


We calculated a dissociation constant of 1.7 µM and in vitro RA was able to dose-dependently displace ANS from Bos d 5. Incubation of PBMCs with apo-Bos d 5 for 48 hours significantly induced high IFN-γ, IL-13 and IL-10 levels whereas T-cell subsets remained unaltered. In contrast, stimulations with holo-Bos d 5 led to a significant decrease in CD4+ positive cells and to a pronounced decrease in all three cytokines. This phenomenon was dependent on the allergen-RA complex, as treatment with RA alone did not influence T-cell subsets or cytokine levels.


Our data suggest that holo-Bos d 5, with RA in its molecular pocket, has a pronounced immunosuppressive effect. We thus propose that proper loading of this major cows milk allergen may prevent subsequent allergic immune responses to it.