Enzymatic treatment of the cow's milk allergen whey results in differential inhibition of mast cell degranulation compared to T cell activation

Objectives and Study: In cow's milk (CM) allergic infants hydrolysates of CM in the diet are used. In hydrolysates the allergenic epitopes within the CM proteins are diminished by enzymatic treatment. In the current study we examined the immunological and allergenic properties of the whey fraction of CM during hydrolysis to evaluate the effect on established allergic responses. Methods: During hydrolysis of whey, protein samples were obtained at 10, 15, 30, 45, 60, 75 and 90 minutes. Degradation of whey was checked by HPLC analysis. Allergenic potential was analysed by its IgE crosslinking capacity on human FceRIa-transduced rat basophilic leukemia cells (RBL-2B12, a kind gift of Dr. Teshima, Japan) sensitized with a serum pool of CMallergic patients. In addition, whey-sensitized C3H/HeOuJ mice were earchallenged intradermally with whey or hydrolysates. Finally, T cell activating potential was tested on wheyspecific human T cell clones and T cell lines at the level of proliferation and release of IL-4, IL-10, IL-13 and IFN-g. Results: After 10 minutes hydrolysis, the majority of the protein was already degraded resulting in 26% inhibition of mast cell degranulation, whereas 15 minutes treatment resulted in 92% inhibition. In the mouse model, ear swelling was markedly reduced after 10 minutes hydrolysis (68%), and further reduced (85%) at 15 minutes hydrolysis. In contrast, the T cell stimulatory capacity was affected less by the hydrolysis, and needed longer treatment of the whey. The average inhibition of human T cell proliferation was 8%, 11%, 57%, 74% and 79% for the 10, 15, 30, 45 and 60 minutes hydrolysates. The cytokine release by the T cells upon activation with the whey hydrolysate samples was inhibited to the same rate as T cell proliferation. No differential inhibition of the Th1/Th2/Treg cytokines was found. Conclusion: We show using in vitro, as well as in vivo models the differential effects of hydrolysis of a CM allergen. The allergenic capacity of the whey hydrolysates analysed by mast cell degranulation and earswelling in a mouse model is inhibited by a relatively low grade of enzymatic digestion compared to activation of human whey-specific T cells. This indicates an approach to actively induce tolerance in CM allergic patients by hydrolysates with reduced allergenic potential, but almost complete T cell activation properties.