METHODS: PBMCs from alpha-gal allergic subjects and controls were stained with fluorochrome-labeled human CD1d tetramers bound to PBS-57 (analog of canonical iNKT cell immunogen alpha-galactosylceramide) and antibodies against CD3 and the activation marker CD69. In separate experiments, basophils from a control subject were stripped of IgE; primed with plasma from subjects with and without alpha-gal allergy; stimulated with glycolipids PBS-112 (contains galactose-alpha-1,4-galactose) or PBS-113 (contains alpha-gal); and stained with fluorochrome-labeled antibodies against basophil marker CD123 and activation markers CD63 and CD203c.
RESULTS: PBMCs from subjects with alpha-gal allergy (n=8) contained double the frequency of activated CD69+ iNKT cells than control PBMCs (n=5). The frequency of activated CD203c+CD63+ basophils among PBMCs stimulated with IL-3 and PBS-113 increased 9-fold when PBMCs were sensitized with plasma from alpha-gal-allergic versus control subjects.
CONCLUSIONS: Alpha-gal-containing glycolipids robustly activated basophils sensitized with alpha-gal sIgE compared to glycolipid without alpha-gal. Circulating activated CD1d-restricted iNKT cells were present at higher frequencies in alpha-gal allergic subjects than controls, suggesting unique roles for iNKT cells and glycolipid rarely described in IgE-mediated food allergy.