Background A key immunological feature of food allergy (FA) is the presence of a T-helper-2 (Th2)-type cytokine bias. were used to determine food-SL binding to the iNKT-TCR. Results Milk-SM, but not egg-CE, can engage the iNKT-TCR and induce iNKT-proliferation and Th2-type cytokine secretion. FA-children, especially those with MA, had significantly fewer peripheral blood (PB) iNKTs and their iNKTs exhibited a greater Th2-response to Gal and milk-SM compared to iNKTs of healthy controls. Conclusion iNKTs from FA-children, especially those with MA, are reduced in number and exhibit a Th2-bias in response to Gal and milk-SM. These data suggest a potential role for iNKTs in FA. Clinical Implications Milk-SM activate PB-iNKTs to produce Th2-cytokines and this effect is greater in FA-MA-children. Hence, SL contained in milk may promote an iNKT cell-mediated-Th2-type-cytokine bias that facilitates sensitization to food allergens. for iNKTs (CD3+V24+V11+) (A, B) and then Suvorexant inhibitor cultured for 10 days with Gal or DMSO (C, D). (A, C) Dot plot of a representative experiment. (B, D) Mean levels of iNKT cells expressed as CD3+ cells. *p 0.02; **p 0.002. To investigate whether the lower number of iNKTs observed in children with FA, particularly those with FA-MA, was due to a reduced proliferative capacity, we examined iNKT responsiveness to a 10 day culture with the potent iNKT cell agonist Gal. iNKTs from FA-MA children expanded in response to Gal, although the Suvorexant inhibitor percentages of expanded iNKTs in Gal-stimulated PBMC cultures were lower in FA-MA children and mirrored the low initial levels of iNKTs observed in fresh peripheral blood samples (Figure 1C-D, Figure E3). The responsiveness of iNKTs from FA-MA children to Gal was confirmed by measuring the expression of the activation marker CD25 on Gal-stimulated cells. Gal induced upregulation of CD25 equivalently in all study groups (Figure E4). Therefore, although iNKTs are reduced in number in FA-MA patients, they do not appear to differ in their responsiveness to Gal. iNKTs from FA-children produce more Th2-type cytokines iNKTs have the capacity to produce Th1- and Th2-type cytokines, but in some diseases they exhibit Th2-skewing8,18,19. We questioned whether iNKTs from FA-children might Suvorexant inhibitor exhibit a Th2-skewed cytokine profile, as has been observed for the T cells of FA-patients1. To test this hypothesis, we measured Th1- and Th2-type cytokine production by Gal-expanded iNKTs following short-term stimulation with PMA/ionomycin. We observed that a greater percentage of Rabbit Polyclonal to CHRNB1 iNKTs from FA-children, compared to controls, expressed IL-4 or IL-13 after Gal expansion and secondary stimulation, although such difference reached statistical significance only in children with FA-NMA. iNKTs from children with FA-MA have a tendency even when unstimulated to produce more IL-4 compared to the other groups of children although this didn’t reach staticals significance (p=0.06). Such tendency was not enhanced by Gal expansion (Figure 2A-C; Figure E5A-F). In contrast, production of IFN was similar in the iNKTs of all patient cohorts (Figure 2A,D; Figure E5G-I). Collectively, these data indicate that iNKTs from FA-children produce a Th2-skewed cytokine profile in response to Gal. Open in a separate window Figure 2 Th2-skewed cytokine responses of iNKTs from children with FA compared to non-allergic controlsPBMCs from 15 FA-MA, 12 FA-NMA and 13 Non-FA-children were cultured for 10 days with Gal or DMSO, then stimulated for 4 hours with PMA and ionomycin and stained for intracellular cytokines. (A) Dot plot Suvorexant inhibitor of a typical experiment. (B, C, D) Mean percentages of iNKTs staining for IL-13, IL-4 or IFN. *p 0.05; **p 0.002. hCD1d tetramers loaded with milk-SM specifically bind to iNKTs SL are abundant in milk and egg20. Since the iNKTs from FA-children.