Invariant natural killer T cells from children with versus without food allergy exhibit differential responsiveness to milk-derived sphingomyelin

Abstract

Background: A key immunologic feature of food allergy (FA) is the presence of a T(h)2-type cytokine bias. Ligation of the invariant natural killer T cell (iNKT) T-cell receptor (TCR) by sphingolipids presented via the CD1d molecule leads to copious secretion of T(h)2-type cytokines. Major food allergens (eg, milk, egg) are the richest dietary source of sphingolipids (food-derived sphingolipids [food-SLs]). Nonetheless, the role of iNKTs in FA is unknown.

Objective: To investigate the role of iNKTs in FA and to assess whether food-SL-CD1d complexes can engage the iNKT-TCR and induce iNKT functions.

Methods: PBMCs from 15 children with cow's milk allergy (MA), 12 children tolerant to cow's milk but with allergy to egg, and 13 healthy controls were incubated with α-galactosylceramide (αGal), cow's milk-sphingomyelin, or hen's egg-ceramide. iNKTs were quantified, and their cytokine production and proliferation were assessed. Human CD1d tetramers loaded with milk-sphingomyelin or egg-ceramide were used to determine food-SL binding to the iNKT-TCR.

Results: Milk-sphingomyelin, but not egg-ceramide, can engage the iNKT-TCR and induce iNKT proliferation and T(h)2-type cytokine secretion. Children with FA, especially those with MA, had significantly fewer peripheral blood iNKTs and their iNKTs exhibited a greater T(h)2 response to αGal and milk-sphingomyelin than iNKTs of healthy controls.

Conclusion: iNKTs from children with FA, especially those with MA, are reduced in number and exhibit a T(h)2 bias in response to αGal and milk-sphingomyelin. These data suggest a potential role for iNKTs in FA.

Figure 1. FA-MA children have significantly fewer PB- iNKTs that respond to αGal

Fresh PBMCs from 15 FA-MA, 12 FA-NMA and 13 Non-FA-children were stained ex vivo for iNKTs (CD3+Vα24+Vβ11+) (A, B) and then cultured for 10 days with αGal 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.

Figure 2. Th2-skewed cytokine responses of iNKTs from children with FA compared to non-allergic controls

PBMCs from 15 FA-MA, 12 FA-NMA and 13 Non-FA-children were cultured for 10 days with αGal or DMSO, then stimulated for 4 hours with PMA and ionomycin and stained for intracellular cytokines. (A) Dot plot of a typical experiment. (B, C, D) Mean percentages of iNKTs staining for IL-13, IL-4 or IFNγ. *p<0.05; **p<0.002.

Figure 3. Human CD1d tetramers loaded with milk-SM specifically bind to iNKTs

αGal-expanded PBMCs were stained with lipid-loaded hCD1d tetramers and anti-CD3 antibodies, or where indicated, were blocked by pre-incubation with non-fluorescently conjugated PBS57-hCD1d tetramers and then washed and stained with food-lipid-loaded-hCD1d tetramers and anti-CD3 antibodies. (A) Dot plot of a representative experiment. (B) Mean percentage of CD3+ cells stained with the indicated tetramers from 7 ND. *p<0.05; ** p<0.005.

Figure 4. Milk-SM induces iNKT activation and expansion

PMBCs were cultured for 10 days with the indicated reagents. (A) Dot plot of a representative experiment. (B) Mean percentage of iNKTs following culture with lipids averaged from 25 ND. (C) Mean percentage of CD25+ iNKTs averaged from 25 ND. (D) CFSE-labeled PBMCs were cultured with the indicated reagents. After 5 days, CFSE-content in CD3+PBS57-hCD1d+ cells was assessed by flow cytometry (n=3 experiments). A representative experiment is shown. *p<0.03; **p<0.001.

Figure 5. More milk-SM-activated iNKTs produce IL-4 compared to iNKTs activated by αGal

PBMCs from 25 ND were cultured for 10 days with the indicated reagents and stimulated for 4h with PMA/ionomycin. (A, B) Mean percentage of iNKTs expressing IL-4 or IFNγ. (C) Expansion of sort-enriched iNKTs cultured for 2 weeks with APC pre-pulsed with the indicated reagents. (D) Cytokine-mRNA expression of sorted iNKTs reported as relative expression of milk-SM or αGal vs. DMSO treated iNKTs (average from 5 different donors). *p<0.05; ** p<0.005.

Figure 6. iNKTs from FA-MA children expand more and have a stronger Th2 response to milk-SM compared to those from Non-FA-children

PMBCs from 15 FA-MA, 12 FA-NMA and 13 Non-FA-children were cultured for 10 days with food-SL or DMSO. (A, B) Mean percentage of total iNKTs and CD25+ iNKTs when cultured with the indicated reagents. (C, D) Mean percentage of iNKTs staining for IL-13 or IL-4. *p<0.005.