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

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Presentation transcript:

Invariant natural killer T cells from children with versus without food allergy exhibit differential responsiveness to milk-derived sphingomyelin Soma Jyonouchi, MD, Valsamma Abraham, PhD, Jordan S. Orange, MD, PhD, Jonathan M. Spergel, MD, PhD, Laura Gober, MD, Emily Dudek, BS, Rushani Saltzman, MD, Kim E. Nichols, MD, Antonella Cianferoni, MD, PhD Journal of Allergy and Clinical Immunology Volume 128, Issue 1, Pages 102-109.e13 (July 2011) DOI: 10.1016/j.jaci.2011.02.026 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Children with FA-MA have significantly fewer PB-iNKTs that respond to αGal. Fresh PBMCs from 15 children with FA-MA, 12 children with FA-NMA, and 13 non-FA children were stained ex vivo for iNKTs (CD3+Vα24+Vβ11+; A and B) and then cultured for 10 days with αGal or DMSO (C and D). A and C, Dot plot of a representative experiment. B and D, Mean levels of iNKT cells expressed as CD3+ cells. ∗P < .02; ∗∗P < .002. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Th2-skewed cytokine responses of iNKTs from children with FA compared to with controls without allergy. PBMCs from 15 children with FA-MA, 12 children with 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-D, Mean percentages of iNKTs staining for IL-13, IL-4 or IFN-γ. ∗P < .05; ∗∗P < .002. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 hCD1d 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 preincubation with nonfluorescently 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 NDs. ∗P < .05; ∗∗P < .005. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 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 after culture with lipids averaged from 25 NDs. C, Mean percentage of CD25+ iNKTs averaged from 25 NDs. 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 < .03; ∗∗P < .001. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 More milk-SM–activated iNKTs produce IL-4 compared with iNKTs activated by αGal. PBMCs from 25 NDs were cultured for 10 days with the indicated reagents and stimulated for 4 hours with PMA/ionomycin. A and B, Mean percentage of iNKTs expressing IL-4 or IFN-γ. C, Expansion of sort-enriched iNKTs cultured for 2 weeks with APCs prepulsed with the indicated reagents. D, Cytokine-mRNA expression of sorted iNKTs reported as relative expression of milk-SM or αGal versus DMSO-treated iNKTs (average from 5 different donors). ∗P < .05; ∗∗P < .005. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 iNKTs from children with FA-MA expand more and have a stronger Th2 response to milk-SM than those from non-FA children. PMBCs from 15 children with FA-MA, 12 children with FA-NMA, and 13 non-FA children were cultured for 10 days with food-SL or DMSO. A and B, Mean percentage of total iNKTs and CD25+ iNKTs when cultured with the indicated reagents. C and D, Mean percentage of iNKTs staining for IL-13 or IL-4. ∗P < .005. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Gating strategy for iNKT detection Gating strategy for iNKT detection. Representative profiles of iNKT frequencies detected in PBMCs (A) in a region made up of the lymphocytes by using (B) PBS57-loaded tetramer (upper dot plot) or unloaded human CD1d tetramer (lower dot plot), or (C) in combination with anti-CD3 or anti-Vα24 and anti-Vβ11 antibodies (right dot plot) or matched isotype control antibodies (left dot plot). FSC, Forward scatter; SSC, side scatter light. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Children with FA-MA have fewer PB-iNKTs Children with FA-MA have fewer PB-iNKTs. Fresh PBMCs from 15 children with FA-MA, 12 children with FA-NMA, and 13 non-FA children were stained ex vivo for iNKTs (CD3+Vα24+Vβ11+). A, Median (line in the box), 25th and 75th percentiles (lower and upper limit of the box), and range (upper and lower limits of whiskers) of the percentages of iNKTs. B, Mean absolute number (#) of iNKTs per mL blood ± SEM. C, Median, 25th and 75th percentiles, and range of absolute number of iNKTs per mL blood ± SEM. ∗P < .02; ∗∗P < .002. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

iNKTs from children with FA children expand to αGal iNKTs from children with FA children expand to αGal. PBMCs were cultured for 10 days with αGal or DMSO. A, Median, 25th and 75th percentiles, and range of the percentages of iNKTs. B, Mean fold induction of the percentages of iNKTs treated with αGal compared with those treated with DMSO. C, Mean absolute number (#) of iNKTs in PBMCs pretreated with αGal. D, Median, 25th and 75th percentiles, and range of the absolute number of iNKTs per 1 × 106 PBMCs. ∗P < .02; ∗∗P < .002. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

iNKTs from children with FA are activated by αGal iNKTs from children with FA are activated by αGal. PBMCs were cultured for 10 days with αGal or DMSO. A, Typical experiment showing percentage of iNKTs expressing CD25. B, Mean percentage of iNKTs expressing CD25. C, Median, 25th and 75th percentiles, and range of the percentages iNKTs expressing CD25. D, Mean fold induction of the percentage of CD25+ iNKTs after culture with αGal compared with those cultured with DMSO. ∗P < .02. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

More iNKTs from children with FA express IL-4 and IL-13 than iNKTs from non-FA patients. After 10 days in culture with αGal or DMSO, PBMCs were stimulated with PMA and ionomycin and stained for intracellular cytokines. A, D, G, Median, 25th and 75th percentiles, and range of percentage of iNKTs expressing IL-13, IL-4, and IFN-γ in αGal-stimulated cultures. B, E, H, Absolute number (#) ± SEM of iNKTs per 1 × 106 PBMCs expressing IL-4, IL-13, and IFN-γ, respectively. C, F, I, Median , 25th and 75th percentiles, and range of the absolute number of iNKTs per 1 × 106 PBMCs expressing IL-13, IL-4, and IFN-γ in αGal-stimulated cultures.∗P < .02; ∗∗P < .002. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Human CD1d tetramers loaded with milk-SM specifically bind iNKTs Human CD1d tetramers loaded with milk-SM specifically bind iNKTs. αGal-expanded PBMCs from 7 NDs were stained with lipid-loaded hCD1d tetramers and anti-CD3 antibodies, or where indicated, were blocked with PBS57-hCD1d and stained with food-lipid–loaded hCD1d tetramers and anti-CD3 antibodies. A, Median, 25th and 75th percentiles, and range of percentage iNKTs stained with the indicated tetramers. B, Mean absolute number (#) of iNKTs per 106 PBMCs that stained with the indicated tetramers. C, Median, 25th and 75th percentiles, and range of the absolute number of iNKTs per 106 PBMCs that stained with the indicated tetramers. ∗P < .05. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Milk-SM induces iNKT activation and expansion Milk-SM induces iNKT activation and expansion. PMBCs from 25 NDs were cultured for 10 days with indicated reagents and examined for expansion and CD25 expression. A and E, Median, 25th and 75th percentiles, and range of percentage iNKTs. B and F, Mean absolute number (#) of iNKTs (per 1 × 106 PBMCs). C and G, Median, 25th and 75th percentiles, and range of the absolute number of iNKTs (per 1 × 106 PBMCs). D, Mean percentage of iNKTs. H, Median, 25th and 75th percentiles, and range of percentage of CD25+ iNKTs. ∗P < .05; ∗∗P < .005. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

More milk-SM–expanded iNKTs produce IL-4 compared with iNKTs expanded by αGal. PBMCs from 25 NDs were cultured with the indicated reagents for 10 days, stimulated with PMA and ionomycin, and examined for cytokine production. A and D, Median, 25th and 75th percentiles, and range of the percentage of iNKTs. B and E, Mean absolute number (#) of iNKTs. C and F, Median and range of the absolute number of iNKTs. Fluorescence-activated cell sorting–sorted iNKTs from 5 different NDs were cultured with lipid-pulsed APCs for 72 hours, and release of IFN-γ (G) and IL-5 (H) was measured. ∗P < .05; ∗∗P < .003. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

iNKTs from children with FA-MA expand more in response to milk-SM compared with iNKTs from non-FA children. PMBCs were cultured for 10 days with/without food-SL and then examined for expansion and CD25 expression. A, A typical experiment. B, Median, 25th and 75th percentiles, and range of the percentage of iNKTs. C, Mean fold induction of the percentage ± SEM of iNKTs pretreated with indicated reagents compared with those pretreated with DMSO. D, Median, 25th and 75th percentiles, and range of the percentage of CD25+ iNKTs. E, Mean fold induction of iNKTs expressing CD25 after culture with the indicated reagents compared with culture with DMSO. ∗P < .05. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

iNKTs from children with FA-MA exhibit a stronger Th2 response to milk-SM than those from non-FA children. PMBCs were cultured for 10 days with/without food-SL and then stimulated with PMA and ionomycin. A, B, D, Median, 25th and 75th percentiles, and range of the percentages of IL-4+, IL-13+ or IFN-γ+ iNKTs, respectively. C, Mean percentage of iNKTs staining for IFN-γ. ∗P < .05; ∗∗P < .005. Journal of Allergy and Clinical Immunology 2011 128, 102-109.e13DOI: (10.1016/j.jaci.2011.02.026) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions