1. Selective ablation of mast cells or basophils reduces peanut-induced anaphylaxis in mice 
Laurent L. Reber, PhD, Thomas Marichal, DVM, PhD, Kaori Mukai, PhD, Yoshihiro Kita, PhD, Suzumi M. Tokuoka, PhD, Axel Roers, MD, Karin Hartmann, MD, Hajime Karasuyama, PhD, Kari C. Nadeau, MD, PhD, Mindy Tsai, DMSc, Stephen J. Galli, MD 
Journal of Allergy and Clinical Immunology 
Volume 132, Issue 4, Pages e11 (October 2013)
DOI: /j.jaci
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Effect of treatment with anti-Ly6G antibodies on PIA in WT and KitW-sh/W-sh mice. A, Peanut (PN)–induced hypothermia in peanut-sensitized C57BL/6J mice (WT; n = 19) and MC-deficient C57BL/6J-KitW-sh/W-sh mice (KitW-sh/W-sh; n = 18). B-E, Peanut-sensitized WT mice (n = 5) and MC-deficient KitW-sh/W-sh mice (n = 12-15) were treated intraperitoneally with 500 μg of neutrophil-depleting anti-Ly6G (α-Ly6G) or isotype control antibodies 24 hours before the challenge. Fig 1, B, Representative flow cytometric plots showing blood neutrophils (Siglec-F− [not shown], Gr-1high, and CD11b+). Fig 1, C, Percentage of blood neutrophils. Fig 1, D and E, Peanut-induced hypothermia in WT mice (Fig 1, D) and KitW-sh/W-sh mice (Fig 1, E) treated with an anti-Ly6G antibody (α-Ly6G) or isotype control antibody. Data are means ± SEMs or means + SEMs. *P < .05, **P < .01, or ***P < .001 versus the corresponding WT group (in Fig 1, A) or isotype control antibody–treated group (in Fig 1, C and E). #P < .05 versus the indicated group (in Fig 1, C). Each crossbones symbol indicates the death of 1 WT mouse.
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Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 2
Effect of DT-mediated CTMC depletion on PIA in Mcpt5-Cre;iDTR mice. A, MC numbers in the peritoneal lavage fluid from DT-treated Mcpt5-Cre−;iDTRfl/+ mice (n = 9) and Mcpt5-Cre+;iDTRfl/+ mice (n = 5). B and C, Toluidine blue staining for MCs (Fig 2, B) and MC numbers (Fig 2, C) in sections of ear skin, back skin, forestomach, and glandular stomach in DT-treated Mcpt5-Cre−;iDTRfl/+ mice (n = 6-9) and Mcpt5-Cre+;iDTRfl/+ mice (n = 5). D, Peanut-induced hypothermia in DT-treated peanut-sensitized Mcpt5-Cre−;iDTRfl/+ mice (n = 15) and Mcpt5-Cre+;iDTRfl/+ mice (n = 13). Data are means ± SEMs or means + SEMs. *P < .05, **P < .01, or ***P < .001 versus the corresponding Mcpt5-Cre−;iDTRfl/+ group. The crossbones symbol indicates the death of 1 mouse.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4. Fig 3
Effect of antibody-mediated basophil depletion on PIA. Peanut (PN)–sensitized C57BL/6J mice were treated intraperitoneally with 50 μg of the basophil-depleting antibody Ba103 (n = 9) or isotype control antibodies (n = 7) 48 hours before peanut challenge. A, Peanut-induced hypothermia. B and C, Representative flow cytometric plots (Fig 3, B) and percentages (Fig 3, C) of blood basophils (CD49b+ and IgE+). D, Numbers of MCs in the peritoneal lavage fluid. Data are means ± SEMs or means + SEMs. *P < .05 or ***P < .001 versus the isotype control antibody–treated group. Each crossbones symbol indicates the death of 1 mouse.
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5. Fig 4
Effect of DT-mediated basophil depletion on PIA in Mcpt8DTR mice. Peanut (PN)–sensitized Mcpt8+/+ (n = 10) and Mcpt8DTR/+ (n = 10) mice were treated with DT 48 hours before challenge with peanut. A, Peanut-induced hypothermia. B and C, Representative flow cytometric plots (Fig 4, B) and percentages (Fig 4, C) of blood basophils (CD49b+ and IgE+). D, Numbers of MCs in the peritoneal lavage fluid. Data are means ± SEMs or means + SEMs. *P < .05, **P < .01, or ***P < .001 versus the corresponding Mcpt8+/+ group. The crossbones symbol indicates the death of 1 mouse.
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Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6. Fig 5
PIA in Cpa3-Cre+;Mcl-1fl/fl MC- and basophil-deficient mice. A, Peanut (PN)–induced hypothermia in peanut-sensitized Cpa3-Cre+;Mcl-1+/+ (n = 6) and Cpa3-Cre+;Mcl-1fl/fl (n = 7) mice. B and C, Toluidine blue staining for MCs (Fig 5, B) and numbers of MCs (Fig 5, C) in sections of ear skin, forestomach, and glandular stomach. D, Numbers of MCs in the peritoneal lavage fluid. E and F, Representative flow cytometric plots (Fig 5, E) and percentages (Fig 5, F) of blood basophils (CD49b+ and IgE+). G-J, Mice were sensitized with cholera toxin and peanut (n = 5-7) or cholera toxin only for control mice (Ctr; n = 3-4) and killed 20 minutes after challenge with peanut to measure levels of histamine and PAF. Fig 5, G and H, Levels of histamine in plasma (Fig 5, G) and peritoneal lavage fluid (Fig 5, H). Fig 5, I and J, Levels of PAF in plasma (Fig 5, I) and spleen samples (Fig 5, J). Data are means ± SEMs or means + SEMs. *P < .05, **P < .01, or ***P < .001 versus the corresponding control group. N.S., Not significant.
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7. Fig E1
Levels of peanut (PN)–specific IgE and IgG1 in serum from peanut-sensitized WT and KitW-sh/W-sh mice. Serum from peanut-sensitized C57BL/6J mice (black circles, n = 15) and MC-deficient KitW-sh/W-sh mice (white circles, n = 13-15) was collected 24 hours before peanut challenge. Levels of peanut-specific IgE (A) and peanut-specific IgG1 (B) were measured by means of ELISA. Data are means ± SEMs.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8. Fig E2
Effect of treatment with an anti-Ly6G antibody on blood monocytes, eosinophils, and basophils in WT and KitW-sh/W-sh mice. Peanut-sensitized C57BL/6J mice (WT) and MC-deficient KitW-sh/W-sh mice were treated intraperitoneally with 500 μg of neutrophil-depleting anti-Ly6G antibody (α-Ly6G, n = 5-8) or an isotype control (n = 5) 24 hours before peanut challenge. Blood was isolated from all groups of mice 1 hour before peanut challenge, and blood leukocytes were analyzed by using flow cytometry. A, Gating strategy used to distinguish neutrophils (Gr-1+, CD11b+, and Siglec-F−), monocytes (Gr-1low, CD11b+, and Siglec-F−), eosinophils (SSChigh and Siglec-F+), and basophils (CD49b+ and IgE+). B, Percentage of blood monocytes (Gr-1low, CD11b+, and Siglec-F−). C, Percentage of blood eosinophils (SSChigh and Siglec-F+). D, Percentage of blood basophils (CD49b+ and IgE+). Data are means + SEMs. *P < .05 or ***P < .001 versus the corresponding isotype control–treated group. #P < .05 versus the indicated group (unpaired Student t test).
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9. Fig E3
Effect of treatment with DT on blood basophils, monocytes, eosinophils, and neutrophils in Mcpt5-Cre;iDTR mice. Mcpt5-Cre−;iDTRfl/+ (Cre−; n = 9) and Mcpt5-Cre+;iDTRfl/+ (Cre+; n = 5) mice were treated intraperitoneally with 500 ng of DT once a week starting at the first oral sensitization with peanut. Blood was isolated from all groups of mice 1 hour before peanut challenge, and blood leukocytes were analyzed by means of flow cytometry. A, Percentage of blood basophils (CD49b+ and IgE+). B, Percentage of blood monocytes (Gr-1low, CD11b+, and Siglec-F−). C, Percentage of blood eosinophils (SSChigh and Siglec-F+). D, Percentage of blood neutrophils (Gr-1high, CD11b+, and Siglec-F−). Data are means + SEMs. N.S., Not significant (P > .05, unpaired Student t test).
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10. Fig E4
Levels of peanut (PN)–specific IgE and IgG1 in serum from DT-treated peanut-sensitized Mcpt5-Cre−;iDTR+ and Mcpt5-Cre+;iDTR+ mice. Serum from peanut-sensitized Mcpt5-Cre−;iDTR+ mice (black circles, n = 11-13) and MC-depleted Mcpt5-Cre+;iDTR+ mice (red circles, n = 5-13) was collected 24 hours before peanut challenge. Levels of peanut-specific IgE (A) and peanut-specific IgG1 (B) were measured by means of ELISA. Data are means ± SEMs.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11. Fig E5
Effect of treatment with the basophil-depleting antibody Ba103 on blood monocytes, eosinophils, and neutrophils. Peanut-sensitized C57BL/6J mice were treated intraperitoneally with 50 μg of a basophil-depleting Ba103 antibody (n = 9) or an isotype control (n = 7) 48 hours before peanut challenge. Blood was isolated from all groups of mice 1 hour before peanut challenge, and blood leukocytes were analyzed by means of flow cytometry. A, Percentage of blood monocytes (Gr-1low, CD11b+, and Siglec-F−). B, Percentage of blood eosinophils (SSChigh and Siglec-F+). C, Percentage of blood neutrophils (Gr-1high, CD11b+, and Siglec-F−). Data are means + SEMs. N.S., Not significant (P > .05, unpaired Student t test).
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12. Fig E6
Effect of treatment with DT on blood monocytes, eosinophils, and neutrophils in Mcpt8DTR mice. Peanut-sensitized Mcpt8+/+ (n = 9) and Mcpt8DTR/+ (n = 9) mice were treated intraperitoneally with 500 μg of DT 48 hours before peanut challenge. Blood was isolated from all groups of mice 1 hour before peanut challenge, and blood leukocytes were analyzed by means of flow cytometry. A, Percentage of blood monocytes (Gr-1low, CD11b+, and Siglec-F−). B, Percentage of blood eosinophils (SSChigh and Siglec-F+). C, Percentage of blood neutrophils (Gr-1high, CD11b+, and Siglec-F−). Data are means + SEMs. N.S., Not significant (P > .05, unpaired Student t test).
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13. Fig E7
Levels of peanut (PN)–specific IgE and IgG1 in serum from peanut-sensitized Cpa3-Cre+;Mcl-1+/+ and Cpa3-Cre+;Mcl-1fl/fl mice. Serum from peanut-sensitized Cpa3-Cre+;Mcl-1+/+ mice (black circles, n = 6-7) and MC- and basophil-deficient Cpa3-Cre+;Mcl-1fl/fl mice (gray circles, n = 5-6) was collected 24 hours before the challenge. Levels of peanut-specific IgE (A) and peanut-specific IgG1 (B) were measured by means of ELISA. Data are means ± SEMs.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14. Fig E8
Responses of WT mice, MC-deficient KitW-sh/W-sh mice, and KitW-sh/W-sh mice engrafted with BMCMCs in a moderate model of PIA. C57BL/6J mice (WT), MC-deficient C57BL/6J-KitW-sh/W-sh mice (KitW-sh/W-sh), and C57BL/6J-KitW-sh/W-sh mice engrafted with WT BMCMCs (2 × 106 cells administered intraperitoneally plus 107 cells administered intravenously 12 weeks before the first sensitization with peanut; WT BMCMCs→KitW-sh/W-sh) were sensitized orally once a week for 4 weeks with 10 mg of peanut plus 10 μg of cholera toxin. Mice were challenged with 2.5 mg of peanut administered intraperitoneally 2 weeks after the last sensitization. A, Peanut (PN)–induced hypothermia. The crossbones symbol indicates the death of 1 mouse. B, MCs in the peritoneal lavage fluid or mesentery (“mesenteric windows”) are indicated by arrows in photomicrographs of cells from the peritoneal cavity (May-Grunwald-Giemsa stain) or of the mesenteric windows (Csaba stain). C and D, MC numbers in the peritoneal lavage fluid (Fig E8, C) and mesenteric windows (Fig E8, D). E and F, Toluidine blue staining for MCs (Fig E8, E) and MC numbers (Fig E8, F) in sections of forestomach, glandular stomach, lung, spleen, and back skin. Data in Fig E8, A, C, D, and F are means ± SEMs or means + SEMs from 8 to 14 (Fig E8, A) or 4 to 6 (Fig E8, C, D, and F) mice per group. #P < .05, ##P < .01, or ###P < .001 versus the KitW-sh/W-sh group (in Fig E8, A), *P < .05, **P < .01, and ***P < .001 versus the WT group (in Fig E8, A) or for comparisons indicated (in Fig E8, C, D, and F). N.S., Not significant (P > .05, unpaired Student t test).
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

15. Fig E9
Effect of basophil depletion in a moderate model of PIA. All mice were sensitized orally once a week for 4 weeks with 10 mg of peanut plus 10 μg of cholera toxin and challenged with 2.5 mg of peanut (PN) administered intraperitoneally 2 weeks after the last sensitization. A and B, Percentage of blood basophils (CD49b+ and IgE+) in blood samples collected 1 hour before challenge with peanut (Fig E9, A) and peanut-induced hypothermia (Fig E9, B) in peanut-sensitized C57BL/6J mice treated intraperitoneally with 50 μg of the basophil-depleting antibody Ba103 (n = 7) or isotype control antibodies (n = 8) 48 hours before peanut challenge. C and D, Percentage of blood basophils (CD49b+ and IgE+) in blood samples collected 1 hour before challenge with peanut (Fig E9, C) and peanut-induced hypothermia (Fig E9, D) in peanut-sensitized Mcpt8+/+ (n = 6) and Mcpt8DTR/+ (n = 7) mice treated with 500 ng of DT 48 hours before challenge with peanut. P > .05, unpaired Student t test for all time points. The crossbones symbol indicates the death of 1 isotype control antibody-treated mouse. ***P < .001.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions