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, FIG 1. IVIG induces activation and cytokine secretion in IL-3-primed basophils. A-C, 660

, Isolated basophils from the human circulation were cultured either alone or with IVIG. (A 661 and B) Representative dot plots and expression (% positive cells and mean fluorescence 662 intensity (MFI)) of CD69 on the basophils (mean±SEM, n=6 donors). (C) Amount of 663 secretion of IL-4, IL-6 and IL-8 (mean±SEM

. Mann-whitney-test.-d-f, Basophils were cultured either alone or with IL-3. IVIG or HSA 665 were added following 2 hours stimulation with IL-3. (D) Representative histogram 666 overlays and MFI of CD69 expression on the basophils (mean±SEM

, Representative dot plots and % of basophils (mean±SEM, n=4 donors) positive for CD63, 668 (F) Effect of IVIG on the secretion (pg/ml) of IL-4, IL-6 and IL-8 (mean±SEM, n=12 669 donors

. **p<0, , p.1

*. P<0,

*. P<0, 0001; ns, 670 not significant, one-way ANOVA with Tukey's multiple comparison tests. G, Expression 671 of CD69 on the basophils of myopathy patients, before (Pre-IVIG) and Post-IVIG therapy

, Expression of type II FcRs, Siglecs and C-type lectin receptors on basophils, and 684 the effects of F(ab')2-and Fc-fragments of IVIG on basophil activation, FIG 3, vol.685

, Representative dot plots of CD23 and CD22 expression on the basophils. C and D, p.686

, E-H, Basophils were cultured either alone or with IL-688 3 for 24 hours. IVIG, F(ab')2 or Fc-fragments were added following 2 hours stimulation 689 with IL-3. (E and F) The expression of CD69 (mean±SEM, n=6 donors). (G and H) The 690 amount of secretion of IL-4, Representative dot plots and expression (% positive cells and MFI) of DCIR on the 687 basophils (mean±SEM, n=3 donors)

. **p<0, , p.1

*. P<0,

*. P<0, 001; ns, not significant, one-way ANOVA with Tukey's multiple

. Basophil-fc?ri-bound-ige, A and B, Modulation of Fc?RI expression (Representative 696 histogram overlays and mean±SEM, n=10 donors) in IL-3-primed basophils by IVIG. C, p.697

, Percentage of 698 basophils positive for the surface IgE and its intensity (MFI) (mean±SEM, n=5 donors). E 699 and F, Percentage of basophils positive for IVIG-binding (Representative dot plots and 700 mean±SEM, n=4 donors). G-I, The effect of anti-IgE-reactivity-depleted IVIG on (G, H) 701 the expression of CD69, Representative dot plots showing the basophils positive for surface IgE. D

, and (I) IL-4, and IL-8 secretion (mean±SEM, n=4 donors). *P<0, p.5

. **p&lt;0, , p.1

*. P&lt;0,

*. P&lt;0, 0001; ns, not significant, two-tailed Mann-Whitney test or one-way 704 ANOVA with Tukey's multiple comparison tests

, Activating and inhibitory CD32/Fc?RII are dispensable for the regulation of 707 basophil activation by IVIG. Basophils were cultured either alone or with IL-3 for 24 708 hours. IVIG or HSA were added following 2 hours stimulation with IL-3. A and B, FIG 5, vol.709

, Representative histogram overlays and mean fluorescence intensity (MFI) of expression 710 (mean±SEM, n=8 donors) of Fc?RII and Fc?RIIB on the basophils. C and D, Repercussion 711 of Fc?RIIB blockade on the (C) expression of CD69 and (D) amount of IL

, E and F, Repercussion of Fc?RIIA blockade on the 713 (E) expression of CD69 and (F) amount of IL-4 and IL-8 secretion (mean±SEM, n=4 714 donors

. **p&lt;0,

*. P&lt;0,

*. P&lt;0, 0001; ns, not significant, one-way 715 ANOVA with Tukey's multiple comparison tests

, FIG 6. Inhibition of Syk pathway abrogates IVIG-induced activation of basophils. A and 718

B. , Representative histogram overlays and mean±SEM (n=6 donors) of phosphorylated Syk 719 (pSyk) expression in basophils stimulated with IL-3 or IL-3 plus IVIG. C and D, The 720 effect of Syk inhibition by R406 towards IVIG-induced expression of CD69 721 (Representative histogram overlays and mean±SEM, n=5 donors). E, Syk inhibition 722 abrogates IVIG-induced IL-4, and IL-8 secretion (mean±SEM

. **p&lt;0, , p.1

*. P&lt;0,

*. P&lt;0, 0001; ns, not significant, one-way ANOVA with

, Inflammatoires rares et des Amyloses

, Hôpital Pitié-Salpêtrière

, HP

, Sorbonne Paris Cité, Paris, F-75006, France 19 5 Research Department, p.3014

, Immunologie et d'Allergologie; CHU d'Angers; Université d'Angers; INSERM 21 Unité1232; LabEx IGO "Immuno-Graft-Onco

F. Paris and F. ,

, Unit of Antibodies in Therapy and Pathology, 25 List of antibodies for flow cytometry and functional assays

. Cd63-pe, Clone:H5C6), CD13-APC (Clone:WM15), p.69

. Apc/cy7, Clone:FN50), CD209-APC (Clone:DCN46)

. Cd62l-fitc, Clone:DREG-56) were from BD Biosciences. Fc?RI?-FITC (Clone:CRA-1)

. Siglec3-fitc, Clone:REA214), SIGLEC8-APC (Clone:7C9), anti-IgE-APC (clone:MB10-5C4) MAbs 56 were obtained from Miltenyi Biotec. CD203c-PE (Clone:NP4D6), p.23

. Cd107a-bv421, Clone:9E8) MAbs were from BioLegend. Anti-IgE MAb (Clone:GE-1) was from Sigma

A. , Unconjugated and FITC-labelled Fc?RIIA MAb (Clone:IV.3) was purchased from

, Stem Cells Technologies. Human ST2/IL-33R-PE polyclonal goat IgG and Isotype control

, Anti-hFc?RIIB (Clone:2B6 variant N297D) MAbs 63 were coupled to Alexa Fluor 647 by using ThermoFisher Scientific kit and IVIG was labelled 64 with the Lightning-Link® Rapid DyLight® 650 kit, MAbs for blocking experiments were from R&D Systems